Dissertation Camelia Vlad .pdf Sequence - uni-konstanz.de

Oligomerization, Degradation and Aggregation Reactions and Products of Synuclein Polypeptides Related to

Parkinson’s Disease

Dissertation

zur Erlangung des akademischen Grades

eines Doktors der Naturwissenschaften

an der Universität Konstanz

Fachbereich Chemie

vorgelegt von

Camelia Vlad

Konstanz 2011

Tag der mündlichen Prüfung: Mittwoch, den 27. Juli 2011 1. Referent: Prof. Dr. Dr. h.c. Michael Przybylski 2. Referent: Prof. Dr. Bastian Hengerer

Vorsitzender der Prüfungskommission: Prof. Dr. Jörg Hartig

http://nbn-resolving.de/urn:nbn:de:bsz:352-147880

“But although, at present, uninformed as to the precise nature of the disease, still it ought not to be considered as one against which there exists no countervailing remedy.”

James Parkinson (1817), An Essay on the Shaking Palsy

I dedicate this work to my wonderful parents Maricica and Alexandru Vlad,

and to my loving husband Stefan.

The present work has been performed in the time from October 2007 to

December 2010 in the Laboratory of Analytical Chemistry and Biopolymer

Structure Analysis, Department of Chemistry of the University of Konstanz,

under the supervision of Prof. Dr. Dr. h. c. Michael Przybylski. This thesis has

been also carried out within the Konstanz Research School Chemical Biology of

the University of Konstanz.

Special thanks to:

Prof. Dr. Dr. h. c. Michael Przybylski for giving me the opportunity to work in his

group, for the interesting research topic and discussions concerning my work

and for his entire support;

Prof. Dr. Bastian Hengerer, for writing the second evaluation of my thesis;

Prof. Dr. Bastian Hengerer for giving me the possibility to work for a short-term

research stay at Boehringer Ingelheim, Biberach, Germany and supporting and

encouraging me during this time;

Dr. Karin Danzer and Dr. Thomas Ciossek, Boehringer Ingelheim, Biberach,

Germany, for providing the α-synuclein, antibodies and mouse brain

homogenates employed in this work;

Prof. Marcel Leist and Christiaan Karreman for providing the recombinant

α-synuclein fragments (72-140) and (1-120);

Nick Tomczyk, Dr. John Rontree and Dr. James Langridge, Waters Ltd.,

Manchester, for help and assistance with IMS-MS analysis;

Konstanz Research School Chemical Biology for the financial and scientific

support;

Gleichstellungsrat, University of Konstanz for the financial support;

All former and present members of the group for the wonderful atmosphere,

scientific discussions and advices during my work;

Last but not least I wish to thank my family and all my friends for their entire

support.

This dissertation has been published in part, and presented at the following

conferences:

Publications

1 Dragusanu, M., Petre, B.A., Slamnoiu, S., Vlad, C., Tu, T. and Przybylski, M. (2010) On-line bioaffinity-electrospray mass spectrometry for simultaneous detection, identification, and quantification of protein-ligand interactions. J. Am. Soc. Mass Spectrom., 21, 1643-1648.

2 Vlad, C., Iurascu, M.I., Slamnoiu, S., Hengerer, B. and Przybylski, M.

(2011) Characterization of oligomerization-aggregation products of neurodegenerative target proteins by ion mobility mass spectrometry. Meth. Mol. Biol., - in press.

3 Vlad, C., Lindner, K., Karreman, C., Schildknecht, S., Leist, M.,

Tomczyk, N., Rontree, J., Langridge, J., Danzer, K., Ciossek, T., Hengerer, B. and Przybylski, M. (2011) Proteolytic Fragments as Intermediates in the Oligomerization-aggregation of Parkinson’s Disease Protein Alpha-Synuclein Revealed by Ion mobility Mass Spectrometry. Angew. Chem. Int. Ed., - submitted.

4 Vlad, C., Slamnoiu, S., Ciossek, T., Hengerer, B. and Przybylski, M.

(2011) Identification and quantification of synucleins by combination of bioaffinity and mass spectrometry, - in preparation.

Conferences presentations Oral presentations

1 Vlad, C., Slamnoiu, S., Ciossek, T., Hengerer, B. and Przybylski, M. (2010, 28th – 30th July) Affinity binding studies of α-synuclein and anti-α-synuclein antibodies by online combination of SAW biosensor and ESI-MS. 2nd KoRS-CB Retreat, Hornberg, Germany

2 Vlad, C., Lindner, K., Slamnoiu, S., Tomczyk, N., Langridge, J.,

Hengerer, B. and Przybylski, M. (2011, 20th – 22nd May) New approaches for characterization of α-Synuclein oligomerization-aggregation products using ion mobility mass spectrometry and on-line SAW-ESI-MS. 3rd Annual PhD Retreat, Feldberg, Germany

Poster presentations

1 Vlad, C., Danzer, K., Hengerer, B., Otto, M., and Przybylski, M. (2007) Structural characterization of α-synuclein and epitope identification with an anti α-synuclein antibody by mass spectrometric epitope excision and extraction. 40th Annual Meeting of the German Society for Mass Spectrometry, Bremen, Germany

2 Vlad, C., Danzer, K., Hengerer, B., Otto, M., and Przybylski, M. (2008)

Structural characterization and antibody- epitope identification of Parkinson’s disease target protein α-synuclein using affinity- mass spectrometry. 56th ASMS, Denver, USA

3 Vlad, C., Danzer, K., Hengerer, B., Tomczyk, N., Rontree, J., and

Przybylski, M. (2009) Structural and biochemical characterization of α-synuclein truncation and oligomerization products related to Parkinson's disease using electrospray mass spectrometry and ion mobility mass spectrometry. 42th Annual Meeting of the German Society for Mass Spectrometry, Konstanz, Germany

4 Vlad, C., Danzer, K., Hengerer, B., Otto, M., and Przybylski, M. (2009)

Structural characterization and affinity- mass spectrometric identification of α-synuclein epitopes related to Parkinson’s disease. 9th International Conference on Alzheimer’s and Parkinson’s Diseases, Prague, Czech Republic


Przybylski, M. (2009) Structural and biochemical characterization of α-synuclein truncation and oligomerization products using ion-mobility mass spectrometry. 1st KoRS-CB Retreat, Hornberg, Germany


Przybylski, M. (2009) Identification and characterization of α-synuclein truncation and oligomerization products related to Parkinson's disease using affinity- and ion-mobility mass spectrometry. 18th International Mass Spectrometry Conference, Bremen, Germany


Przybylski, M. (2009) Identification and characterization of α-synuclein truncation and oligomerization products related to Parkinson's disease using affinity- and ion-mobility mass spectrometry. Proteome Binder Workshop / DGMS: Affinity-Mass Spectrometry, Konstanz, Germany

8 Vlad, C., Danzer, K., Hengerer, B., Tomczyk, N., Rontree, J., and Przybylski, M. (2010) Identification and characterization of α-synuclein truncation and oligomerization products related to Parkinson's disease using affinity- and ion-mobility mass spectrometry. 1st International Conference of the RSMS, Sinaia, Romania

9 Vlad, C., Slamnoiu, S., Hengerer, B. and Przybylski, M. (2010) Affinity

binding studies of synuclein and anti-synuclein antibodies by online combination of SAW biosensor and ESI-MS. 31st European Peptide Symposium, Copenhagen, Denmark

10 Vlad, C., Lindner, K., Rontree, J., Tomczyk, N., Langridge, J., Leist, M., Hengerer, B. and Przybylski, M. (2010) New approaches for characterization of reaction intermediates and conformation-dependent α-Synuclein aggregation using ion-mobility mass spectrometry, Thioflavin T assay and online SAW-ESI-MS. 2nd International Workshop Affinity- Mass Spectrometry – Biochemical and Medical Applications, DGMS - Fachgruppe Affinity-Mass Spectrometry, Konstanz, Germany

11 Vlad, C., Danzer, K., Tomczyk, N., Rontree, J., Hengerer, B. and

Przybylski, M. (2011) Identification and characterization of α-Synuclein truncation and oligomerization products related to Parkinson's disease using affinity- and ion- mobility mass spectrometry. 10th International Conference on Alzheimer’s and Parkinson’s Diseases, Barcelona, Spain

12 Vlad, C., Slamnoiu, S., Dragusanu, M., Ciossek, T., Hengerer, B. and Przybylski, M. (2011) Affinity binding studies of α-synuclein and derived peptides to anti-α-synuclein antibodies by an online combination of surface acoustic wave biosensor and mass spectrometry. 10th International Conference on Alzheimer’s and Parkinson’s Diseases, Barcelona, Spain

13 Vlad, C., Lindner, K., Slamnoiu, S., Tomczyk, N., Langridge, J.,

Hengerer, B. and Przybylski, M. (2011) New approaches for characterization of reaction intermediates and conformation-dependent α-Synuclein aggregation using ion-mobility mass spectrometry, Thioflavin T assay and online SAW-ESI-MS. 3rd KoRS-CB Retreat, Blaubeuren, Germany

14 Vlad, C., Lindner, K., Ciossek, T., Hengerer, B., Tomczyk, N., Rontree,

J., Leist, M. and Przybylski, M. (2011) Chemically modified and proteolytic intermediates in the oligomerization-aggregation pathway of alpha-synuclein related to Parkinson’s disease. Gordon Research Conferences, Andover, NH, USA

I

TABLE OF CONTENTS

1 INTRODUCTION I

1.1 Intrinsically disordered proteins: Discovery, structural classification and chemical basis of aggregation 1

1.2 Neurodegenerative aspects of protein aggregation: Chemistry and reaction pathways 8

1.3 Synucleins: Relationship to Parkinson’s disease 12

1.4 Analytical methods for protein structure analysis and characterization of aggregates 18

1.5 Scientific goals of the dissertation 24

2 RESULTS AND DISCUSSION 27

2.1 Structure determination of synucleins 27

2.1.1 Primary structure determination of synucleins by mass spectrometry 27

2.1.2 Secondary structure characterization of synucleins by circular dichroism 31

2.1.3 Primary structure characterization of synucleins by mass spectrometric peptide mapping 33

2.2 Formation, electrophoretic separation and isolation of oligomerization-aggregation products of synucleins 39

2.2.1 In vitro oligomerization-aggregation products of synucleins 40

2.2.1.1 Separation of oligomerization-aggregation products of synucleins using Tris-tricine gel electrophoresis 40

2.2.1.2 Immunoanalytical characterization of oligomerization-aggregation products of synucleins 43

2.2.2 In vivo oligomerization-aggregation products of synucleins 46

2.2.2.1 Isolation of α-synuclein oligomerization-aggregation products from human α-synuclein transgenic mouse brain homogenates using 1D and 2D-gel electrophoresis 48

2.2.2.2 Biochemical characterization of oligomerization-aggregation products from human α-synuclein transgenic mouse brain homogenates by Western blot analysis 50

II

2.2.2.3 Separation and biochemical characterization of α-synuclein oligomerization-aggregation products from human neuroblastoma cell culture by gel electrophoresis and Western blot analysis 54

2.3 Structure identification of oligomerization and degradation products of α-synuclein by ion-mobility mass spectrometry 55

2.3.1 Primary structure characterization of in vitro oligomers by electrospray ionization mass spectrometry 56

2.3.2 Detection and sequence determination of α-synuclein oligomers and degradation products 60

2.3.3 Elucidation of oligomers and proteolytic products in vitro by ion-mobility mass spectrometry 63

2.3.3.1 Methodology of ion-mobility mass spectrometry 63

2.3.3.2 Identification of full-length and proteolytic fragments of α-synuclein 66

2.4 Epitope identification of synuclein- antibodies by affinity-mass spectrometry 73

2.4.1 Preparation of immobilized anti-α-synuclein specific antibodies 73

2.4.2 Binding characterization of α-synuclein to antibody columns by affinity- mass spectrometry 74

2.4.3 Epitope identification of α-synuclein-specific antibodies 76

2.4.3.1 Methodology of mass spectrometric epitope analysis 76

2.4.3.2 Epitope identification of α-synuclein by mass spectrometric epitope excision and epitope extraction 79

2.5 Synthesis and structural characterization of synuclein polypeptides 84

2.5.1 Synthesis and structural analysis of α-synuclein peptides and peptide models 84

2.5.2 Structural characterization of synthetic α-synuclein polypeptides 92

2.5.3 Preparation and characterization of chemically modified α-synuclein 97

2.6 Identification and quantification of synucleins by combination of bioaffinity and mass spectrometry 101

III

2.6.1 Interactions of synuclein proteins and peptides with anti-synuclein specific antibodies 104

2.6.2 Determination of dissociation constants of anti-synuclein antibody-ligand complexes 107

2.6.3 Interaction studies of synucleins and anti-synucleins antibodies by online bioaffinity- MS 112

2.6.4 Isolation and structure determination of in vivo oligomerization-aggregation products 121

2.6.4.1 Structure characterization of in vivo oligomers by affinity mass spectrometry 121

2.6.4.2 Online SAW-MS identification of in vivo oligomerization products of αSyn from mouse brain homogenate 127

2.7 Characterization of oligomerization-aggregation of synucleins and synuclein peptides 130

2.7.1 Oligomerization-aggregation analysis by gel electrophoresis and immunoanalytical methods 130

2.7.2 Oligomerization-aggregation analysis by ion-mobility mass spectrometry 134

2.7.3 Oligomerization-aggregation analysis by Thioflavin-T assay 136

2.7.4 Characterization of oligomerization-aggregation of chemically modified α-synuclein 140

3 EXPERIMENTAL PART 142

3.1 Materials and reagents 142

3.1.1 Buffers and stock solutions 142

3.1.2 Proteins 145

3.1.3 Antibodies 145

3.2 Sample preparation for proteome analysis 146

3.2.1 Expression of recombinant α-synuclein 146

3.2.2 α-Synuclein oligomers preparation 147

3.2.3 Succinylation of amino groups on α-synuclein 148

3.2.4 Preparation of mouse brain homogenates 148

IV

3.2.5 Human SH-SY5Y-A53T neuroblastoma cell culture 151

3.2.6 BCA assay for protein quantification 152

3.2.7 Acetone precipitation for removal of contaminants 153

3.3 Solid phase peptide synthesis 153

3.4 Chromatographic and electrophoretic separation methods 155

3.4.1 High performance liquid-chromatography (HPLC) 155

3.4.2 ZipTip clean up procedure 156

3.4.3 One dimensional gel electrophoresis 157

3.4.4 Two-dimensional gel electrophoresis 159

3.4.5 Colloidal Coomassie Brilliant Blue Staining 161

3.4.6 Silver Staining 163

3.4.7 Bioanalyzer gel reader for native fluorescence detection 163

3.5 Proteolytic digestion 164

3.5.1 Digestion in solution 164

3.5.2 Proteolytic in gel digestion 166

3.6 Immunological assays 166

3.6.1 Dot blot and Western blot 166

3.6.2 Preparation of antibody columns 169

3.6.3 Affinity-mass spectrometry methods 170

3.6.4 Epitope excision and extraction experiments 171

3.6.1 Enzyme-linked immunosorbent assay 173

3.7 Thioflavin-T assay 175

3.8 Circular dichroism spectroscopy 176

3.9 Mass spectrometric methods 177

3.9.1 MALDI-TOF mass spectrometry 178

3.9.2 FTICR mass spectrometry 181

V

3.9.2.1 MALDI-FTICR mass spectrometry 182

3.9.2.2 Nano-ESI-FTICR mass spectrometry 182

3.9.3 ESI-ion trap mass spectrometry 183

3.9.4 LC-ESI-ion trap mass spectrometry 187

3.9.5 Linear trap quadrupole (LTQ) Orbitrap mass spectrometry 188

3.9.6 Ion-mobility mass spectrometry 190

3.10 Programs for mass spectrometry 191

3.10.1 GPMAW 191

3.10.2 Data Analysis 192

3.10.3 Mascot 192

3.10.4 HyperChem 6.0 193

3.10.5 BallView 1.1.1 193

3.10.6 PDQuest software 193

3.11 SAW biosensor 193

3.12 Online of SAW-ESI-MS 196

3.13 Edman sequence determination 199

4 SUMMARY 202

5 ZUSAMMENFASSUNG 206

6 BIBLIOGRAPHY 210

7 APPENDIX 236

7.1 Appendix 1 236

7.2 Appendix 2 238

7.3 Appendix 3 240

7.4 Appendix 4 241

1 INTRODUCTION 1

1 INTRODUCTION

1.1 Intrinsically disordered proteins: Discovery, structural classification and chemical basis of aggregation

The post- genome era brought many challenges and raised new questions

in the fields of biochemistry and molecular biology, most important being the

identification and characterization of the functions of the encoded protein

sequences. An understanding of functions, as well as molecular causes of

malfunction, can be obtained by casting light on protein interactions with

themselves and with other biomolecules. For explaining such weak interactions,

(also called bioaffinities), a major paradigm, introduced more than 100 years

ago by the “lock-and-key” model formulated for explaining the specificity of

enzymatic hydrolysis of glycosides [1], is the strong correlation with the three-

dimensional (3D) structure of proteins. However, in recent years, this paradigm

has been challenged by the discovery of the intrinsically disordered or natively

unstructured proteins (IDPs), which are receiving increased attention because

of the functional importance inherent to their flexible nature [2-10]. In structural

terms, IDPs resemble denatured states of ordered proteins, characterized by an

ensemble of rapidly interconverting alternative conformations [11]. Structural

disorder confers many advantages on proteins, such as an increased speed of

interaction, specificity without excessive binding strength and adaptability in

binding [11, 12]. The number of full-length proteins and domains that have been

classified as IDPs already exceeds > 1000, increasing rapidly, and a database

of protein disorder has been established (DisProt) [3, 13]. IDPs differ from normal

ordered proteins at structural, functional and conformational levels. Both

ordered and disordered proteins are polypeptides. The functional role of IDPs

has been established in important areas such as transcriptional regulation,

translation, cellular signal transduction, gene expression and chaperone action [4, 8, 12, 14-16]. Importantly, it was emphasized that the majority of IDPs undergo a

disorder-to-order transition upon functioning [17].

1 INTRODUCTION 2

In 2002, Uversky challenged the “Protein Trinity” model introduced in 2001

by Dunker by extending it to a new concept called the “Protein Quartet”, in

which globular proteins (native intracellular proteins), stabilized by non-covalent

interaction (conformational forces), exist in at least four different equilibrium

conformations: folded (ordered, native), molten globule, pre-molten globule and

unfolded (random coil) [6] (Figure 1). Furthermore, the IDPs were divided into

three subclasses: native molten globules with native- like secondary structure

but disordered tertiary structure, native premolten globules with a different

phase state of the protein which are separated by the first-order phase

transition [18] and native coils [9]. In practice, no protein is ever a completely

random coil, but the term is a convenient shorthand for the ensemble of

conformations that occur for an unfolded protein.

Ordered

Random coil

Pre-MoltenGlobule

MoltenGlobule

Figure 1: Extension of the “Protein Trinity” to the “Protein Quartet” model of protein

functioning, showing the four specific conformations of the polypeptide chain and transitions between any of the states. In blue is the fourth introduced state: pre-molten globule [6].

A prime argument that IDPs differ from globular proteins in vivo relates to

the low sequence complexity, with amino acid compositional bias and high

predicted flexibility [19, 20]. The IDPs amino acid composition is enriched in

disorder- promoting amino acids (Ala, Arg, Gly, Gln, Ser, Pro, Glu, Lys) and

depleted in order-promoting amino acids, including bulky hydrophobic (Ile, Leu,

Val) and aromatic residues (Trp, Tyr, Phe) which would normally form the

hydrophobic core of a folded protein. Likewise, the IDPs possess low content of

Cys and Asn residues [17]. A high proline (P) content is linked with lack of

structure and disfavors a rigid secondary structure. PEST sequences (rich in

1 INTRODUCTION 3

Pro, Glu, Ser, and Thr) are often present as unstructured regions of a protein

and located preferentially in the C-terminal regions. The average of amino acid

frequencies in IDPs extracted from the DisProt database have been recently

illustrated [9]. Moreover, it has been found that the combination of low mean

hydrophobicity, defined as the sum of the normalized hydrophobicity of all

residues divided by the number of residues in a polypeptide, and relatively high

net charge, defined as the net charge at pH 7 divided by the total number of

residues, constitutes an important precondition for the lack of compact structure

in extended IDPs [7, 21].

IDPs are characterized by low conformational stability, which is reflected

in the presence of large concentrations of strong denaturants, such as urea or

guanidinium chloride (GdmCl) showing low cooperativity (or the complete lack

thereof) of the denaturant- induced unfolding. The analysis of the temperature

effects on structural properties of IDPs revealed that native coils and pre-molten

globules possess so-called “turned out” response to heat. An increase in

temperature induces the partial folding of IDPs, rather than the typical unfolding

of ordered globular proteins, due to the increased strength of the hydrophobic

interaction at high temperature, leading to a stronger hydrophobic attraction,

which is the major driving force for folding. A key experimental method for

obtaining information regarding the temperature effects on IDPs is far-UV

circular dichroism (CD). It had been shown that at high temperature around 30-

50°C, proteins like α-synuclein [22-25], caldesmon 636-771 fragment [26], α-casein [27] and receptor extracellular domain of nerve growth factor [28] adopt a partially

folded conformation. Partial folding of extended IDPs is also characterized by

the “turned out” response to changes in pH. A decreased pH from 5.5 to 3.0

induces changes in the far-UV CD spectra of proteins leading to the formation

of partially folded premolten globule-like conformation [22-25, 29, 30]. Under

physiological pH, extended IDPs are essentially unfolded because of the strong

electrostatic repulsion between the non-compensated charges of the same sign

and because of the low hydrophobic attraction. Furthermore, the presence of

various counter ions provides ordered conformations to extended IDPs. Many

cations (monovalent, bivalent and trivalent) were shown to induce

1 INTRODUCTION 4

conformational changes in IDPs by transforming them into partially folded

conformation. The validity of this assumption was illustrated for several IDPs

using far-UV CD [9, 22-25].

The membrane surface is considered as one of the protein structure

modifying factors of the living cell and actually, several ordered proteins lose the

rigid 3D structure upon interaction with membranes. Additionally, many IDPs

are able to gain pronounces α-helical structures as a result of efficient binding

to artificial and natural membranes, as well as under model conditions

mimicking the effect of the membrane field (organic solvents). The effectiveness

of proteins such as human α-synuclein [31, 32] or human Jagged-1 [33] (one of the

five ligands to Notch receptors) interactions with vesicles is highly dependent on

the phospholipid composition, the protein to phospholipid ratio, and the size of

the vesicles. Therefore, the binding of these proteins to the negatively charged

vesicles was shown to be accompanied by a dramatic increase in α-helical

content. The increase in helicity was detected by far-UV CD and Fourier-

transform infrared (FTIR) spectroscopy [34, 35]. α-Synuclein (αSyn), key

polypeptide in the pathogenesis of Parkinson’s disease and other related

synucleinopathies, was found in the free cytosolic fraction of the cell. The amino

acid sequence of αSyn consists of 140 residues with 7 copies of an unusual 11-

residue repeat, followed by a hydrophilic tail (Figure 2a). The primary sequence

of human αSyn can be divided into 3 domains (Figure 2a): (1), residues 1-60 of

the N-terminal end shown in black; (2), the central region (green) with residues

61-95 and (3), the carboxy terminal region with residues 96-140 shown in

brown. Based on sequence analysis, it was suggested that αSyn interacts with

lipid membranes through its repeat region [36, 37] and interactions with small

unilamellar vesicles (SUVs) and micelles preferentially containing negatively

charged head groups have been documented in vitro [36, 38]. The structure and

dynamics of αSyn in the micelle-bound form were determined by solution NMR

spectroscopy related to the vesicle-bound state [34]. Figure 2b shows the

structure and dynamics of micelle-bound αSyn in which Val3-Val37 and Lys45-

Thr92 form curved α-helices, connected by a well ordered, extended linker in an

1 INTRODUCTION 5

unexpected anti-parallel arrangement, followed by another short extended

region (Gly93-Lys97), and a highly mobile tail (Asp98-Ala140).

aMDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH

GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL

GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA

1

51

101 140

b

- N C-

Figure 2: (a), Schematic representation of the primary structure of α-synuclein (αSyn)

divided in three regions: N-terminal (residues 1-60) shown in black, central region (residues 61-95) in green and C-terminal region (residues 96-140) in brown; (b), Ribbon diagram of the NMR structure of micelle-bound αSyn adapted from Ulmer, T. (2005) (PDB accession number, 1XQ8) [34]. Helix-N (Val3-Val37) and helix-C (Lys45-Thr92) are connected by a short linker, followed by another short extended region (Gly93-Lys97) and a predominantly unstructured tail (Asp98-Ala140). (Figure prepared using the BallView 1.1.1. program).

Nevertheless, the membrane-bound protein has been suggested to play an

important role in fibril formation [39]. This protein may exist in two structurally

different isoforms in vivo: a helix-rich, membrane-bound form and a disordered,

cytosolic form, with the membrane-bound protein generating nuclei that seed

the aggregation of the more abundant cytosolic form [39]. The effect of organic

solvents on proteins conformation has been shown for αSyn [40] and many other

IDPs revealing the dramatic conformational changes in the presence of different

concentrations of fluorinated alcohols [41-43]. Further, the conformation IDPs

adopt is largely defined by their interacting partners and not so much by their

amino acid sequence as for globular proteins. Thus, a unique feature of IDPs is

their capability to fold in the presence of natural binding partners; i.e., being

1 INTRODUCTION 6

involved in one-to-many interactions [44] which is commonly characterized by the

IDP polymorphism in bound state. An example is the intrinsically disordered C-

terminal fragment (residue 374-388) of p53 transcription factor which adopts

four completely different crystal structures when bound to different partners [45,

46]. p53 is a key player in a large signalling network involving the expression of

genes carrying out such processes as cell cycle progression, apoptosis

induction, DNA repair, response to cellular stress. Loss of p53 function, either

directly through mutations or indirectly through several other mechanisms, is

often accompanied by cancerous transformation [47]. Figure 3 shows the four

complexes (crystal structures) of the C-terminal p53: helix when bound to

S100ββ [45], a sheet when bound to sirtuin [48], and a coil with distinct backbone

trajectories when bound to CBP [49] and cyclin A2 [46, 50].

365 HSSHLKSKKGQSTSRHKKLMFKTEGPDSD-COO -

S100ββ-p53complex

Sirtuin-p53complex

CBP-p53complex

Cyclin A2-p53complex

Figure 3: Sequence and structure comparison for the four overlapping complexes in the

C-terminus of p53. The four complexes display all three major secondary structure types: primary, secondary, and quaternary structure. The core span becomes a helix when binding to S100ββ, a sheet when binding to sirtuin, and a coil with two distinct backbone trajectories when binding to CBP and cyclin A2. Modified from [46].

In terms of the ensuing functional modes, IDPs can be classified into six

functional classes [5, 11]. In the first class, IDPs function stems from the ability of

1 INTRODUCTION 7

proteins to fluctuate over an ensemble of structural states (entropic chains). In

the other five classes, IDPs function is realized via permanently or transiently

binding to one or several partner molecules. Of those permanently binding their

partner(s) belong three subclasses of effectors (bind and modify the activity of

their partner enzyme with an inhibitory and, occasionally, activatory effect),

assemblers (assemble multi-protein complexes and/or target the activity of

attached domains) and scavengers (store and/or neutralize small ligands) [2, 4].

Of those transiently binding their partner(s) belong two subclasses of display

sites (sites of post-translational modification) and chaperones (assist the folding

of RNA or protein) [15, 16].

Another typical feature of the conformational behaviour of IDPs is their

capability as naturally occurring osmolytes; e.g. trimethylamine-N-oxide (TMAO)

to force folding and regaining high functional activity of unstable proteins due to

osmophobic effect [51, 52]. The conformational behaviour of α-synuclein [22-25] and

of the intrinsically disordered transactivation domain AF1 of the Glu-Cocorticoid

receptor [53], in presence of high TMAO concentrations (> 3 M) have been

analyzed using biophysical analysis. In this condition, the IDPs showed in far-

UV CD spectra typical characteristics of the well-folded protein. Another feature

of IDPs is based on their response to the crowded environment (restricted

amounts of free water). Thus, IDPs are classified in foldable (gain of structure

inside living cells) and non-foldable (remain unstructured in crowded

environment) [54-56]. Some of these non-foldable through crowding IDPs may

require another protein (or DNA, or RNA, or some other natural binding

partners) to provide a framework for structure formation.

A number of intrinsically disordered proteins are associated with human

diseases such as cancer, cardiovascular disease, amyloidoses,

neurodegenerative diseases, diabetes and others. Moreover, there is an

interesting interconnection between intrinsic disorder, cell signalling and human

diseases, which suggests that protein conformational diseases may result not

only from protein “misfolding”, but also from misidentification and missignaling.

Intrinsically disordered proteins, such as αSyn, tau protein, p53 and other

disease-associated proteins represent attractive targets for drugs modulating

1 INTRODUCTION 8

protein-protein interactions. Therefore, novel strategies for drug discovery are

based on intrinsically disordered proteins.

1.2 Neurodegenerative aspects of protein aggregation: Chemistry and reaction pathways

“Misfolding” and subsequent self-assembly of proteins to aggregates

(pathogens) is a common molecular mechanism for neurodegenerative

diseases [57-60]. Neurodegeneration is a complex and multifaceted process

leading to many chronic disease states [61]. For a long time, neurodegenerative

diseases were obscure regarding causes, symptoms, mechanism, evolution

and treatment. In the past years however, numerous studies have brought

some light into their biochemical mechanisms, and have provided new

strategies for developing drugs with high curative potential. Several IDPs are

known as key proteins and putative biomarkers associated with

neurodegenerative diseases. α-Synuclein (αSyn) is involved in Parkinson’s

disease [62, 63], prion protein (PrP) in prion disorders (PrD) such as Creutzfeldt-

Jakob disease (CJD), Gerstmann-Straussler-Scheinker (GSS) syndrome and

Fatal Familial Insomnia (FFI) [64, 65], and tau protein in Alzheimer’s disease (AD)

and related tauopathies [66]. The paradigm of a loss of function accompanied by

fibril toxic action is probably valid for all neurodegenerative processes involving

IDPs.

Many age-related neurodegenerative diseases, also known as

“proteinopathies” share a common pathogenic process, abnormal accumulation

and processing of modified and damaged proteins [67, 68]. The presence of

protein aggregates in tissue serves as a hallmark of the above mentioned

disorders and has been used to imply a causative relationship between the

pathologies and the observed aggregates [69]. These diseases include

conditions in which the proteins are predominantly cytosolic (αSyn in PD),

intranuclear, aggregated in the endoplasmatic reticulum and secreted

extracellularly (β-amyloid in AD) [70]. Aggregated proteins characteristic of AD,

PrD, PD and Huntington’s disease (HD) lack significant primary sequence

homology, but have a high similarity in their ability to undergo conformational

1 INTRODUCTION 9

transition (α-helix to β-sheet) and resist proteolysis in vitro and in vivo. They

may have comparable pathways of aggregation, making it tempting to postulate

a common mechanism of neurotoxicity [67, 71, 72].

Aggregation is a highly complex self-assembly process. Proteins

aggregate in various species, e.g. soluble amyloid oligomers, amyloid fibrils and

amorphous aggregates that are present in several morphologically and

structurally different forms. The aggregates, termed amyloid, usually consist of

fibers containing misfolded protein with a β-sheet conformation. The fibrillar

amyloid deposits represent an end stage of a molecular cascade of several

steps, while earlier steps (oligomeric intermediates) in the cascade may be

more directly tied to pathogenesis [73]. Indeed, increasing evidence suggests

that amyloid intermediates exist that have high toxicity [74]. Neurotoxicity assays

on PC12 cells confirm that β-sheet intermediates show higher toxicity than

fibrils, indicating that the β-sheet formation may trigger neurotoxicity [74]. This

hypothesis would be consistent with findings of a poor correlation between

clinical symptoms of AD patients and the number of plaques observed in their

brain [75]. Some elderly patients with abundant amyloid deposits do not show

any neurodegenerative symptoms, suggesting that mature amyloid fibrils do not

cause the onset of amyloidosis-related neurodegenerative diseases. Recent

studies indicated that small soluble oligomers are the neurotoxic species [76-80].

The term “soluble oligomers” is used to describe any non-monomeric form of an

amyloidogenic protein that is soluble in aqueous solutions and remains in

solution after high- speed centrifugation, indicating that it is not an insoluble

fibrillar or aggregated species [81].

Alzheimer’s disease is a late-onset illness, with progressive loss of

memory, tack performance, speech and recognition of people and objects [82].

Extracellular amyloid plaques, which contain amyloid-β peptide (Aβ) derived

from proteolytic processing of the amyloid precursor protein (APP), and

intracellular neurofibrillary tangles (NFTs), consisting mainly of microtubule-

associated protein tau, are two proteinaceous pathological lesions observed in

the brain of AD patients [83-86]. In solution, Aβ is capable of adopting several

different conformations depending on the solvent type [87, 88]. Aβ oligomeric

1 INTRODUCTION 10

forms ranging from dimers to 24-mers have been reported for natural and

synthetic Aβ-peptides [78, 89-91]. These oligomers are highly diverse with respect

of their structure, size and shape. The analysis of soluble fractions of human

brain and amyloid plaque extracts revealed the presence of SDS-stable dimers

and trimers, suggesting that these oligomeric species could play a key role in

the formation of larger oligomers or insoluble amyloid fibrils [92-94]. This concept

was supported by in vitro studies, which have shown that Aβ dimers are three

times more toxic than monomers, while Aβ tetramers were 13 times more toxic [95]. In addition, the formation of soluble tau oligomers rather than that of mature

fibrils play a crucial role for cell death [96]. Recently, it has been shown that tau

assembly involves two distinct dimers (cysteine-dependent and cysteine-

independent) that differ in resistance to reduction. Interestingly, increased levels

of granular tau oligomers have been found in brains with a very early

neuropathology stage at which clinical symptoms of AD are not visible,

suggesting that these Tau oligomeric species may represent a very early sign of

NFT formation and AD [97, 98].

Amyloid diseases are a group of progressive disorders including AD, PrD,

PD and HD. All of the diseases involve selective neuronal vulnerability with

degeneration in specific brain regions, and deposits of abnormal protein in

neurons and other cells, or extracellularly [99-104]. They share common pathways

of protein aggregation, protease resistance and fibrillar structure thus making it

a generic property of several polypeptide chains (Table 1).

Table 1: Neurodegenerative diseases: proteins and pathology:

Disease Protein Aggregation Conformation in

aggregates

Oligomerization Fibrillar

morphology

Alzheimer Aβ + β-sheet + +

Prion prion + β-sheet + +

Huntington huntingtin + β-sheet ? +

Parkinson αSyn + β-sheet + +

1 INTRODUCTION 11

Prion diseases (PrD) are fatal neurodegenerative disorders of infectious,

genetic, or sporadic origin, which are caused by a pathogenic isoform (PrPSc) of

a normal cellular protein (PrPC) [105-108]. The cellular PrPC is a water soluble

membrane glycoprotein with increased α-helix content [109, 110]. By analogy with

other conformational diseases, prion protein oligomers and/or prefibrillar

aggregates might be cytotoxic [111, 112]. Huntington’s disease (HD) is a

progressive neurodegenerative disorder caused by the expansion of a CAG

repeat coding for polyglutamine (polyQ) located in exon 1 of the gene encoding

huntingtin protein (htt) mapped to the chromosome 4 [113]. Studies of htt

aggregation reveal globular and protofibrilar intermediates, the latter high in β-

structure [114]. In contrast to AD and PD, where presence of protein aggregates

is a pathological hallmark, the link of the aggregates and HD is much more

controversial [100]. Inclusions containing htt are observed in regions of the brain

that degenerate. Nevertheless, the neurons with inclusions do not correspond

exactly to the neurons that degenerate [115]. Parkinson’s disease is the second

most common neurodegenerative disease and the most frequent

neurodegenerative movement disorder. α-Synuclein (αSyn) forms spherical

protofibrils that bind to brain-derived membrane fractions much more tightly

than monomeric or fibrillar species. The annular oligomers (amyloid pores) are

also able to bind membrane which affects cells viability. Thus, αSyn spheroidal

and annular oligomers were proposed to be cytotoxic [116]. In addition, formation

of deposits could have protective properties resulting from cellular self-defense [117].

Research on the initiation of misfolding suggests several possible

mechanisms which may act independently, additively or synergistically [118].

Aging accompanied by oxidative modifications of proteins [119, 120]; abnormal

post-translational modifications (e.g. hyperphosphorylation of tau protein in AD [121]; phosphorylation of αSyn at Ser129 present in Lewy Bodies in PD [122]);

proteolytic cleavage of the precursor protein (β-amyloid precursor protein in

AD); single amino acid mutations (familial forms of AD and PD); exposure to

environmental agents (pesticides, herbicides, heavy metals in αSyn); and

increased protein concentration (triplication of αSyn gene in familial PD).

1 INTRODUCTION 12

Protein self-aggregation can be described by two distinctive models: 1) a

nucleation mechanism [123, 124]; 2) a template model [81]. The former suggests

that the rate limiting step is the initial step of nucleus formation, followed by a

rapidly growing elongation step, while the latter postulates that presence of

aggregate induces conformational changes of the non-β monomer to

aggregate- prone β conformer. An idealized model of amyloid fibril formation

was formulated by Uversky (2010) and shows that fibrillization is a directed

process with a series of consecutive steps, including the formation of several

different oligomeric species [81]. It has been shown that heterogeneous mixtures

of aggregated forms, rather than homogeneous mixtures are more often

observed. In addition, each aggregated form can have multiple morphologies

and monomers comprising morphologically different aggregated forms can be

structurally different, suggesting that aggregation is a complex and dynamic

process.

1.3 Synucleins: Relationship to Parkinson’s disease

Parkinson’s Disease (PD) is a common neurodegenerative disorder- a

synucleinopathy which afflicts about 1% of a population over the age of 65 [125,

126]. A study of mortality among PD patients showed a likelihood ratio of 2.5

compared with age-matched subjects [127]. The diagnosis of PD continues to be

based on presenting signs and symptoms. Motoric disabilities, muscle rigidity,

rest tremor, slowing of physical movement (bradykinesia), and in extreme

cases, loss of physical movement (akinesia), depression, autonomic and

dementive abnormalities are most common clinical characteristics for this

disease [128, 129]. The pathological hallmarks of PD are cell loss within the

substantia nigra (SN) and increased appearance of intracellular inclusions, so-

called Lewy bodies (LBs) and Lewy neurites (LNs), which are located in many

brain regions including SN [130]. Neither cell loss nor LBs is absolutely specific

for PD but both are required for a diagnosis of PD under current definitions [131].

The LBs have been found not only in PD, but also in other neurodegenerative

disorders such as AD [132], dementia with LBs (DLB) [133] and multiple system

atrophy [134].

1 INTRODUCTION 13

α-Synuclein (αSyn), a small protein (140 amino acids) with a molecular

mass of 14.5 kDa, first identified as a presynaptic protein in rat brain [62], is a

major fibrilar component of LBs and LNs intracytoplasmatic inclusions. αSyn is

part of a gene family including β- and γ-synucleins and synoretin. The name

“synuclein” was selected as a result of the protein localization within synapses

and the nuclear envelope [62]. The αSyn gene has been mapped to human

chromosome 4q21.3-q22 [135], βSyn to human chromosome 5q35 [136], and γSyn

to human chromosome 10q23.2-q23.3 [137]. The αSyn gene is organized as 7

exons, 5 of which are protein-coding, while the βSyn gene has 6 exons (5

protein-coding) and γSyn gene has 5 exons (all protein-coding) [138, 139]. αSyn

(140 amino acids) and βSyn (134 amino acids) proteins are localized

predominantly at presynaptic nerve terminals and are largely absent from

peripheral tissues [140, 141], while γSyn (127 amino acids) protein is abundant in

the peripheral nervous system and is expressed in other tissues, including brain [142], olfactory epitheliumn [143], and has also been found in breast and ovarian

cancers [144].

All three synucleins are intrinsically unstructured when isolated under

physiological conditions and their physiological functions are unknown.

Biophysical studies indicate that they have a natively unfolded structure and

thus may potentiate protein-protein interactions and/or play a role in cell

regulation [145]. In addition to their overlapping expression patterns, the

sequences of all synucleins consist of a highly conserved amino-terminal

domain that includes a variable number of 11-residue repeats and a less-

conserved carboxy-terminal domain that includes a preponderance of acidic

residues (Figure 4). Within the N-terminal domain, the most important sequence

difference between the proteins is the deletion of 11 residues in the βSyn

sequence that correspond to parts of repeats 6 and 7 of αSyn and γSyn, and

are located in the most hydrophobic region of the proteins, which is referred to

as the non-amyloid component (NAC) [146] region in αSyn. Within the C-terminal

domain, both αSyn and βSyn contain two 16 residue imperfect repeats, while

γSyn has a relatively shorter C-terminal tail that does not contain these repeats.

1 INTRODUCTION 14

αSyn MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVH βSyn MDVFMKGLSMAKEGVVAAAEKTKQGVTEAAEKTKEGVLYVGSKTREGVVQ γSyn MDVFKKGFSIAKEGVVGAVEKTKQGVTEAAEKTKEGVMYVGAKTKENVVQ

**** **:* ******.*.*******:*** ******:***:**:*.**:

αSyn GVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLβSyn GVASVAEKTKEQASHLGGAVFS-----------GAGNIAAATGLVKREEF γSyn SVTSVAEKTKEQANAVSEAVVSSVNTVATKTVEEAENIAVTSGVVRKEDL

.*::********.. :. **.: * .**.::*.*:::::

αSyn GKNEEG-----APQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA βSyn PTDLKPEEVAQEAAEEPLIEPLMEPEGESYEDPPQEEYQEYEPEA γSyn R------------PSAPQQEGEASKEKEEVAEEAQSGGD------

. : . : * .:. : Figure 4: Multiple sequence alignment of human αSyn, βSyn and γSyn by CLUSTALW.

'*' indicates positions that have a single, fully conserved residue; ':' indicates that one of the following 'strong' groups is fully conserved and '.' indicates that one of the following 'weaker' groups is fully conserved. Human αSyn shares 60.4% and 54.3% similarity with human βSyn and γSyn. βSyn, which lacks 11 central hydrophobic residues (grey background), fails to assemble into filaments [147].

Among the three synucleins, αSyn has been intensively studied because

it is linked to both familial and sporadic Parkinson's disease. The primary

structure of human αSyn can be divided into 3 domains (Figure 5a): (1),

residues 1-60 of the N-terminal composed of α-helix forming capability including

the sites of PD mutations (A30P, E46K, A53T); (2), a central region with

residues 61-95 comprising the highly amyloidogenic NAC sequence and (3), the

carboxy terminal region with residues 96-140 which is rich in acidic residues

suggesting that it can adopt a disordered conformation. The highly conserved

amino-terminal repeat domain of αSyn is thought to mediate both lipid binding

and dimerization [148], while the C-terminal appears to be primarily involved in

the solubilization of the high molecular weight complexes [149] and may regulate

the aggregation of full-length protein, since C-terminally truncated fragments

aggregates faster than the full-length protein [150-152]. Moreover, its acidic

domain 125-140 appears to be critical for the chaperone activity of αSyn [153].

1 INTRODUCTION 15

Monomer Soluble oligomers

Lewy bodies

Protofibril Amyloid like- fibril

Truncation/ Degradation

H2N COOH

1 14061 95

A30P E46K A53T

Amphipathic region Acidic region

KTKEGV repeats

NAC domain

b

a

A30P a A53T b A53T b

A30P a - promotes the formation of oligomersA53T b - promotes the formation of fibrils

Figure 5: (a), Alpha-synuclein’s domains. Black highlighted: N-terminal domain. Green highlighted: NAC or non-Aβ (amyloidogenic) component of αSyn, responsible of protein-protein interactions. Brown highlighted: the unstructured C-terminal domain. Mutations A30P, E46K and A53T are enhanced. The seven 11 aa repeats are shown in light blue; (b), Possible pathway(s) of αSyn aggregation and degradation. αSyn is a protein with natural tendency to aggregate into oligomers that are then further aggregate into fibrils that are deposited as Lewy bodies. A53T mutation promotes the formation of such fibrillar species, A30P does not. In fact A30P slows the rate of fibril accumulation but strongly promotes the formation of oligomeric species. The pathway including truncated species of αSyn may be of crucial importance in the process. Immunohistochemistry for αSyn is showing positive staining (brown) of an intraneural Lewy body in the Substantia nigra in Parkinson's disease [154].

The physiological function of αSyn is poorly understood. The localization

of this protein in synaptophysin- immunoreactive presynaptic terminals suggests

a role of αSyn in the regulation of synaptic vesicles [140, 155]. However, αSyn

knockout mice show only subtle abnormalities in neurotransmission, suggesting

that this protein plays a non-essential function at the synapse [156-158]. Under

physiological conditions, αSyn is believed to be involved in the development of

1 INTRODUCTION 16

neuronal differentiation and regulation of dopamine synthesis. It could be shown

that this protein is able to provide a certain protection against oxidative stress

on overexpression [159].

The main evidence of a relevant role of αSyn in PD came from the

discovery of three point mutations in the αSyn gene: A53T (change Ala in

position 53 to Thr) [160], A30P (change Ala in position 30 to Pro) [161] and E46K

(change Glu in position 46 to Lys) [162] in a few families with autosomal dominant

Parkinson’s disease and that αSyn accumulates in LBs and other pathological

inclusions in conditions inducing Parkinson’s disease [163]. LBs contain many

proteins in addition to αSyn, including neurofilaments and other cytoskeletal

proteins, suggesting that there are coprecipitants that might be important in

aggregation. However, fibrils can be formed in vitro from αSyn alone, indicating

that this protein is sufficient to form inclusions. αSyn is also the most sensitive

biomarker for Lewy bodies. All three mutations occur within the N-terminal side

of the protein and are able to accelerate the αSyn oligomeric aggregation

process and protofibril formation [164] faster than wild type (wt) αSyn. Therefore,

the fibrillization rate was also higher (with the exception of the A30P mutation)

than of the wt variant [165, 166], leading to pathologic inclusions, such as LBs and

LNs.

In its native state, αSyn is a soluble unfolded protein. Owing to the

central hydrophobic amyloidogenic region, αSyn has a high propensity to

aggregate and initially forms an intermediate annular structure called oligomer

or protofibril and ultimately forms insoluble polymers or fibrils [147]. Although the

cause of neurodegeneration in PD is not well understood, recent studies

suggest that small oligomers rather than the fibrillar amyloid deposits of αSyn

represent the principal toxic species [167]. Overlooked in most previous research

on the αSyn aggregation, the pathway including truncated species of αSyn may

be of crucial importance (Figure 5b). Some studies demonstrated that C-

terminally truncated low molecular mass αSyn species with aggregation-

promoting properties are normally generated in vivo (cells and brain),

suggesting that this species may be of pathogenic significance [168]. Truncated

αSyn species were found using low concentrations of proteinase K (PK). The

1 INTRODUCTION 17

fragments were assigned as N-terminally truncated products, because the

bands were detected with αSyn antibodies against a C-terminal epitope [169].

Other experiments using PK digestion of intact αSyn fibrils identified a 7 kDa

core peptide region corresponding to residues 31 to 109 [170], and similar studies

using trypsin and Glu-C digestion identified a slightly different fragment αSyn

(32-102) [171]. More recently, short peptide fragments located between residues

76 and 96 were identified from protease- resistant fibril core structure of αSyn

using a combination of Edman degradation and MALDI-TOF-MS analyses [172].

In addition to mutations that promote aggregation [166], a variety of factors

contribute to the formation of oligomeric but not fibrillar species: post-

translational covalent modifications of αSyn such as phosphorylation [173],

nitration [174, 175] and glycosylation [120]; stabilization of protofibrils by forming a

dopamine-αSyn adduct [119]. The major phosphorylation sites of αSyn were

identified at Ser129 and at Ser87. It has been determined that more than 90%

of insoluble αSyn in LBs is phosphorylated. By contrast, phosphorylation

involves only about 4% of normal αSyn, suggesting that phosphorylation is a

relevant pathogenic event [173, 176]. Additionally, phosphorylation at Ser129

increases fibril formation [173]. Subsequent coexpression of S129A αSyn showed

an important decrease in cytoplasmic inclusions [177], demonstrating that

phosphorylation at Ser129 enhances the formation of inclusion bodies and is a

crucial step in the development of LBs. Transgenic mouse models that

overexpress αSyn have shown neurodegeneration accompanied by

phosphorylation at Ser129 and apoptosis [178].

Moreover, two pathways supporting the degradation of misfolded proteins

are the ubiquitin- proteasome [179] and macroautophagy systems. Generally, the

ubiquitin- proteasome pathway (UPS) system is more efficient than basal levels

of macroautophagy, so for proteins that have access to both pathways,

proteasomes are the favored and dominating clearance route. αSyn is targeted

to proteasomal degradation after being modified with ubiquitin which is

conjugated through its carboxy- terminus to ε-amino groups of lysine residues.

In particular regarding αSyn, a specific substrate for ubiquitination by the E3

ligase parkin is O-glycosylated αSyn with possible glycosylation at Ser129 [180].

1 INTRODUCTION 18

αSyn ubiquitination occurs in vivo at Lys-6, -10 and -12. Nevertheless, it

remains unclear whether monomeric αSyn requires ubiquitination, since αSyn is

a natively unfolded protein and may not require ubiquitination and unfolding.

Instead, it could directly enter the 20S proteasome [181]. Mutations in the parkin

gene are known to cause a large portion of early onset autosomal recessive

parkinsonism [182]. The loss of E3 ligase activity and substrate binding is

observed [183, 184] inducing the UPS failure and the structurally deformed,

aggregated αSyn cannot be degraded and recycled by the cell. The second

pathway of αSyn clearance is autophagy, a process mediating bulk degradation

of cytoplasmic proteins or organelles in the lytic compartment. Autophagy

involves the formation of double-membrane structures called autophagosomes,

which fuse with primary lysosomes to become an autophagolysosome where

their content is degraded and then either disposed of or recycled back to the

cell [185]. αSyn is predominantly degraded by the lysosomal pathway [186, 187],

including chaperone-mediated autophagy (CMA) [188], and the lysosomal

cathepsins are important in proteolysis [189]. Cell death is a significant part of the

pathology of PD with the prime suspect for toxicity αSyn. Moreover, soluble

oligomers of this protein might be more toxic than the insoluble fibrils found in

Lewy bodies. Therefore, most of the therapeutic strategies are aimed to prevent

aggregation. Since little is known about the function of αSyn in normal vs.

neurodegenerative conditions; the elucidation of the molecular features of αSyn

is of crucial importance for understanding the mechanism of Parkinson’s

disease.

1.4 Analytical methods for protein structure analysis and characterization of aggregates

Neurodegenerative diseases of the central nervous system display a

common feature in their pathogenesis: a “misfolding” and a progressive

polymerization of soluble proteins. Amyloid diseases are characterized by the

deposition of insoluble aggregates and comprise over 30 diseases, including

Alzheimer’s disease, Parkinson’s disease, Huntington’s, and Prion diseases. In

vitro studies showed the existence of not only soluble monomers but also

1 INTRODUCTION 19

partially folded intermediates that lead to the formation of the amyloidogenic

nucleus and fibrils [22]. Because of the metastable nature of these prefibrillar

assemblies and the noncrystalline nature of fibrillar protein aggregates, the

structural study of amyloid proteins is difficult.

Analytical methods used to study the secondary, tertiary, and quaternary

structures, and morphology of prefibrillar and fibrillar assemblies of amyloid-

forming proteins are summarized in Table 2 [190].

Table 2: Analytical methods used to study amyloid protein structure, folding and assembly.

Monomer Oligomer Protofibril Fibril Amyloid

(in vivo)

Solid-state NMR Solid-state NMR

X-Ray crystallography

Atomic structure

X-Ray absorption

Circular dichroism spectroscopy

Fourier transform infrared microscopy

X-Ray fiber diffraction

Neutron scattering

Congo red binding/ Thioflavin S fluorescence

Secondary structure

Thioflavin T fluorescence

Transmission electron microscopy

Scanning transmission electron microscopy

Scanning tunneling microscopy

Morphology/

Topology

Atomic force microscopy

Electron spin resonance

Hydrogen-Deuterium exchange

Limited proteolysis

Fluorescence methodologies

Tertiary/ quaternary structure

Ion-mobility mass spectrometry

Gel electrophoresis

Size-exclusion chromatography

Ultracentrifugation

Light scattering

Electrospray mass spectrometry

Supramolecular assembly size

Ion-mobility mass spectrometry

1 INTRODUCTION 20

X-ray crystallography and nuclear magnetic resonance (NMR)

spectroscopy are key sources of information about the protein tertiary structure [191]. They are of limited use in the amyloid field, though recent advances in

studies of soluble monomers and oligomers and of insoluble fibrils are

encouraging. Solution-state NMR is a powerful high-resolution method for

studying 3D structures of soluble samples. This method was successfully

applied to full-length Aβ(1-40) and Aβ(1-42) in aqueous solution at neutral pH [192]. Solid state NMR determinations performed on a short Aβ(10-35) suggested

that the peptide forms a parallel β-sheet structure [193]. It was subsequently

found that full-length Aβ(1-42) forms β-sheet with the same registry and

orientation [194]. Using X-ray microcrystallography techniques, the structures of

30 short (6 – 7 residues) segments of amyloidogenic proteins, including Aβ, tau,

PrP, αSyn, were found to be organized in a ‘‘steric zipper’’ [195, 196]. X-ray

absorption spectroscopy (XAS) is useful for studying amino acid residues

coordinating metal ions [61, 197].

The secondary structure analysis of proteins provides first information

about their three-dimensional structure. It is most easily assessed by lower

resolution methods, including X-ray fiber diffraction and neutron scattering;

spectroscopic methods, including circular dichroism (CD), Fourier transform

infrared spectroscopy (FTIR); tinctorial methods such as Congo Red (CR)

binding, and changes in fluorescence of thioflavin dyes. X-ray fiber diffraction

revealed the presence of cross-β structure in amyloid fibrils [198]. Neutron

scattering is used, often in combination with X-ray diffraction techniques, to

study the structure of amyloid fibers and provides information about size, shape,

and extent of aggregation of the species under consideration and measurement

of mass per unit length. Aβ(1-40) protofibrils have been found by Small-angle

neutron scattering (SANS) to be cylindrical structures with 24 Å cross-sectional

radii and ~110 Å length [199]. Each cylindrical unit was reported to comprise 30

Aβ(1-40) monomers. Aggregation of amyloidogenic proteins into protofibrils and

fibrils is accompanied by abundant formation of β-sheet conformation, which is

revealed by a negative band of an absorption minimum at 215 – 218 nm in the

far–UV CD spectrum. IR spectroscopy is complementary to CD and widely used

1 INTRODUCTION 21

for the characterization of proteins with β-sheet or β-turn structures. Congo Red

(CR) staining is commonly used for the identification of amyloid aggregates in

tissue sections [200, 201]. Furthermore, CR binding prevents formation of mature

fibrils [114, 202, 203]. The aggregation profile monitored by ThT typically is a

sigmoidal curve consisting of three different regions: (1) lag phase, in which β-

sheet structures are absent and ThT binding does not occur; (2) burst phase

that marks the commencement; (3) β-sheet formation. Eventually, a saturation

phase denoting fibril maturation is observed [204]. Similar to ThT, ThS binds

amyloid fibrils but not monomers, and undergoes fluorescence intensity

changes and distinct spectral shift upon binding with emission maxima of ThS

~45 nm [205].

Microscopic techniques including transmission electron microscopy

(TEM), scanning transmission electron microscopy (STEM) [206], scanning

tunneling microscopy (STM), and atomic force microscopy (AFM) [207] examine

the morphology of prefibrillar and fibrillar protein assemblies. TEM was used to

assess metal-induced fibril formation in αSyn showing that Fe and Cu ions have

differential effects on αSyn fibrillization [208]. Moreover, incubation of αSyn with

different metals produced spherical and annular oligomers that were proposed

to be cytotoxic [116, 209]. STEM is an excellent tool for characterizing the

homogeneity and structural properties of transient quaternary structure

intermediates in the fibril-formation pathway of amyloid proteins like Aβ and

αSyn [206]. STM studies of Aβ showed ribbon like filamentous nature or right-

handed twist of Aβ fibrils [210-212]. AFM has been used to investigate the

assembly dynamics of several amyloidogenic proteins and its advantage is that

it allows continuous monitoring of the growth of oligomers [213] and fibrils in

solution [214]. In situ AFM analysis showed that the formation of αSyn globular

oligomers precedes the appearance of amyloid fibrils and is systematically

observed under conditions for accelerated fibrillization, potentially indicating that

oligomers can act as on-pathway intermediates during amyloidogenesis [215].

Electron spin resonance (ESR), hydrogen–deuterium (H/D) exchange,

limited proteolysis, and intrinsic fluorescence are applied to characterize tertiary

and quaternary structures. ESR, also called electron paramagnetic resonance

1 INTRODUCTION 22

(EPR) has been applied to αSyn, which has been shown to generate hydroxyl

radicals in the presence of small amounts of Fe(II) [216]. For protein analysis, the

degree and rate of backbone amide proton exchange is useful for distinguishing

regions that are exposed to the solvent and therefore is carried out using H/D

exchange, followed by monitoring by NMR or mass spectrometry (MS) [217].

H/D-NMR analysis has been applied to study the structure of Aβ fibrils and

showed that the N- and C-termini of the peptide are accessible to the solvent in

the fibril state, while most of the middle domain was involved in a β-sheet

structure [218]. Limited proteolysis is a complementary method to H/D exchange

applied to amyloid-forming proteins. This method in combination with LC–MS

revealed a protease-resistant region (Ala21–Ala30) in both Aβ(1-42) and Aβ(1-

40) [219]. The intrinsic fluorescence of Tyr10 residue in Aβ(1-42) and Aβ(1-40)

showed that the C-terminal area underwent substantial conformational

rearrangement during aggregation, whereas Aβ(17–21) was essential for

controlling Aβ aggregation [220]. Fluorescence resonance energy transfer

(FRET) measurements were used to study the structure and dynamics of αSyn

under different conditions. Under acidic (pH 4.4) conditions, the C-terminus of

the protein became more compact, while in the presence of SDS micelles the

N-terminus was more compact and the C-terminus was more extended [221-223].

Another method used to monitor protein aggregation is fusion of an

amyloidogenic protein to a fluorescent protein. The method is based on the slow

folding of green fluorescent protein (GFP) and the requirement for the fully

folded protein for fluorescence [224-226].

Finally, the methods used to study assembly size and distributions

include gel electrophoresis, cross-linking, size-exclusion chromatography

(SEC), analytical ultracentrifugation (AUC), dynamic light scattering (DLS),

electrospray ionization mass spectrometry (ESI-MS) and ion-mobility

spectrometry–mass spectrometry (IMS–MS). A frequently used method for

protein analysis is Sodium-Dodecylsulfate- Polyacrylamide- gel electrophoresis

(SDS-PAGE) where molecules are separated based on their electrophoretic

mobility and their molecular weight is estimated by using standard calibration

proteins (markers) [227]. Using 2D SDS-PAGE it is possible to separate up to

1 INTRODUCTION 23

10000 proteins based by their isoelectric point within a broad pH gradient [228].

Tris-tricine is another technique used to separate proteins in the mass range 1 –

100 kDa [229]. It is the preferred electrophoretic system for the resolution of

proteins smaller than 30 kDa. Moreover, analytical and preparative Size-

Exclusion Chromatography (SEC) methods are used extensively to distinguish

between protofibrils (PF) and small oligomers of aggregation-prone proteins [230]. Analytical ultracentrifugation (AUC) is based on equilibrium and non-

equilibrium thermodynamics and is re-emerging as a versatile tool for the study

of proteins and protein complexes. Usually, two types of AUC experiments are

performed: sedimentation velocity (SV) and sedimentation equilibrium (SE),

which are used to analyze size distribution of various components of

amyloidogenic assemblies under different solution conditions [231]. Dynamic

Light-Scattering Spectroscopy (DLS) is used for measurements of sub-micron

particle sizes, diffusion coefficients, viscosities, molecular weights of

amyloidogenic proteins. This technique confirmed the existence of significant

amounts of oligomeric species of insulin prior to the appearance of mature fibrils

and throughout the fibril elongation process [232].

In recent years, mass spectrometry (MS) has become a major analytical

tool in biochemistry and structural biology [233]. ESI-MS is able to provide

quantitative information on dimensions of natively folded proteins and their

assemblies in solution. ESI is a “soft ionization” technique that allows non-

covalent biomacromolecular complexes to be ionized intact. A most useful

attribute of ESI is its ability to couple MS and liquid-separation techniques. For

the development of ESI mass spectrometry of biomolecules, John B. Fenn

received the 2002 Nobel Prize in Chemistry. ESI-MS is an ionization method

that produces gaseous ions from molecules in solution [234] by creating a fine

spray of charged droplets in an electric field [235, 236]. An illustration of the ESI

process is shown in Figure 6 and in more detail in 3.9.3.

1 INTRODUCTION 24

Capillary Spray

Solution with analyte

Massspectrometer

Figure 6: Electrospray ionization principle: a high positive potential is applied to the

capillary (anode), causing positive ions in solution to drift towards the exit, where the liquid surface is distorted, forming a cone (“Taylor cone”); from the tip of the cone is emitted a spray of droplets with an excess of positive charge; gas phase ions are formed from charged droplets in a series of ion emission steps accompanied by solvent removal.

Ion-mobility mass spectrometry (IMS-MS) (see 2.3.3.1) is most recently

emerging as a powerful tool for analysis of conformation- dependant

interactions of proteins and reaction intermediates, which are not feasible to

conventional mass spectrometry (MS), such as electrospray-MS (ESI-MS) [237-

241]. IMS–MS studies of Aβ have shown that freshly prepared Aβ(1-40)

contained monomers, dimers, trimers, and tetramers, whereas similarly

prepared solutions of Aβ(1-42) comprised oligomers up to a dodecamer [242, 243].

1.5 Scientific goals of the dissertation

Parkinson’s disease (PD) is one of the most common disorders leading to

progressive neurodegeneration, for which the 14 kDa presynaptic αSyn has

been identified as a key protein in brain. The intracellular accumulation of high

molecular weight αSyn aggregation products as fibrils is a characteristic

pathological feature of PD and related neurological disorders. Recently, soluble

oligomeric aggregates of αSyn have been suggested to be more toxic than the

insoluble fibrils found in Lewy bodies. At present, little is known about the

structure, composition and mechanism of formation of αSyn oligomers. The

elucidation of chemical structures of intermediates is expected to contribute to

1 INTRODUCTION 25

the development of lead structures for new molecular therapeutic and

diagnostic tools of Parkinson’s disease.

The major objectives of the present thesis are summarized as follows:

• Isolation and structure identification of oligomerization-aggregation

products in vitro.

Proteolytic degradation studies of synucleins were carried out for

identifying the cleavage specificity and the preferred cleavage sites of

different proteases, by using mass spectrometric methods. In addition,

first molecular studies of αSyn degradation products, previously detected

but never identified, were carried out by affinity mass spectrometry and

the new ion-mobility mass spectrometry.

• Identification of epitope(s) to αSyn specific antibodies using affinity mass

spectrometry.

The major goals of this study were (i) the immobilization of synuclein

specific antibodies on Sepharose; (ii) the epitope elucidation of α-

synuclein using synuclein specific antibodies by epitope excision and

extraction methods in combination with mass spectrometry.

• Synthesis and characterization of specific peptides: a) as models for

proteolytic fragments in vitro; b) as models for epitopes.

Synuclein peptides were prepared by stepwise synthesis, and structurally

and immuno- analytically characterized.

• Affinity characterization of synucleins by a combination of SAW

biosensor and mass spectrometry.

The combination of affinity biosensor with mass spectrometry (SAW-ESI-

MS) was successfully applied to anti-synuclein antibodies – synuclein

proteins and peptides complexes. SAW-ESI-MS was also applied to the

direct analysis of αSyn (A30P) from mouse brain homogenate, indicating

this method as a powerful new tool to the molecular characterization of

1 INTRODUCTION 26

intermediates of protein aggregation. In addition, molecular interaction

kinetics between synuclein and anti-synuclein mono/ polyclonal

antibodies were determined by SAW biosensor and the obtained KD

values were in the low nano-molar range.

• Chemical stability studies of αSyn.

Chemical modification of αSyn by specific acylation was suitable to

stabilize the structure of αSyn and inhibit degradation and

oligomerization.


2 RESULTS AND DISCUSSION

2.1 Structure determination of synucleins

Structurally, α-synuclein (αSyn) is a natively unfolded protein with

unknown function and unspecific conformational heterogeneity. In general, a

protein is characterized not only by its amino acid sequence but also by any

post-translational modification. However, determination of the primary structure

is generally the first step in the complete characterization of higher order

structure and function of a protein.

2.1.1 Primary structure determination of synucleins by mass spectrometry

Mass spectrometry has been developed and improved dramatically over

the last years; particularly new ionization techniques and new structure

determination tools have been developed, allowing intact peptides and large

proteins to be analyzed. Two most important methods concerning peptides and

proteins are electrospray ionization (ESI) and matrix-assisted laser desorption

ionization (MALDI); these methods have been awarded with the 2002 Nobel

Prize in Chemistry [234, 244].

MALDI-TOF-MS (TOF; time of flight) for analysis of large biomolecules

was first described in 1988. Laser light is used to irradiate a UV- or IR-

absorbing matrix, while the peptide or protein of interest is embedded in a large

excess of this matrix. Energy is transferred onto the target molecules and

ionization and desorption takes place. Analyte ions are emitted and fly towards

the detector. Different masses cause different flight times and ions can be

detected subsequently. A second technique for analyzing proteins by mass

spectrometry uses electrospray ionization (ESI) [234, 241, 245, 246]. An aqueous

protein or peptide solution passes with a flow rate of 1 - 10 µL min-1 through a

fused silica capillary which ends in a stainless steel capillary held at a potential

of several kV. The resulting electrostatic field generates positively or negatively


charged droplets. These droplets shrink by an ion emission process

(accompanied by solvent evaporation) that may be supported by heating or by

passing a curtain of dry gas until the charge accumulation on the liquid surface

is high enough for ion emission. These ions enter into the mass analyzer and

their mass-to-charge ratio can be determined. Electrospray ionization mass

spectrometry generates multiply charged ions, thus direct molecular weight

analysis of even large proteins (up to hundred kDa) with high sensitivity and

precision is possible. Electrospray ionization delivers singly and multiplies

charged ions with little excess of energy, which results in stable ions and little

fragmentation. To obtain structural information, the kinetic energy has to be

converted into vibrational energy, typically by collision of the ions with a neutral

gas (for example, helium or argon) that induces fragmentation of the analyzed

ions. One major benefit of ESI-MS is its applicability to the determination of the

molecular mass of proteins with intact structure. In positive ion ESI analysis,

protonation of basic amino acid residues and the terminal amino group in

proteins leads to the generation of multiprotonated ions of the general

composition (M+nH)n+. Similarly, abstraction of protons from acidic amino acids

of a protein results in the formation of multiply charged negative ions such as

(M-nH)n-. The molecular mass M of a macromolecule is determined from m/z

values and charge state of any two multiply charged ions in a consistent series.

Here, m1 is the m/z of the (M+nH)n+ ion (here, H = 1.007829 is the mass of the

proton) and m2 (m1 > m2) of the adjacent ion such that

m1 =M + nH

nm1 =M + nH

n and

m2 =M + (n + 1)H

n + 1m2 =M + (n + 1)H

n + 1 These two simultaneous equations can be solved for n and M to give:

n =m2 – Hm1 – m2

n =m2 – Hm1 – m2

and

M = n (m1 – H)


In the present work, αSyn was purified prior to use by analytical reversed

phase high performance liquid chromatography (RP-HPLC) on a UltiMate 3000

system (Dionex, Germering, Germany), equipped with a Vydac C4 column.

Linear gradient elution (0 min - 0% B; 5 min - 0% B; 50 min - 90% B) with

solvent A: (0.1% TFA in water) and solvent B: (0.1% TFA in ACN-water 80:20

v:v) was employed at a flow rate of 1 mL min-1. Further, the protein was

solubilized in 1% formic acid, 30% methanol and analyzed by nano-ESI-FTICR-

MS. The resulting fine droplet spray was dried by evaporation in a stream of dry

gas, and the spectrum was obtained in positive ion mode. In the spectrum, the

multiply charged ions were obtained, with the most intensive peak

corresponding to the ion +10 (Figure 7).

MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH



1

51

101 140

b

a

20 30 40 500

10

20

30

40

Abs

orba

nce

(220

nm

)

Time (min)

29.8

20 30 40 500

10

20

30

40

Abs

orba

nce

(220

nm

)

Time (min)

29.8

Mw exp : 14452.2668 DaMw calc : 14452.2263 Da∆m = 2.8 ppm

c

1118.179813+

1207.860612+

1317.297511+

1448.929510+

1607.58649+

1811.02658+

1100 1200 1300 1400 1500 1600 1700 1800 m/z

1118.179813+

1207.860612+

1317.297511+

1448.929510+

1607.58649+

1811.02658+

1100 1200 1300 1400 1500 1600 1700 1800 m/z

Figure 7: (a), Schematic representation of the primary structure of α-synuclein (αSyn);

(b), Analytical RP-HPLC profile of αSyn with retention time (Rt) of 29.8 min; (c), nano-ESI-FTICR-MS of αSyn in 1% formic acid, 30% methanol showing the 8+ to 13+ charged molecular ions.

The protein was also analyzed by MALDI-TOF-MS. The mass spectrum of αSyn

is presented in Figure 8, showing the formation of [M+3H]3+, [M+2H]2+ and


[M+H]+ ions with a mass accuracy between of approximately 42 ppm for the

average- isotope mass. The most intensive peak corresponds to the [M+2H]2+.

This result confirmed the primary structure and homogeneity of αSyn.




1

51

101 140

6000 8000 10000 12000 14000 m/z

4820.6

7230.3

14460.5

[M + 2H] 2+

[M + H] +

[M + 3H] 3+


b

a

Figure 8: (a), Schematic representation of the primary structure of αSyn; (b), MALDI-

TOF mass spectrum of αSyn with the most abundant ion of [M+2H]2+.

Further, the two missense mutants of the αSyn, αSyn (A53T) and αSyn (A30P),

which have been linked to familial PD [160, 161], were purified prior to use, by

analytical RP-HPLC on a UltiMate 3000 system and analyzed by direct infusion

ESI-ion trap-MS. The ESI mass spectrum of αSyn (A30P) in 1% aqueous formic

acid at a concentration of 30 µM showed the 13+ to 21+ charged molecular

ions, while the spectrum of αSyn (A53T) showed the 13+ to 23+ charged

molecular ions. Furthermore, βSynuclein (βSyn) that co-localizes with αSyn in

some brain regions [140] was also analyzed by ESI-ion trap-MS. In the spectrum,

the 13+ to 21+ charged molecular ions were obtained with the most intensive

peak corresponding to the +16 ion. The HPLC chromatograms and the [M+H]+

ions of the synuclein proteins are summarized in Table 3. The amino acid


variations in the sequence relative to the wild type αSyn (wt-αSyn) are

highlighted in red in 7.3.

Table 3: Characterization of synucleins by RP-HPLCa and ESI-MSb:

Code Protein HPLC

Rt (min)a

[M+H]+ exp b [M+H]+ calc

c ∆m (ppm)

1 αSyn 29.8 14461.3 14461.1 14

2 βSyn 33.8 14288.1 14288.9 56

3 αSyn (A30P) 36 14487.5 14487.2 21

4 αSyn (A53T) 36.8 14491.3 14491.2 7 a RP-HPLC purification (UltiMate 3000, Dionex) using a C4 Vydac column b Mass spectrometric analysis by ESI-ion trap-MS using an Esquire 3000 + mass

spectrometer (Bruker Daltonics) c GPMAW software 5.0 (Lighthouse Data, Denmark)

2.1.2 Secondary structure characterization of synucleins by circular dichroism

The secondary structure analysis of proteins provides initial information

about their three-dimensional structure. It is most readily assessed by

spectroscopic methods, particularly circular dichroism (CD). CD spectroscopy is

an optical technique that provides the detection and quantification of the

chirality of molecular structures. The measurements were carried out in water at

25°C in quartz cells of 0.1 cm path-length. αSyn was also solubilized in PBS

buffer pH 7.5, preserving the physiological conditions. The concentration of the

protein used for CD analyses was 30 µM which approximates the range of

concentrations of αSyn found in the cytoplasm of neurons [247]. The spectra

were obtained as an average of four scans between 180 and 260 nm and

results were expressed as molar ellipticities. The CD spectra of αSyn showed a

strong negative band around 198 nm, characteristic of an unordered structure.

Furthermore, βSyn, αSyn (A30P) and αSyn (A53T) were solubilized in water at

comparable concentrations of 30 μM. The spectra, each obtained as an


average of four scans, are shown in Figure 9. All three proteins adopt random

coil structures at the conditions employed.

180 200 220 240 260

-3000000

-2000000

-1000000

0

1000000

2000000

Mol

. Elli

ptic

ityΘ

(deg

cm2

dmol

-1

Wavelength (nm)

2, βSyn3, αSyn (A30P)4, αSyn (A53T)

1, αSyn

Figure 9: CD spectra of synucleins recorded in water at a final concentration of 30 µM.

All proteins adopt a random coil conformation with a minimum at 198 nm.

Additional information about the αSyn structure was obtained by

molecular dynamic simulation. For this analysis, the HyperChem 6.0 and

BallView programs for molecular modelling were used to assign a structure of

the protein as shown in Figure 10. The amino acid sequence of αSyn is divided

into three domains: (1), residues 1-60 of the N-terminal part are shown in black;

(2), the central domain (green) with residues 61-95 and (3), the C-terminal

region with residues 96-140 shown in brown.


NH2

COOH

H2N COOH

1 14061 95

Amphipathic region Acidic regionNAC domain

Figure 10: Molecular modelling of the structure of αSyn using the HyperChem and

BallView programs. Ribbon diagram of the structure of αSyn contains in black the N-terminal domain, in green the NAC or non-Aβ (amyloidogenic) component and in brown the unstructured C-terminal domain. The insert in the upper part contains a schematic representation of αSyn regions.

2.1.3 Primary structure characterization of synucleins by mass spectrometric peptide mapping

Proteases or peptidases are enzymes that catalyze the cleavage of

peptide bonds which are widely distributed in cells, where they serve a large

variety of different tasks and functions such as degrading misfolded proteins,

performing regulatory pathways, and being involved in cell growth and

apoptosis. The cleavage reaction of many proteases renders them important

tools for peptide mapping and elucidation of primary and higher-order protein

structures [248]. Proteases important for protein characterization studies can be

divided into endo- and exoproteases. Endopeptidases attack the protein

backbone, generating peptide fragments. They are mainly used for protein

analytical studies, taking advantage of the high specificity to cleave at specific


residues. In contrast, exopeptidases attack the N- or C-terminal amino acids of

a polypeptide.

The proteolytic cleavage of synucleins was studied in the presence of

trypsin and Glu-C protease. Wild type (wt) αSyn and the two mutants, αSyn

(A53T) and αSyn (A30P), were first digested using trypsin with an enzyme:

substrate ratio of 1:50 at 37°C. The digestion was performed in PBS buffer (5

mM Na2HPO4, 150 mM NaCl, at pH 7.5) and with different reaction times (30

min, 2 h, 4 h, 6 h, 8 h, 16 h). Endoproteiase Glu-C from Staphylococcus Aureus

was also used for the digestion of wt-αSyn and corresponding mutants αSyn

(A53T), αSyn (A30P), as Glu-C cleaves the peptide bonds at the C-terminus of

glutamic and aspartic residues. The resulting peptide mixtures were analyzed

by MALDI-TOF and LC-ESI-MS. The MALDI-TOF mass spectra of wt-αSyn

after trypsin and Glu-C digestion are presented in Figure 11. The digestions of

αSyn proteins with trypsin and Glu-C lead to the formation of multiple fragments

with m/z values within a large region of the spectrum, between 500 and 5000.

The calculated and experimental average masses of the tryptic and Glu-C

fragments in Table 4a and Table 4b, respectively. Almost all expected

proteolytic fragments were identified in the mass spectrum, covering mostly the

central and C-terminal regions.

1000 2000 3000 4000 5000 m/z1000 2000 3000 4000 5000 m/z

(81-96)

(1-6)

(35-43)

(46-58)

(81-97)(61-80)

(59-80)

(59-102)(33-80)

(97-140)(11-21)

a


1000 2000 3000 4000 5000 m/z

(1-13)

(124-140)

(84-104)

(84-105)

(58-83)

(84-114)

(46-83)(36-46)

(132-140)(47-57)

b

Figure 11: MALDI-TOF mass spectra of αSyn digested in solution: (a), In solution

digestion of αSyn with trypsin at 37°C for 2 h; (b) In solution digestion of αSyn with Glu-C at 37°C for 2 h.

Table 4a: αSyn tryptic peptides identified by LC-ESI-MS and MALDI-TOF-MS.

Nr. Sequence Peptide ion Mexp Mcalc

1 11 - 21 (536.7)2+ a) 1071.4 1071.6

2 13 - 21 (437.2)2+ a) 872.7 872.4

3 46 - 60 (509.1)3+ a) 1523.4 1523.8

4 46 - 58 (432.6)3+ a) 1295.1 1295.6

5 35 - 45 (950.8)2+ a) 1179.5 1179.6

6 35 - 43 (476.2)2+ a) 950.5 950.5

7 1 - 6 (385.6)2+ a) 769.3 769.3

8 81 - 97 (536.4)3+ a) 1606.2 1606.8

9 59 - 80 (720.1)3+ a) 2157.2 2157.4

10 81 - 96 (740.2)2+ a) 1478.5 1478.6

11 61 - 80 (643.6)3+ a) 1927.9 1928.1

12 59 -102 (4288.7)1+ b) 4287.7 4287.8

13 33 - 80 (4826.7)1+ b) 4825.7 4827.0

14 97 -140 (4959.1)1+ b) 4958.1 4958.1


Table 4b: αSyn proteolytic (Glu-C) peptides identified by LC-ESI-MS and MALDI-TOF-MS.

Nr. Sequence Peptide ion Mexp Mcalc

1 36 - 46 (590.9)2+ a) 1079.8 1080.3

2 132 -140 (1071.3)1+ b) 1070.3 1071.0

3 124 -140 (502.7)4+ a) 2007.8 2008.0

4 47 - 57 (519.8)2+ a) 1037.6 1037.5

5 1 - 13 (495.4)2+ a) 1483.2 1482.7

6 84 -105 (548.8)4+ a) 2191.2 2190.1

7 53 - 83 (654.7)4+ a) 2614.8 2614.9

8 59 - 80 (720.1)3+ a) 2157.2 2157.4

9 62 - 83 (710.3)3+ a) 2127.9 2127.1

10 84 -114 (3086.3)1+ b) 3085.3 3086.4

11 47 - 83 (3635.2)1+ b) 3634.2 3635.1a Mass spectrometric analysis by LC-ESI-MS (Esquire 3000 +, Bruker Daltonics) b Mass spectrometric analysis by MALDI-TOF-MS

Moreover, in solution digestion of βSyn was performed with trypsin and Glu-C

proteases. The substrates were dissolved in PBS ELISA (pH 7.5), and

digestions performed for 2 h at 37°C. The sample containing the mixture of

digested peptides with Glu-C was analyzed by LC-ESI ion trap-MS and the

results presented in the Figure 12. The obtained molecular average masses are

summarized in Table 5 together with the calculated values, using the GPMAW

program. As in the case of digestion with trypsin, the results showed a good

agreement with the predicted structure.


1

51

101 134

aMDVFMKGLSM AKEGVVAAAE KTKQGVTEAA EKTKEGVLYV GSKTREGVVQ

GVASVAEKTK EQASHLGGAV FSGAGNIAAA TGLVKREEFP TDLKPEEVAQ

EAAEEPLIEP LMEPEGESYE DPPQEEYQEY EPEA

244.9 374.0

401.0504.9

724.1

844.1

1167.21323.2

400 600 800 1000 1200 m/z

y2 y3

b4

y4

715.1y6

b7

y7

853.1b8 y10

1194.2b11

b12y8

957.1

1296.2y11

1063.2b10

611.0b6

b MS/MS, m/z 784.5 (2+)102A A E E P L I E P L M E P E115

y3y4y6y7y8y10y11 y2

b12b11b10b8b6b4 b7

102A A E E P L I E P L M E P E115y3y4y6y7y8y10y11 y2

b12b11b10b8b6b4 b7

Figure 12: (a), Total ion chromatogram by LC-ESI-MS of the in solution digestion of

βSyn with Glu-C shows the presence of twelve peptides which are depicted in Table 5; (b), LC-MS/MS fragmentation mass spectrum of ion 784.5 (2+) (with y and b ions assigned), which led to the identification of peptide (102-115) is also displayed. Peptide (102-115) is in red and highlighted in a black box in the amino acids sequence. The insert in the upper part of the figure contains a schematic representation of the primary structure of βSynuclein (βSyn) that indicates the amino acid residues different than in αSyn sequence. In red are the peptides identified by LC-ESI-MS/MS.


Table 5: βSyn proteolytic Glu-C peptides identified by LC-ESI-MS.

Nr. Sequence Peptide ion Mexp a Mcalc

b

1 14 - 20 (616.1)1+ 615.1 615.3

2 118 - 126 (547.3)2+ 1092.6 1092.4

3 116 - 126 (640.2)2+ 1278.4 1278.4

4 14 - 28 (496.6)3+ 1487.0 1486.8

5 127 - 134 (514.3)2+ 1026.6 1027.4

6 47 - 57 (508.3)2+ 1014.6 1014.5

7 118 - 129 (757.5)2+ 1513.0 1512.5

8 47 - 61 (751.5)2+ 1501.0 1500.8

9 89 -101 (501.7)3+ 1502.1 1501.7

10 1 - 13 (743.5)2+ 1485.0 1485.7

11 62 - 87 (621.3)4+ 2481.2 2481.3

12 102 - 115 (784.5)2+ 1567.0 1566.7a Mass spectrometric analysis (Esquire 3000 +, Bruker Daltonics) b GPMAW software 5.0 (Lighthouse Data, Denmark)

Further characterization of βSyn was performed by SDS-PAGE gel

electrophoresis. The protein (5 µg) was reduced with urea and DTT and then

heated to 95°C for 5 min. The alkylation step was omitted because βSyn does

not have cysteine residues. After staining the gel with Coomassie Blue, a band

corresponding to the βSyn monomer was detected. The intensive band was

excised by cutting out the center (most concentrated part) to minimize

acrylamide contamination, and the peptides extracted after in gel digestion and

subjected to LC-ESI-ion trap mass spectrometric analysis. The identified

peptides were sequenced in the MS/MS mode. The sequences of the peptides

were determined by subjecting the MS/MS results to a database search against

the human proteome database using the MASCOT program.


M 2, β-Syn

40 -50 -

30 -25 -

20 -

15 -

kDa

60 -

85 -

200 -

Match to: gi4507111 Score 278βSynuclein [Homo sapiens]Mass: 14279; pI: 4.41

1

51

101 134

MDVFMKGLSM AKEGVVAAAE KTKQGVTEAA EKTKEGVLYV GSKTREGVVQ

GVASVAEKTK EQASHLGGAV FSGAGNIAAA TGLVKREEFP TDLKPEEVAQ

EAAEEPLIEP LMEPEGESYE DPPQEEYQEY EPEA

a b

Figure 13: (a), SDS-PAGE of the βSyn: Five micrograms of protein were subjected to

SDS–PAGE (12%) and subsequently stained with Coomassie blue; (b), MASCOT browser output depicting the sequence coverage for the human βSyn (gi 4507111) with score of 278. In the amino acids sequence of the protein, the identified tryptic peptides are shown in red and the amino acid residues different than in αSyn sequence are underlined.

2.2 Formation, electrophoretic separation and isolation of oligomerization-aggregation products of synucleins

αSyn possesses a high propensity to aggregate and thus represents a

suitable model for amyloidogenic intrinsically disordered proteins (IDPs). The

aggregation conditions, acidic pH and high temperature have been also shown

to favour the partial folding of αSyn [249]. Besides the amyloidogenic partial

folding, αSyn reveals a diversity of structurally unrelated conformations like the

mainly unfolded state as well as different α-helical or β-structural species with

various folding degrees, equally found as monomeric and oligomeric forms.

Furthermore, the aggregates can be categorized into several morphologically

different types, including oligomers (spheres or doughnuts), amorphous

aggregates, and amyloid-like fibrils [249, 250] (see 1.3, Figure 5b). Since the

protein can adopt different folded conformations, it was named “protein

chameleon”, according to which the structure of αSyn to a high degree depends


on the environment and the preference for different conformations is determined

by the features of protein surroundings [249, 251].

Abnormal αSyn aggregation was found in sporadic and familial PD, the

underlying cause of which is currently not known with certainty [140]. A first step

is the aggregation of typically (3 - 30) αSyn monomers to form oligomers, which

in their transient configuration are termed protofibrils [252, 253]. Some of these

form filamentary fibrils, which together with ubiquitin and some other chemical

components associate to form well structured cytoplasmatic inclusions as they

mature to form the basis of LBs [252, 253]. Interestingly, αSyn fibrils were found to

be radially arranged within the otherwise spherical structures [134], a finding that

allows the assumption that αSyn plays an active role in the aggregation

process. The rate of fibril formation is nucleation dependent and most notably

promoted by excessive concentrations of wild type αSyn itself [254, 255]. Once a

critical concentration is attained, aggregates precipitate [256], which might

contribute to the late-life appearance and rapid progression of PD.

2.2.1 In vitro oligomerization-aggregation products of synucleins

2.2.1.1 Separation of oligomerization-aggregation products of synucleins using Tris-tricine gel electrophoresis

αSyn oligomers were prepared by dissolving the wt-αSyn [250] in sodium

phosphate buffer (pH 7.5), ammonium acid carbonate or ammonium acetate,

followed by incubation at 37°C with shaking for different periods of time. αSyn

oligomers type S2 were obtained by dissolving the lyophilized αSyn in 20 mM

PBS, 0.03% NaN3 [257] up to 30 µM, corresponding to the range of the reported

concentration of αSyn found in the cytoplasm of neurons [247], followed by

shaking at 37°C. Moreover, αSyn oligomers S3, S4 were prepared by dissolving

the recombinant protein in ammonium acetate (NH4Ac) and ammonium acid

carbonate (NH4HCO3) followed by incubation at 37°C with shaking for different

periods of time (2 - 48 h). These buffer solutions were selected because of their

compatibility with mass spectrometry. αSyn oligomers type S5 were prepared


by dissolving the protein in 50 mM sodium phosphate buffer (pH 7.0) containing

20% ethanol to a final concentration of 7 µM [250]. In the case of oligomers type

S6, 10 µM of FeCl3 were additionally added, followed by incubation for 7 days at

25°C. After their preparation, αSyn oligomers were characterized by Tris-tricine

gel electrophoresis, immunoanalytical and mass spectrometric methods.

Table 6: In vitro incubation conditions for the formation of αSyn oligomerization-aggregation products:

Code Buffer Concentration Temperature (°C)

S1 water 30 37

S2 20 mM PBS 30 37

S3 20 mM NH4Ac 30 37

S4 20 mM NH4HCO3 30 37

S5 50 mM PBS 7 25

S6 50 mM PBS, 10 µM FeCl3 7 25

First attempts for identification of αSyn oligomerization-aggregation

products were performed by 1D- Tris-tricine polyacrylamide gel electrophoresis.

Figure 14 shows three 15% Tris-tricine gels run simultaneously that contained

αSyn oligomerization-aggregation products (S2, S3, S4) obtained by incubation

of the protein at 37°C for two days with shaking at a final concentration of 30

µM. The first gel was analyzed using the Gel BioAnalyzer, which uses protein

detection based on native fluorescence of aromatic residues, mainly tryptophan

residues. It has been recently reported that the fluorescence intensity depends

on the amount of tryptophane and aromatic amino acids present in proteins [258,

259]. Wt-αSyn contains four tyrosine (2.86%), two phenylalanine (1.43%) but no

tryptophane residues, therefore the band corresponding to this protein gave a

week fluorescence signal at ~17 kDa (Figure 14a). Figure 14b shows the

Coomassie stained gel and Figure 14c the silver staining. Monomeric αSyn

migrated as a 15 to 20 kDa band [(αSyn)1]. In addition, a number of further

bands were observed: a presumed dimer [(αSyn)2] at 37 kDa, higher molecular

weight aggregates [(αSyn)n], and several truncation and/or degradation

products with 14, 12 and 10 kDa bands [∆N-αSyn].


M S2 S3 S4(2 d) (2 d) (2 d)

40 -50 -

30 -25 -20 -15 -

kDa

10 -

60 -80 -

200 -

40 -50 -

30 -25 -20 -15 -

kDa

10 -

60 -80 -

200 -

M S2 S3 S4(2 d) (2 d) (2 d)

(αSyn)n

(αSyn)1

(αSyn)2

∆N-αSyn

a

40 -50 -

30 -25 -20 -15 -

kDa

10 -

60 -80 -

200 -

M S2 S3 S4(2 d) (2 d) (2 d)

(αSyn)n

(αSyn)1

(αSyn)2

∆N-αSyn

b

c

Figure 14: 15% Tris-tricine gel separation of αSyn oligomerization-aggregation

products (S2, S3, S4) obtained by incubation of the protein at 37°C for two days with shaking at a final concentration of 30 µM: (a), Unstained gel with native fluorescence detection; (b), Coomassie staining; (c), Silver staining.

αSyn oligomers type S5, S6 [250] prepared by dissolving purified protein in

50 mM PBS (pH 7.0) to a final concentration of 7 µM and incubation for seven

days, were separated by Tris-tricine gel electrophoresis using a 15% separation

gel. Proteins were visualized by sensitive colloidal Coomassie blue and the gel

was scanned using a GS-710 calibrated imaging densitometer. In addition to a

monomeric αSyn [(αSyn)1] band visible at approximately 17 kDa, the gel

separated bands of dimeric [(αSyn)2] and oligomeric [(αSyn)n] products as well

as three additional bands below the αSyn monomer, corresponding to truncated


or proteolytic forms ∆N-αSyn, which were identified by mass spectrometry in

2.3.

40 -50 -

30 -

25 -

20 -

15 -

kDa

10 -

60 -

85 -

200 - (αSyn)n

(αSyn)1

(αSyn)2

∆N-αSyn

M S5 S6 (7 d) (7 d)

Figure 15: Coomassie staining of 15% Tris-tricine gel separation of αSyn

oligomerization-aggregation products (S5, S6) obtained by incubation of protein at 25°C for seven days with shaking at a final concentration of 7 µM. Besides the monomeric αSyn [(αSyn)1] band visible at approximately 17 kDa, bands of dimeric (αSyn)2 and oligomeric (αSyn)n products as well as three additional bands below the αSyn monomer, corresponding to truncated or proteolytic forms ∆N-αSyn are visible.

2.2.1.2 Immunoanalytical characterization of oligomerization-aggregation products of synucleins

The αSyn oligomerization-aggregation products were assessed for their

binding affinity to (i), anti-αSyn pASY-1 and (ii), anti-oligomer A11 polyclonal

antibody using Dot blot analysis. The anti-oligomer A11 antibody recognized

preferentially aggregation products (Figure 16b) while the pASY-1 antibody

recognized specifically the monomeric and oligomeric forms of αSyn (Figure

16a). A plausible explanation for these results is the existence of an equilibrium

between monomers and oligomers.


S1 S2 S3 S4 S5 S6

S1 S2 S3 S4 S5 S6

a

Anti-oligomer A11 Ab

Anti-αSyn pASY-1 Ab

b

Figure 16: Dot blot affinity experiments of αSyn oligomer- aggregates with: (a), anti-

αSyn pASY-1 diluted 2500 times in PBS-T (v/v) and (b), anti-oligomer A11 diluted 1000 times in PBS-T (v/v). The pASY-1 antibody recognizes specifically the monomeric form of αSyn found in equilibrium with the aggregation products while anti-oligomer A11 antibody recognizes preferentially αSyn oligomerization-aggregation products.

For immunoblotting, the oligomer-aggregate separations (10 µg total

protein/ gel lane) were first performed using 15% Tris-tricine gels. The gels

were then used for transfer on nitrocellulose membranes and immunoblotting

with the anti-αSyn pASY-1 antibody (Figure 17) and the anti-oligomer A11

antibody (Figure 18). The pASY-1 antibody specifically recognized monomeric

[(αSyn)1], dimeric [(αSyn)2], oligomeric (αSyn)n αSyn bands in oligomerization-

aggregation products and three additional bands below the αSyn monomer that

were assigned to truncation forms [∆N-αSyn]. The anti-oligomer A11 antibody

was subsequently proven to be unsuitable for Western blot analysis.


M S2 S3 S4(2 d) (2 d) (2 d)

(α-Syn)1

(α-Syn)2

Figure 17: Western blot αSyn oligomer- aggregates S2, S3, S4 with anti-αSyn pASY-1

diluted 2500 times in PBS-T illustrating the specific binding to monomeric (αSyn)1 and dimeric (αSyn)2 αSyn.

M S5 S6(αSyn)n

(αSyn)1

(αSyn)2

∆N-αSyn

40 -50 -

30 -25 -

20 -15 -

M S5 S6kDa

40 -50 -

30 -25 -20 -15 -

kDa

ba Anti-oligomer A11 AbAnti-αSyn pASY-1 Ab

Figure 18: Western blot of αSyn oligomer- aggregates S5, S6 with: (a), anti-αSyn

pASY-1 diluted 2500 times in PBS-T and (b), anti-oligomer A11 diluted 1000 times in PBS-T. The pASY-1 antibody specifically recognizes the monomeric αSyn (αSyn)1, dimeric (αSyn)2, oligomeric (αSyn)n products and three additional bands below the αSyn monomer corresponding to truncated forms ∆N-αSyn. The anti-oligomer A11 antibody is not suitable for Western blot analysis.

αSyn oligomerization-aggregation products S5, S6 were subjected to

ELISA to compare for their binding to anti-αSyn pASY-1 and anti-oligomer A11

antibodies. The experiments were performed using antigen dilutions from

1.4×10-5 µM to 2.5 µM (quantitative comparison of binding intensities). After

coating the plates with the aggregates, anti-αSyn pASY-1 antibody and anti-


oligomer A11 were applied in 1:2500 and 1:1000 dilutions, and peroxidase

conjugated goat anti-rabbit IgG and OPD/H2O2 substrate added for analysis.

Figure 19a shows that monomeric αSyn, in equilibrium with higher molecular

weight aggregates, bound to the anti-αSyn polyclonal antibody. After

optimization of the ELISA experimental conditions concerning aggregate

concentration and at increased temperature up to 37°C, the data showed that in

vitro αSyn oligomerization-aggregation products had no affinity to the anti-

oligomer A11 polyclonal antibody (Figure 19b).

0

0.5

1

1.5

2

2.5

OD

(450

nm

)

S5S6

0.000040.0001

0.00030.001

0.0030.01 0.03

0.0920.27 0.83 2.5

-0.02

0

0.02

0.04

0.06

0.08

0.10

S5S6

Antigen concentration (µM)

OD

(450

nm

)

a

b0.00001

0.000040.0001

0.00030.001

0.0030.01 0.03

0.0920.27 0.83 2.5

0.00001

Anti αSyn pASY-1 antibody

Anti-oligomer A11 antibody

Figure 19: ELISA (enzyme-linked immunosorbent assay) of αSyn oligomer-aggregates

(S5, S6) using: (a), anti-αSyn pASY-1 diluted 2500 times in 5% BSA; (b), anti-oligomer A11 diluted 1000 times in 5% BSA. The pASY-1 antibody shows affinity to S5, S6 type aggregates.

2.2.2 In vivo oligomerization-aggregation products of synucleins

Several animal models have been developed based on transgenic

expression of αSyn [255]. Expression of αSyn in transgenic mouse neurons


partially reproduced some features of human Lewy pathology, accumulation of

detergent-insoluble αSyn in neuronal cell bodies and swollen neurites and

cardinal features of PD pathology including proteinase K (PK) resistance [169, 254,

260-264]. Moreover, the prediction sites of α-synucleinopathy in old Thy-1-human

(A30P) αSyn transgenic mice overlapped with the sites of earliest α-

synucleinopathy in humans [265], mainly in hindbrain region. Investigation of

transgenic mouse models suggested a cascade of events that might occur in

human patients as well, hence αSyn transgenic mice could serve as a model

system to study the molecular mechanism of abnormal αSyn aggregation.

The generation of Thy-1-human (A30P) αSyn transgenic mice has been

described elsewhere [261, 266-268]. Thy1 is a brain neuron specific promoter [269]

that was introduced to drive over-expression of human, mutated αSyn (A30P) in

a transgenic animal model of Parkinson’s disease. The two mice showing a

progressive deterioration of locomotors function were sacrificed by cervical

dislocation, and brains harvested and divided into four parts: forebrain left

(FB/L)/ right (FB/R), hindbrain left (HB/L)/ right (HB/R). αSyn (A30P) protein

(hereafter referred to as 3) was extracted from brain tissue using multiple steps

with five different buffers (Figure 93). First, 300 µL buffer 1 (tris buffer saline,

TBS) were added to the brain pieces which were thereafter crash with a tissue-

ruptor. The brain mixture was than rinsed with 200 µL buffer 1 and centrifuged

at 4°C. The soluble fraction was used as elution 1 (E1: TBS soluble fraction).

Further, the pellet was resuspended in 500 µL buffer 2 (TBS- 1% Triton X-100).

After centrifugation, the soluble protein fraction was used as elution 2 (E2) and

the pellet was resolubilized in 500 µL buffer 3 (TBS- 1M sucrose) using the

same procedure as in the previous step. Further, the soluble fraction resulted

after centrifugation was used as elution 3 (E3), while the pellet was dissolved in

500 µL buffer 4 (RIPA buffer). The last centrifugation step provided the elution 4

(E4) and pellet P5 which, after being brought in solution with a 6 M urea/ 5%

SDS in TBS buffer, was also employed as the last αSyn containing fraction. The

BCA-assay for protein quantification of mice brain fractions containing the TBS,

TBS-Triton, TBS-sucrose, RIPA and urea-soluble proteins was carried out and

revealed concentrations > 2 mg mL-1 in E1 and E2, and < 2 mg mL-1 in E3, E4,


P5. A more detailed description of the subcellular fractionation in isolation of

αSyn (A30P) oligomerization-aggregation products from human αSyn (A30P)

transgenic mouse brains is given in 3.2.4.

2.2.2.1 Isolation of α-synuclein oligomerization-aggregation products from human α-synuclein transgenic mouse brain homogenates using 1D and 2D-gel electrophoresis

The soluble mouse brain homogenate proteins were separated on 10%

Tris-tricine separation gel. 20 µg of the forebrain fractions (FB) and 20 µg of the

hindbrain fractions (HB) of two Thy-1-human (A30P) αSyn transgenic mice Syn-

m-130 and Syn-m-131 were first loaded on gel. Prior to Coomassie staining, the

gel was analyzed using the Gel BioAnalyzer, which showed a weak

fluorescence signal (Figure 20a). In Figure 20b the Coomassie stained gel is

shown revealing a complex mixture of proteins extracted from Thy-1-human

(A30P) αSyn transgenic mice.

M

Syn-m-130 Syn-m-131

HB FB HB FB kDa M

Syn-m-130 Syn-m-131

HB FB HB FB 3

40 -50 -

30 -25 -20 -15 -

kDa

10 -

85 -

200 -

a b

40 -50 -

30 -25 -20 -15 -

10 -

85 -

200 -

a HB hindbrainb FB forebrain

Figure 20: 10% Tris-tricine gel separation of 20 µg of elution 1 (E1) containing TBS soluble mouse brain homogenate proteins: (a), Unstained gel with native fluorescence detection; (b), Coomassie staining showing in lane 6 protein 3 (recombinant αSyn A30P) used as control.


Due to the complexity of the mouse brain homogenate protein mixtures, 1D

electrophoresis could not provide efficient resolution, so that 2D gel

electrophoresis was mainly employed as a separation tool. 2D Electrophoresis

was carried out to evaluate the isolation and localization of proteins spots

containing TBS soluble mouse brain homogenate proteins present in hindbrain

left (HB/L) and hindbrain right (HB/R) region. After intensive optimization

experiments concerning the protein amounts and percentage of the separation

gels to be used, satisfactory gel maps were obtained with approximately 1 mg

total protein loaded on each 15% separation gel. The proteins were loaded on a

3-10 linear immobilized pH gradient (IPG) strip (17 cm), separated and

visualized with sensitive colloidal Coomassie staining. The protein spots

separated at these conditions are shown in Figure 21. Using the Bio-Rad

PDQuest software comparable protein spots were illustrated in both hindbrain

left (HB/L) and right (HB/R) regions, hence in the following experiments the left

and right regions were combined.

SDS

IEF60 kDa

5 kDapI 3 10 pI 3 10

60 kDa

5 kDa

ba

SDS

IEF

Figure 21: 2D gel electrophoresis separation of elution 1 (E1) containing TBS soluble

mouse (Syn-m-131) brain homogenate proteins within pI 3–10: (a), hindbrain left (HB/L) and (b) hindbrain right (HB/R). 1 mg total protein were loaded on a 3-10 linear IPG strip (17 cm), separated using 15% separation gel and visualized with sensitive colloidal Coomassie staining. The protein spots revealed by 2D are identically present in both samples.


2.2.2.2 Biochemical characterization of oligomerization-aggregation products from human α-synuclein transgenic mouse brain homogenates by Western blot analysis

The expression of an αSyn (A30P) mutant isoform (hereafter referred to

as 3) was detected by Western blot analysis in the transgenic mouse brain

homogenates (Figure 23). To assess the solubility of the protein 3, differential

extractions were performed. Most of the protein was highly soluble in TBS

aqueous buffer (E1) (Figure 22) and the rest easily extractable with TBS- 1%

Triton X-100 (E2), TBS– 1M sucrose (E3), RIPA buffer (E4). However,

detergent- insoluble αSyn (A30P) monomers and aggregates were detected in

urea extracts from the transgenic mouse brains.

kDa

(3)1

40 -50 -

30 -

15 -20 -

25 -

(3)n

M E1 E2 E3 E4 P5

Figure 22: Western blot analysis of 20 µg of elution fractions (E1, E2, E3, E4) and

pellet (P5) mouse (Syn-m-130) brain homogenate proteins Thy-1-human (αSyn A30P) using anti-αSyn pC20 diluted 1:500 (v/v). The antibody specifically recognizes the monomer 3 and additionally several oligomeric bands.

The soluble mouse brain homogenate proteins (elution E1) were further

characterized by Western blot using anti-synuclein specific antibodies (Figure

23). First, the proteins (20 µg) were separated on a 10% Tris-tricine separation

gel. Separated proteins were then transferred onto nitrocellulose membranes,

and the blots incubated in 10% Rotiblock in water prior to probing with: (a),

mouse anti-α/βSyn antibody at 1:3000 (v/v) dilution; (b), mouse monoclonal

anti-αSyn m4B12 at 1:5000 (v/v) dilution; (c), mouse anti-αSyn mBD at 1:500


(v/v) dilution; (d), rabbit polyclonal anti-αSyn pC20 at 1:500 (v/v) dilution and (e),

rabbit polyclonal anti-αSyn pASY-1 at 1:2500 (v/v) dilution for 2 h at 25°C. After

three washes with PBS-T (10 min), membranes were probed with a 1:5000

dilution of horseradish peroxidase labeled goat anti-rabbit or rabbit anti-mouse

immunoglobulin secondary antibody. After washing, enhanced

chemiluminescence (ECL) substrate solution was used to generate the

chemiluminescent signal. The anti-α/βSyn recognized the monomeric (3)1 of 3,

αSyn (A30P) protein and oligomeric (3)n forms. Both anti-αSyn m4B12 and anti-

αSyn mBD antibodies bound to 3 monomer, oligomer (3)n and, additionally, to

the truncation products ∆N (3). As shown in Figure 23d, the anti-αSyn pC20

antibody recognized specifically the monomer as well as multiple bands in the

35- to 120- kDa range corresponding to putative oligomers of 3. The polyclonal

anti-αSyn pASY-1 antibody had affinity to multiple bands in the 55- to 120- kDa

range.

kDa M

Syn-m-130 Syn-m-131

HB FB HB FB

b

40 -50 -

30 -

15 -10 -

(3)n

(3)1

∆N (3)

M

Syn-m-130 Syn-m-131

HB FB HB FB

40 -50 -

30 -

15 -

kDa

10 -

a

(3)n

(3)1


M

Syn-m-130 Syn-m-131

HB FB HB FB kDa M

Syn-m-130 Syn-m-131

HB FB HB FB

40 -50 -

15 -

kDa

10 -

c d

40 -50 -

30 -

15 -

kDa M

Syn-m-130 Syn-m-131

HB FB HB FB

e

40 -50 -

25 -

(3)n

(3)1

∆N (3)(3)1

Figure 23: Western blot analysis of 20 µg of elution 1 (E1) containing TBS soluble

mouse brain homogenate proteins Thy-1-human (αSyn A30P) using anti-αSyn antibodies: (a), no.1 anti-α/βSyn diluted 1:3000 in PBS-T recognizes the monomeric (3)1 of 3, αSyn (A30P) protein and oligomeric (3)n forms; (b), no.2 anti-αSyn m4B12 diluted 1:5000 in PBS-T binds to 3, αSyn (A30P) monomer, oligomer and related ∆N (3), truncation products; (c), no.3 anti-αSyn mBD diluted 1:500 has an affinity similar to no.2 anti-αSyn m4B12 antibody; (d), no.4 anti-αSyn pC20 diluted 1:500 recognizes specifically the monomer 3 and additionally several oligomeric bands; (e), no.5 anti-αSyn pASY-1 diluted 1:2500 binds to oligomeric forms of 3 protein.

Figure 24 shows the 2D gel electrophoresis and Western blot of TBS

soluble mouse brain homogenate proteins present in hindbrain left (HB/L). An

amount of 1 mg total protein was applied on a linear IPG strip (3-10, 17 cm) and

run in the first dimension (IEF). After equilibration, the IPG strips were applied

on the top of 15% SDS polyacrylamide gels for the second separation step. Two

2D gels were run simultaneously and processed under identical conditions. The

first gel was stained with colloidal Coomassie blue and scanned using a GS-710

calibrated imaging densitometer, and the second gel subjected to

immunoblotting using the anti-αSyn mBD antibody. The monoclonal antibody

recognized protein spots corresponding to monomeric (3)1 of 3, αSyn (A30P), at

~15 kDa and a pI of 4.5.


SDS

IEF60 kDa

5 kDapI 3 10 pI 3 10

60 kDa

5 kDa

ba

SDS

IEF

(3)1

Figure 24: (a), 2D gel electrophoresis separation of elution 1 (E1) containing TBS

soluble mouse (Syn-m-131) hindbrain left (HB/L) homogenate proteins within pH 3–10; (b) Western blot using anti-αSyn mBD at 1:500 dilution in PBS-T. The antibody recognizes the monomeric (3)1 of αSyn (A30P) protein.

The detection of detergent- insoluble αSyn (A30P) monomers and

aggregates in urea extracts from transgenic mouse brains was confirmed by

Western blot using the anti-αSyn mBD antibody. Figure 25 shows the Tris-

tricine gel containing E1 (TBS soluble proteins) and P5 (detergent- insoluble

proteins) stained with colloidal Coomassie blue. In Figure 25b, the immunoblot

revealed monomeric (3)1, which migrated as a 17 kDa band and limited N-

terminal degradations ∆N (3) in the 7- to 15 kDa range.


ba

(3)1

∆N (3)

kDa

76 -

52 -

38 -31 -

24 -17 -

12 -

225 -

40 -50 -

30 -

25 -20 -15 -

kDa

10 -

85 -

200 -

M E1 P5 M E1 P5

70 -

Figure 25: (a), 1D gel electrophoretic separation of elution 1 (E1) containing TBS

soluble proteins and pellet (P5) containing urea/SDS insoluble proteins from mouse (Syn-m-130) hindbrain right (HB/R) homogenate; (b) Western blot using anti-αSyn mBD at 1:500 dilution in PBS-T. The antibody recognizes the monomeric (3)1 and ∆N (3) truncation products of αSyn (A30P) protein.

The identification of specific fragments, which were observed in low amounts as

proteolytic bands by gel electrophoresis and Western blot, was obtained by ion-

mobility mass spectrometry, due to its separation capability of proteins with

different topology and conformation, which showed this method as a powerful

tool for the direct characterization of intermediates in protein aggregation.

2.2.2.3 Separation and biochemical characterization of α-synuclein oligomerization-aggregation products from human neuroblastoma cell culture by gel electrophoresis and Western blot analysis

The human neuroblastoma cell line SH-SY5Y is widely used as a neural

cellular model system. The cells were prepared into a collagen flask using 500

mL cell growth medium containing high glucose supplemented with 15% fetal

calf serum (FCS), 4 mM glutamine and incubated at 37°C in the presence of 5%

CO2. Further, SH-SY5Y cells were split, scraped, washed by centrifugation in

cold PBS and resuspended in Triton-lysis buffer and incubated on ice for 15

min. After centrifugation the protein concentration of the supernatant was

determined to 3296 mg mL-1 using the BCA assay. Lysates (10 µg of protein)

were resolved by electrophoresis on a 4-12% Bis-Tris gradient gel (NuPAGE,


Invitrogen). After transfer, the nitrocellulose membrane was boiled for 5 min in

PBS and the membrane blocked in Odyssey Blocking Buffer for 1 h at 25°C.

The blot was probed with anti-αSyn mBD antibody for 1 h at 25°C or overnight

at 4°C. Bands were detected using fluorescence alkaline phosphatase-

conjugated goat anti-mouse 1:15000 (absorbance – 770 nm, emission – 794

nm) secondary antibody for 1 h at 25°C in the dark and imaged with Odyssey

Infrared Imager (Li-COR Biosciences). This imager introduces a direct infrared

fluorescence detection which provides a quantitative analysis and wide linear

dynamic range.

In Figure 26 the Western blot of human neuroblastoma SH-SY5Y αSyn

(A53T) cell extracts without acetone precipitation (-) and after acetone

precipitation (+) is shown. The monoclonal antibody recognized specifically the

4, αSyn (A53T) monomer and related ∆N (4), truncation products.

baM SH-SY5Y α-Syn (A53T)

- +M SH-SY5Y α-Syn (A53T)

- +

76 -

52 -

38 -31 -

24 -17 -

12 -

kDa

(4)1

∆N (4)

kDa

(4)1

∆N (4)

225 -

76 -

52 -

38 -31 -

24 -17 -

12 -

225 -

Figure 26: Western blot of SH-SY5Y αSyn (A53T) cell extracts without acetone

precipitation (-) lane 2 and after acetone precipitation (+) lane 3 using anti-αSyn mBD: (a), green florescence; (b), grey scale image of the green fluorescence. The monoclonal antibody recognizes specifically the 4, αSyn (A53T) monomer and related ∆N (4), truncation products.

2.3 Structure identification of oligomerization and degradation products of α-synuclein by ion-mobility mass spectrometry

Protein amyloidogenesis is generally considered to be a major cause of

two most severe neurodegenerative disorders, Parkinson’s disease (PD) and

Alzheimer’s disease (AD). Formation and accumulation of fibrillar aggregates


and plaques derived from α-synuclein (αSyn) and β-amyloid (Aβ) polypeptide in

brain have been recognized as features of Parkinson’s disease and Alzheimer’s

disease. Oligomeric aggregates of αSyn and Aβ are considered as neurotoxic

intermediate products leading to progressive neurodegeneration. However,

molecular details of the oligomerization and aggregation pathway(s) and the

molecular structure details are still unclear. Nevertheless, chemical structures of

αSyn oligomers and its possible intermediates have not been hitherto identified.

The slow rates of formation and low concentrations of aggregating

intermediates have been suggested as major reasons for the failure of

conventional methods of mass spectrometry to identify oligomerization-

aggregation products.

2.3.1 Primary structure characterization of in vitro oligomers by electrospray ionization mass spectrometry

In vitro oligomerization products of αSyn type S2, S3, S4, S5, S6 were

prepared by incubation [250] in sodium phosphate buffer, ammonium acid

carbonate or ammonium acetate followed by incubation at 37°C with shaking for

different periods of time as described in 2.2.1 and 3.2.2 and summarized in

Table 21. The formation of oligomers was monitored by one-dimensional Tris-

tricine polyacrylamide gel electrophoresis (Figure 27) which revealed bands

corresponding to monomeric (band 1) and oligomer-like αSyn at approximately

37 kDa (band 5) and 200 kDa (band 6). Furthermore, three bands (2, 3, 4) of

minor abundances with molecular weights lower than full-length αSyn indicated

the formation of truncation or degradation products.


40 -

50 -

30 -

25 -

20 -

15 -

kDa

10 -

60 -

85 -

200 -

120 -

1, 1a

2

34

5

6M S1 S2 S3 S4 S5 S6

Figure 27: Tris-tricine PAGE of αSyn oligomerization in vitro stained with Coomassie

Blue: left lane molecular weight marker proteins; (S1) αSyn freshly prepared (lane 2); (S2) αSyn incubated in PBS buffer at 37°C, 2 days (lane 3); (S3) αSyn in ammonium acetate at 37°C, 2 days (lane 4); (S4) αSyn in ammonium hydrogen carbonate at 37°C, 2 days (lane 5); (S5) αSyn incubated in PBS buffer at 25°C, 7 days (lane 6). 1, 1a, intact wt-αSyn and truncation product αSyn (7-140); 2, 3, 4, αSyn truncation and proteolytic products (Table 9); 5, dimer of full-length αSyn; 6, oligomer of αSyn.

Direct mass spectrometric analysis of monomer (band 1) was performed

with an Esquire 3000+ ion trap mass spectrometer. The protein was solubilized

in 1% formic acid as a volatile solvent, to a final concentration of 30 µM, pH 2.5.

The ESI spectrum was obtained in the positive ion mode, and showed a series

of multiply charged ions with the most abundant ion at charge state +19 (data

not shown). The bands due to αSyn monomer 1 and “oligomer” 5 were excised

from the gel, digested with trypsin and analyzed by LC-ESI-MS/MS. For the LC

separation, a binary gradient system consisting of solvent A (0.1% formic acid in

water) and solvent B (80% acetonitrile, 0.1% formic acid in water) was

employed and the spectra were recorded in positive ion mode. The resulting

peptides (Table 7) were sequenced in the MS/MS mode (Figure 28c). Peptide

sequences were determined by subjecting the MS/MS results to database

search using the MASCOT program, and were searched against the human

proteome database. The combination of gel electrophoresis with in gel tryptic


digestion, LC-ESI-ion trap-MS/MS and database search was found to be an

excellent tool for the structure determination of αSyn. ESI-MS and tandem-MS

of the tryptic peptides revealed the presence of monomeric and dimeric αSyn

with full-length sequences, respectively; additional structural characterization

was obtained by Edman sequencing after transfering the proteins onto a PVDF

membrane (2.3.2).

9 10

2

3

46

8

9

10

711

10 20 30 40 50 60 70 Time [min]

1

5

493.6

739.9

300 500 700 900 1100 m/z

3+

2+

493.6

739.9

300 500 700 900 1100 m/z

3+

2+

1081TVEGAGSIAAATGFVK96

G T A A A I/L S GF

200 400 600 800 1000 1200 m/z

y2

y3

y4

y5 y6y7

y8

y9 y10

b15

b14

y13

b13

b12b11

b10

b9

b8

540.1

720.0

300 500 700 900 1100 m/z

3+

4+540.1

720.0

300 500 700 900 1100 m/z

3+

4+

959TKEQVTNVGGAVVTGVTAVAQK80

822.4

857.4

906.4

943.4

1006.0

A V A T

200 400 600 800 1000 1200 m/z

y2

y3y4

y5y6

b

c

a

692.8

Figure 28: LC-ESI-MS/MS of αSyn (S1) tryptic peptides: (a), Total Ion Chromatogram

(TIC); (b), ESI mass spectra of the fraction 30.2 - 30.4 min and of the fraction 30.7 - 30.8; (c), MS/MS fragmentation of the double charged ion at m/z 739.9 (with y and b ions assigned) and of the triple charged ion at m/z 720.0 (with y and b ions assigned) led to the identification of peptides (59-80) and (81-96) that are also displayed.


Table 7: Identification of αSyn proteolytic (tryptic) peptides by a) LC-MS/MS and b) MALDI-TOF-MS for protein bands 1 and 5 from αSyn oligomerization-aggregation products (for band numbers see Figure 27).

Nr. Peptide sequence of

protein band

Molecular ion observed

Mexp Mcalc

Band 1 Band 5

1 11 - 21 (536.7)2+ a) 1071.4 1071.6

2 13 - 21 (437.2)2+ a) 872.7 872.4

3 46 - 60 (509.1)3+ a) 1523.4 1523.8

4 46 - 58 (432.6)3+ a) 1295.1 1295.6

5 35 - 45 (950.8)2+ a) 1179.5 1179.6

6 35 - 43 (476.2)2+ a) 950.5 950.5

7 1 - 6 (385.6)2+ a) 769.3 769.3

8 81 - 97 (536.4)3+ a) 1606.2 1606.8

9 59 - 80 (720.1)3+ a) 2157.2 2157.4

10 81 - 96 (740.2)2+ a) 1478.5 1478.6

11 61 - 80 (643.6)3+ a) 1927.9 1928.1

12 59 -102 (4288.7)1+ b) 4287.7 4287.8

13 97 -140 (4959.1)1+ b) 4958.1 4958.1a Mass spectrometric analysis by LC-ESI-MS (Esquire 3000 +, Bruker Daltonics) b Mass spectrometric analysis by MALDI-TOF-MS

Moreover, direct ESI-MS of αSyn upon incubation in ammonium acid carbonate

for 3 h at 37°C (sample S4) provided the identification of an initial N-terminal

truncated peptide sequence αSyn (7-140) of 13 kDa (band 1a). In contrast to

these results, attempts to identify the truncation or degradation products (bands

2, 3, 4 in Figure 27) by direct mass spectrometric analysis and HPLC-MS were

unsuccessful, presumably due to their low concentrations.


807.3

857.6

914.8

980.1

1033.7

1055.3

1113.1

1143.3

1205.8

1247.0

1315.31371.6

1446.8 1523.9

1607.3

800 900 1000 1100 1200 1300 1400 1500 1600 m/z

11+

12+

13+

17+

16+

15+

14+

14+

13+

12+

11+

965.015+

14+904.6

851.517+

10+

9+10+9+

1 (1-140)1a (7-140)

1MDV A140

7GLS A140

Mw exp 1: 14460.5 Da

Mw exp 1a: 13708.6 Da

Figure 29: ESI-ion trap-MS of the αSyn incubated in 20 mM NH4HCO3 for 3 h at 37°C

(S4). The protein was solubilized in 0.2% formic acid at a final concentration of 25 µM, pH 2.5 using an Esquire 3000 + ion trap mass spectrometer. In black is the amino acids sequence of αSyn; in blue is the truncated peptide sequence 1a, αSyn (7-140).

2.3.2 Detection and sequence determination of α-synuclein oligomers and degradation products

In the most useful method of N-terminal residue identification, the Edman

degradation occurs in three stages, each requiring different conditions [270].

Amino acid residues can therefore be sequentially removed from the N-terminus

of a polypeptide in a controlled stepwise approach (3.13). Subsequent to

electrophoretic separation, the bands of αSyn oligomerization-aggregation

products (S2, S3, S4, S5, S6) were transferred onto polyvinylidene difluoride

(PVDF) membranes and subjected to Edman sequencing in order to provide the

identification of possible N-terminal truncations of αSyn. For this purpose, Tris-

tricine gels containing αSyn oligomerization-aggregation products (Figure 27)

were electroblotted onto membranes using a semi-dry transfer procedure.

Membranes were stained with 0.1% Coomassie Brilliant Blue R-250 in 40%

methanol in water until protein bands became visible, and subsequently air-

dried The membranes were then destained in destaining solution (50%

methanol in water) until the bands were visible. The visible bands 1, 2, 3, 4 and


5 (Figure 27) were excised and destained with 100% methanol and applied into

the sequencing cartridge. Figure 30 shows an example of a PVDF membrane

stained with 0.1% Coomassie Brilliant Blue R-250 of αSyn aggregates S5 and

S6 obtained by incubation at 25°C for seven days. The presence of αSyn

molecular species was ascertained by probing the PVDF membrane with anti-

αSyn mBD antibody diluted 1:500 (v/v) in PBS-T, in a following immunoblotting

experiment, which also confirmed the correct excision of the bands used for

sequence determination. This approach (gel bands transfer on membrane,

excision for Edman analysis and immunoblotting of the remaining membrane)

was employed several times, thereby analyzing the bands from the whole range

of molecular weights. The monoclonal anti-αSyn antibody recognized

specifically the αSyn monomer (band 1), the dimer (band 5) and truncation

products (band 3, 4), as well as multiple bands in the 70- to 200 kDa range

corresponding to putative oligomers. The data obtained by Edman degradation

confirmed the presence of αSyn N-terminal amino acid sequences by the

analysis of αSyn dimer (band 5), thus confirming the previous mass

spectrometric analysis (2.3.1), accordingly to which the dimer band consists of

an associate of two intact αSyn polypeptides (Table 8).

40 -50 -

30 -

25 -20 -15 -

kDa

10 -

70 -85 -

M S5 S6 M S5 S6

123, 4

5

a b

123, 4

Figure 30: (a) PVDF membrane stained with 0.1% Coomassie Brilliant Blue R-250 of

S5, S6 αSyn aggregates shows the presence of bands 1, 2, 3 and 4 that were excised and subjected to Edman analysis; (b), Immunoblotting of PVDF membrane with anti-αSyn mBD antibody diluted 1:500 (v/v) in PBS-T, which indicates the presence of multiple bands that were not visible stained on the membrane (Figure 30a).


The bands 1, 2, 3 and 4 were excised and subjected to Edman analysis using

an automated sequencer that sequentially cleaved N-terminal amino acids and

analyzed the resulting phenylthiohyantoin (PTH)-amino acid residues. The

HPLC chromatograms for six PTH-amino acids released during Edman cycles

of band 3 plus 4 are shown in Figure 31. A standard mixture of PTH amino

acids was injected onto the column for separation as the standard cycle of the

sequencing run. The retention times of the amino acids from this chromatogram

are used to identify the amino acids in the sequencing cycles, providing the

identification of N-terminal amino acid sequences of αSyn and a new truncation,

αSyn (5-140), in the band at 13.9 kDa, (Table 8).

G

DN

SQTGE H A R Y P MV dptu W

dpuF I KL

5.0 10.0 15.0 20.0Retention time (min)

K

L

dptu

dptu

dptu

S

dptu

dptu

M dpu

dpu

dpu

dpu

dpu

269

nm

Standard

Residue 1

Residue 2

Residue 3

Residue 4

Residue 5

Residue 6

K

dptu

dpu

Figure 31: N-terminal Edman sequencing profile of band 3 plus 4 of αSyn S5 and S6

oligomerization-aggregation products. The standard amino acids and the peak of residues number 1, 2, 3, 4, 5 and 6 corresponded to the ordering of individual amino acid shown in red, which match with an N-terminally truncation, αSyn (5-140), in the band at 13.9 kDa (Table 8).


The N-terminal sequences of αSyn oligomerization-aggregation products

determined by Edman analysis are summarized in Table 8.

Table 8: N-terminal sequences determined by Edman analysis:

Sample No. Band Cycles Sequence

S2

S3

S4

S5

S6

1 12

10

5

12

9

MDVFMKGLSKAK__

MDVFMKGLSK__

MDVFM__

MDVFMKGLSKAK__

MDVFMKGLS__

S5

S6

2 10 ___

S5

S6

3 + 4 14

25

5MKGLSKAKEGVVAA__

5MKGLSKAKEGVVAAAEKT*QGV__

S2 5 12 MDVFMKGLSKAK__

2.3.3 Elucidation of oligomers and proteolytic products in vitro by ion-mobility mass spectrometry

Conventional “soft-ionization” mass spectrometric methods such as ESI-

MS and HPLC-MS are not suitable to direct “in-situ” analysis of conformational

states and intermediates at different concentrations. The ESI-MS analysis of

oligomer-aggregates αSyn type S4 incubated in ammonium acid carbonate for

3 h at 37°C revealed an initial N-terminal truncated peptide sequence αSyn (7-

140) of 13 kDa (band 1a) (Figure 29). In contrast to these results, attempts to

identify the truncation or degradation products (bands 2, 3, 4 in Figure 27) by

direct mass spectrometric analysis and HPLC-MS were unsuccessful,

presumably due to their low concentrations, and therefore ion-mobility mass

spectrometry (IMS-MS) was employed.

2.3.3.1 Methodology of ion-mobility mass spectrometry

Ion-mobility mass spectrometry (IMS) was first introduced in the 1970s

as a portable economical alternative to MS for detection of airborne volatiles,


e.g., environmental and industrial process contaminants, explosives, chemical

warfare agents, and drugs [271]. Over the past decade, two major technical

developments have raised and broadened the analytical importance of IMS: (i)

the effective coupling of IMS to MS [272], especially the advent of IMS-TOF

instrumentation that enables a parallel dispersion of ion mixtures in mobility and

mass/charge (m/z) dimensions [273, 274], and (ii) interfacing of ESI and MALDI

soft ionization sources to IMS [275] and IMS-MS [276, 277] which has enabled

applications to biopolymer chemistry and biomedicine.

Ion-mobility spectrometry and mass spectrometry share a similar

theoretical background and the historical development of both was presented

recently by Uetrecht and co-workers [278]. Mass spectrometric methods are

limited to the separation of ions measured by mass-to-charge (m/z) ratios and

ion intensities, while ion-mobility separates ions based on their mobility and

measures the time that it takes an ion to migrate through a buffer gas in the

presence of a low electric field (drift time). The velocity of the ion, ν (drift

velocity) is directly proportional to E with the proportionality constant K called

ion-mobility, and is determined by measuring the time required, tD to traverse a

drift cell of known dimensions, d. IMS-MS enables separation of species

according to their collisional cross section (CCS) (Ω) which represents the

orientationally averaged area of the ion capable to interact with a buffer gas. For

large ions (e.g. proteins), Ω can be approximated computationally [279]. Ions with

a larger apparent diameter undergo a greater number of collisions with the

buffer gas, consequently their passage through the drift cell is retarded in

comparison to smaller ions which experience less friction [278]. IMS-MS

implements a new mode of separation that allows the differentiation of protein

conformational states; therefore is ideally suited to the study of protein folding,

unfolding and aggregation [243, 280, 281].

Recently, “traveling wave” ion-mobility mass spectrometry (TWIMS) [282,

283] has been developed as a new tool to probe complex biomolecular

structures, due to the potential of IMS-MS for separation of mixtures of protein

complexes by conformation state, spatial shape and topology [237, 238, 284-286].

Unlike the traditional “drift time” IMS, in which a low electrical field is applied


continuously to the cell, a high electrical field is applied to one segment of the

cell, thus ions are moved through the mobility cell in pulses as waves of the

electrical field pass through them [239, 287, 288]. IMS spectra are obtained in milli

seconds and TOF mass spectra are obtained in micro seconds, so that

thousands of mass spectra can be obtained for each ion-mobility spectrum

producing a two-dimensional array in which both mobility and mass of ions are

recorded [239].

IMS-MS was applied to the direct analysis of mixtures of αSyn

aggregation products in vitro during prolonged incubation times. IMS-MS

determinations were performed at Waters Corporation (Manchester, UK) with a

Synapt High Definition Mass Spectrometry (HDMS) QTOF-MS equipped with a

nano-electrospray ionization source (Figure 32).

ESI sourceDetectorTrap Ion mobility

separation Transfer

T-Wave Ion Guide

Quadrupole

TRIWAVE

TOF

Figure 32: Adapted scheme of ion-mobility MS Waters Synapt high-definition mass

spectrometer (HDMS) system [282]: Ion-mobility MS system consists of an electrospray ionization (ESI) source, quadrupole mass spectrometer, ion gate, drift region (Triwave), time-of-flight mass spectrometer for a two-dimensional analysis

Ions were allowed to first pass through a quadrupole that is set to either

transmit a substantial mass range, or to select a particular m/z before entering

the Triwave ion-mobility cell. Triwave consists of a series of planar electrodes

arranged orthogonally to the ion transmission axis. Opposite phases of radio-

frequency (rf) voltage are applied to adjacent electrodes to provide radial

confinement of the ions, resulting in high ion transmission. A traveling voltage

wave can be incorporated into the rf ion guide to minimize the ion transit time.


The Triwave system is composed of three T-Wave devices (Figure 33a). The

Trap T-Wave accumulates ions which are stored and gated (500 µsec) into the

second device. The final electrode on the trap ion guide is dc only and its

voltage is modulated to periodically gate ions into the IMS. Typically no traveling

wave is used in the trap ion guide. In the ion-mobility T-Wave cell the ions are

separated according to their cross section-dependent mobilities. The transfer T-

Wave cell is used to transfer the mobility separated ions to the MS analysis,

accomplished by an orthogonal acceleration time-of-flight (oa-TOF) analyzer. In

order to ensure that the mobility separation is maintained on transit to the oa-

TOF, the transfer ion guide has a continual running traveling voltage wave. As

both the trap and transfer ion guides operate at approximately standard collision

cell pressures, it is possible to fragment ions in either one of these regions.

They share a common gas supply (typically Argon, 10-2 mbar). Using the

DriftScope feature, it is possible to extract m/z and drift time information for any

ion of interest and thus obtain a much better drift time separation. The extracted

drift time distributions for a two dimensional plot of drift time versus (vs.) mass-

to-charge (m/z) snapshot clearly revealed the separation of components.

Generally, the speed with which an ion traverses the drift region depends on its

CCS (gas phase size); ions with larger CCSs proceed more slowly than ions

with smaller ones.

2.3.3.2 Identification of full-length and proteolytic fragments of α-synuclein

The characterization of the truncated products of αSyn, being

unsuccessful using ESI-MS and HPLC-MS, was obtained by ion-mobility mass

spectrometry (IMS-MS) consisting of a drift region (Triwave). The Triwave

system is composed of three T-Wave devices and is illustrated in Figure 33a.

The DriftScope was used to extract the drift time distributions, which revealed

separation of two components (Figure 33b). Generally, the speed with which an

ion traverses the drift region depends on its collisional cross section CCS (gas

phase size); ions with larger CCSs proceed more slowly than ions with smaller

ones. Peak 1 from Figure 33b contained ions with small CCSs and they have an


arrival time distribution shorter than ions from peak 2. Figure 33c shows the

HDMS analysis (drift time vs. m/z) of αSyn. The intensity of an ion is indicated

using a color scale from least (blue/red) to most (yellow/white) intense.

TOFOrthogonal quadrupole

Trap Ion mobilityseparation Transfer

Peak 1

Peak 2

m/z

b

c

%a

Figure 33: (a), Scheme of Ion-mobility MS system, which incorporates Triwave

technology for ion-mobility separations at the limits of MS detection in the SYNAPT HDMS system; (b), Ion-mobility arrival time distribution for ions observed; (c), Plot of m/z versus drift time of αSyn illustrating the multidimensional nature of the data produced by ion-mobility mass spectrometry.

An ion-mobility plot of the αSyn incubation mixture after 48 hours (sample S2;

see Figure 27) provided two peaks with different drift times for the multiply

charged ion series, which were analyzed by molecular mass determination and

by tandem-MS sequencing (Figure 34). Deconvolution of the multiply charged

ion series to singly charged ions provided identifications of intact, full-length

αSyn monomer (14459.4 Da) and dimer (28919.6 Da) in peak 2, in agreement

with MS data of the gel electrophoretic bands (Figure 27 and Table 9). A most

remarkable fragment was identified in the ion series of peak 1 by cleavage

between residues Val-71 and Thr-72 at the central amyloidogenic domain (61-

95), corresponding to the gel electrophoretic band 4 of ca. 8 kDa (Figure 27).

This fragment was identified as the C-terminal polypeptide αSyn (72-140) by


exact molecular mass determination (7271.1 Da) and tandem-MS sequencing

of the 1455.6 (+5) m/z precursor ion (Figure 34b, d). 3906_T002.raw : 13906_T002.raw : 1

Bufferions

Peak1 Peak2

Drift time (milli secs)

m/z

7292.1343

%

0

100

7250 7260 7270 7280 7290 7300 mass

%

0

100

7250 7260 7270 7280 7290 7300 mass

7271.1191αSyn (72-140)

Peak 2Peak 1

Nab c

Bufferions

a

d

mass10000 14000 18000 22000 26000

%

0

10014459.4

28919.614480.9

massmass10000 14000 18000 22000 26000

%

0

10014459.4

28919.614480.9

αSyn (1-140)

Dimer

Figure 34: Identification of proteolytic truncation products of αSyn by ion-mobility mass

spectrometry: (a), HDMS analysis (driftscope data view) of S2 oligomerization-aggregation products of αSyn incubated in PBS for 2 d at 37°C. Signals corresponding to monomers, dimers and truncated peptide sequences are observed. Each pixel represents one ion with color representing intensity from low (blue) to high (yellow); (b), Extracted deconvoluted spectrum of peak 1 is the truncated peptide sequence αSyn of 7.2 kDa (72-140); (c), Extracted deconvoluted spectrum of peak 2 showing monomer plus dimer of αSyn; (d) MS/MS fragmentation of 1455.6 (+5) m/z precursor ion using elevated collision energy in transfer region of Triwave leads to identification of proteolytic truncation product αSyn 72-140.

Figure 35 compares the ion-mobility- MS drift time profiles (driftscope

data view) of a freshly prepared αSyn solution, and αSyn- oligomers in

incubation mixtures type S5, S6 and S5 + S6 after 7 days of incubation in PBS

with 20% ethanol, 10 µM FeCl3. In the different charge state series of ion


signals, αSyn monomer, dimer as well as proteolytic and truncated peptide

sequences were identified by molecular weight determinations and partial

tandem-MS sequence determinations (Figure 36, Figure 37a and b). The

comparison of the extracted drift time “mobilograms” of the ion at m/z 804.1

(18+) between αSyn oligomers type S5, S6, and S5 + S6 is shown in Figure 36.

In the αSyn oligomers type S5, two drift time (conformational) states are

present, while in type S5 and S5 + S6 even three drift time states are observed

indicating three different structural types.

3282_D023.raw : 1

3282_D024.raw : 1

3282_D022.raw : 1

3282_D025.raw : 1

S1(0 d)

+13

+19+17

+18

+14+15

+16

+12+11

+10

+15+16

+17+18

+12+13

+14

+15+16

+17+18

+14+15

+16+17

+18

+14

S5(7 d)

S6(7 d)

1 50 100 150m/z

Drift Time (scans)

1 50 100 150 1 50 100 150

1 50 100 150

a

c

b

S5 +S6(7 d)

d

Figure 35: Ion-mobility drift time of αSyn after 7 days of incubation in 50 mM sodium

phosphate buffer, containing 20% ethanol to a final concentration of 7 µM (S5); αSyn after 7 days of incubation in 50 mM sodium phosphate buffer, containing 20% ethanol, 10 µM FeCl3 (S6); freshly prepared αSyn. Signals corresponding to monomers, dimers and truncated peptide sequences are observed.


sample alphasyn

Scan25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150 155 160 165

%

0

100

25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150 155 160 165

%

0

100

25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150 155 160 165

%

0

100

25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150 155 160 165%

0

1003282_D025_dt_01 TOF MS ES+

804.074_804.52 0.10Da937

6.30762.1

3282_D024_dt_01 TOF MS ES+ 804.164_804.602 0.10Da

4406.66804.3

3282_D023_dt_01 TOF MS ES+ 804.106_804.602 0.10Da

9016.39804.3

3282_D022_dt_01 TOF MS ES+ 804.106_804.52 0.10Da

4.26e36.12;851.5

S5 +S6(7 d)

S6(7 d)

S5 (7 d)

S1(0 d)

a

c

d

b

Figure 36: Comparison of the extracted mobilograms of the ion m/z 804.1 between

αSyn oligomers type S5, S6, S5 + S6. In αSyn oligomers type S5 two structures are observed; in type S6 and S5 + S6 three main forms are present.

The extracted, deconvoluted mass spectra of the driftscope profile due to the

αSyn oligomers, type S5 revealed the αSyn monomer and the intact full-length

dimer (Figure 37a; Mw 14459.4; 28919.6 Da), while the oligomer peak, type S5

+ S6 showed an additional truncated polypeptide, αSyn (40-140) (Figure 37b;

Mw 10437 Da). Further peptide fragments of αSyn with high aggregating

propensity, corresponding to the bands observed by gel electrophoresis (Figure

27) are summarized in Table 9.


14459.4

mass10000 12000 14000 16000 18000 20000 22000 24000 26000 28000

%

0

100

28919.6

massmass10000 12000 14000 16000 18000 20000 22000 24000 26000 28000

%

0

100

28919.6

αSyn (1-140)monomer

αSyn (1-140)dimer

a

αSyn (1-140)


massmass10000 11000 12000 13000 14000 15000 16000 17000 18000 19000

%

0

100

αSyn (1-140)

αSyn (40-140)

αSyn (1-140)


αSyn (40-140)


14460.1

10437.0

b

Figure 37: Extracted deconvoluted spectra of αSyn oligomers: (a), Type S5 oligomers

are showing αSyn monomer plus dimer; (b), type S5 + S6 oligomers are containing truncated polypeptide sequence at 10.5 kDa (αSyn (40-140)).

In conclusion IMS-MS of the in vitro aggregation of wt-αSyn enabled the

structure elucidation of several hitherto unknown truncation and degradation

products that are summarized in Table 9 and a proteolytic fragment at V71-T72

in the aggregation domain that appears to be a key intermediate in the

aggregation pathway (see 2.7.3).


Table 9: αSyn aggregation products observed by gel electrophoresis and identified by IMS-MS.

αSyn a Gel electrophoresis b IMS-MS

solvent time gel spot Mw gel Mcalc Mexp sequence

S1 water 0 d 1 17 kDa 14460.1 14460.0 1-140

S2 20 mM PBS 2 d 4 < 10 kDa 7270.3 7270.1 72-140

S3 20 mM NH4(Ac) 2 d 2 14 kDa 12161.5 12163.0 14-133

S4 20 mM NH4HCO3 3 h 1a 17 kDa 13708.2 13706.6 7-140

S4 20 mM NH4HCO3 2 d 4 < 10 kDa 7270.3 7270.1 72-140

S5 PBS 7 d 3 10 kDa 10436.4 10436.0 40-140

S6 PBS 7 d 3 10 kDa 10436.4 10436.0 40-140a αSyn aggregation has been studied under various conditions which differ in used

buffer systems and incubation time b Aggregation products of αSyn were observed by Tris-tricine gel electrophoresis

These results, showing IMS-MS as a powerful tool for the analysis of reactive

intermediates due to its “affinity”-like separation ability, of a protein aggregation

pathway suggest proteolytic fragments as a key molecular species involved in

the “misfolding” and aggregation of αSyn.


2.4 Epitope identification of synuclein- antibodies by affinity-mass spectrometry

The major goal of this thesis was the specific detection of different αSyn

amino acid sequences in order to characterize the oligomerization-aggregation

products of this protein. To this goal, it is essential to elucidate and structurally

characterize the αSyn epitopes as the key sequences involved in the affinity

interactions with antibodies. The location of these sequences is of crucial

importance for understanding the oligomerization-aggregation process, the

central event in PD.

2.4.1 Preparation of immobilized anti-α-synuclein specific antibodies

For affinity- mass spectrometry studies as well as proteolytic epitope

excision/ extraction experiments, four anti-αSyn- antibody micro-columns were

prepared with the following antibodies: anti-αSyn mBD (no.3), anti-αSyn pC20

(no.4), anti-αSyn pASY-1 (no.5), anti-oligomer A11 (no.6) (see Table 20 in the

Experimental Part). Approximately 100 µg antibody was dissolved in coupling

buffer (pH 8.3) (see Table 18: Composition of buffer and stock solutions). The

solution was added on dry NHS-activated 6-aminohexanoic acid-coupled

Sepharose 4B and the coupling reaction performed at 25°C. The mixture was

loaded into a micro-column and extensive washing and blocking steps were

carried out. The collected flow-through fraction together with the corresponding

antibody (used as a control) and intravenous immunoglobulins (IVIG) from

Calbiochem (used as a control) were first reduced with a 10000 molar excess of

dithiothreitol (DTT), and then alkylated with a 30000 molar excess of

iodacetamide. SDS-PAGE was carried out in order to exclude that unbound

antibody remained after immobilization on the matrix. An example representing

the immobilization of anti-αSyn pASY-1 antibody is shown in Figure 38. The

presence of a low amount of anti-αSyn pASY-1 antibody indicated that the

immobilization was successful.


Manti-αSynpASY-1 Flow-through IVIG

200 -120 -100 -85 -70 -60 -

40 -

50 -

30 -

25 -

20 -

kDa

Light chain

Heavy chain

Figure 38: SDS-PAGE of the flow-through after antibody micro-column preparation

using a 12% separation gel and visualized by silver staining. As a control, the polyclonal ASY-1 antibody and IVIG were reduced with DTT and alkylated with iodacetamide. The flow-through fraction shows a low amount of anti-αSyn pASY-1 antibody.

2.4.2 Binding characterization of α-synuclein to antibody columns by affinity- mass spectrometry

Affinity- mass spectrometry experiments were carried out on the anti-

synuclein specific antibody micro-columns which contain the Sepharose-

immobilized antibody. Wt-αSyn, mouse brain homogenates and human

neuroblastoma SH-SY5Y cell lysates were added on the antibody micro-

columns in order to prove the specific binding and separation capability of

synucleins to the antibody (see 3.6.3). After 2 h, the unbound proteins were

collected in the supernatant fraction. The supernatants were analyzed by Tris-

tricine gel electrophoresis and mass spectrometry. The columns were washed

with 0.1% aqueous TFA to dissociate the remaining αSyn affinity-bound to

antibodies. The collected solutions, forming the elution fractions, were also

analyzed by MS and separated by gel electrophoresis.

Figure 39 shows the MALDI-TOF mass spectra of supernatant, wash and

elution fractions of αSyn affinity experiment with the immobilized anti-αSyn

pASY-1 antibody. In the spectrum of the elution fraction, αSyn was identified by

the signals of [M+H]+ and [M+2H]2+ ion signals (Figure 39c), which ascertained

that αSyn bound to the prepared antibody column.


8000 10000 12000 14000 m/z

14460.57230.6

8000 10000 12000 14000 m/z

14460.57230.6

8000 10000 12000 14000 m/z8000 10000 12000 14000 m/z

8000 10000 12000 14000 m/z

14460.67230.6

8000 10000 12000 14000 m/z

14460.67230.6[M + 2H] 2+ [M + H] +


b

a

[M + 2H] 2+ [M + H] +c

Figure 39: MALDI-TOF mass spectra of the αSyn affinity experiment with the

immobilized anti-αSyn pASY-1 antibody: (a), Supernatant; (b), Wash test; (c), Elution fraction showing [M+H]+ and [M+2H]2+ ion signals.

The polyclonal anti-αSyn pC20 antibody micro-column was used first to

prove the specific binding of wt-αSyn, and in subsequent experiments for the

separation of αSyn (A30P) and αSyn (A53T) mutants from mouse brain

homogenates and human neuroblastoma cell lysates after binding to the

antibody. Wt-αSyn was bound to the antibody column for 2 h at 25°C. The ESI-

mass spectrum of the unbound protein showed the multiply charged ions with

the most intensive +16 ion (Figure 40a). After washing, the bound wt-αSyn was

dissociated by acidification, and ESI-MS spectrum of the elution fraction (Figure

40b) proved that the anti-αSyn pC20 antibody column was specific for αSyn.


658.522+689.8

21+

724.220+

762.219+

804.518+

851.717+

904.916+

965.215+

1034.114+

1113.513+

1206.112+

0

2

4

6

Intens

500 600 700 800 900 1000 1100 1200 1300 m/z

689.621+

724.220+

762.319+

804.518+

851.817+

905.016+

965.215+

1034.014+

1113.413+

0.2

0.4

0.6

0.8

1.0.

500 600 700 800 900 1000 1100 1200 1300 m/z

X10 8

IntensX10 6


b

a

Figure 40: ESI mass spectra of the αSyn affinity experiment with the immobilized anti-

αSyn pC20 antibody: (a), Supernatant; (b), Elution fraction showing multiple charged ions of αSyn with the most intensive peak corresponding to the ion +17.

2.4.3 Epitope identification of α-synuclein-specific antibodies

Efficient analytical methods for the molecular characterization of binding

sites in proteins and peptides have been developed since the early 1990’s in

our laboratory, by combining the proteolytic isolation of antibody-bound peptides

using immuno-affinity techniques followed by structural identification of epitope

peptides by mass spectrometry [289-291].

2.4.3.1 Methodology of mass spectrometric epitope analysis

For the epitope elucidation and structural characterization of αSyn, direct

methods of mass spectrometric epitope mapping (proteolytic epitope excision/

extraction) were performed using an immobilized anti-αSyn specific antibody on


an affinity matrix. The epitope excision approach (Figure 41) consists of three

distinct steps: (1), a solution containing the intact antigen is exposed to the

immobilized antibody and allowed for formation of the immune complex. The

remaining antigen is collected as non-binding fraction and analyzed by MS,

SDS-PAGE or Western blot; then, the affinity matrix is washed until complete

removal of free antigen. (2), A proteolytic enzyme is applied and allowed to

cleave the amino acid residues that are not shielded by the interaction with the

antibody. The proteolytic fragments released in the supernatant fraction are

collected and analyzed by MS. (3), The affinity matrix is washed extensively and

the last volume of the washing buffer (referred to as washing fraction) is

collected and analyzed by MS. The peptides remaining bound to the antibody

are then eluted under acidic conditions (elution fraction) by addition of 0.1%

TFA (aqueous solution, pH 2.5) and analyzed by MS. For re-using the affinity

column, the affinity matrix is regenerated by washing with the elution solution

and finally with a neutral buffer (pH 7).

non-binding fraction

immobilizedAb micro-column

complex formation

MS analysisSDS-PAGEWestern Blot

supernatant

MS analysis

eluted epitope peptides

removal of non-bound antigen

addition of enzyme

removal of non-epitope fragments

proteolytic digestion

washing

0.1% TFA

MS analysis

complex dissociation

addition of antigen

Figure 41: Schematic representation of the methodology for epitope excision. The

antigen is added to the immobilized antibody column. After separation of the proteolytic unbound peptides from the epitope-containing peptide bound to the antibody, the epitope is dissociated by addition of 0.1% TFA (aqueous solution, pH 2.5) and characterized by mass spectrometry.


Complementary to epitope excision, the epitope extraction approach (Figure 42)

approach consists of three distinct steps: (1), the peptide antigen is cleaved in

solution using a specific protease; (2), the mixture of proteolytic fragments is

applied to the immobilized antibody and mixed together with the affinity matrix.

All unbound fragments are removed by washing the column. The collected

solution is the supernatant fraction which contains all peptide fragments not

bound to the antibody column; (3), the epitope fraction is eluted from the affinity

column and the epitope peptides are then analyzed and identified by MS

immobilized

Ab micro-columncomplex formation

supernatant

MS analysis

eluted epitope peptides

removal of non-epitope fragments

washing

0.1% TFA

MS analysis

complex dissociation

addition ofmixture containingepitope peptides

Figure 42: Schematic representation of the methodology for epitope extraction. The

antigen is digested in solution, and then the peptide mixture is added to the antibody immobilized column. After separation of the proteolytic unbound peptides from the epitope-containing peptide bound to the antibody, the epitope is dissociated by addition of 0.1% TFA (aqueous solution, pH 2.5) and characterized by mass spectrometry.

Proteolytic epitope excision and extraction mass spectrometry methods

have been successfully used by a number of laboratories in the last years for

the determination of epitopes and interaction structures [290-295] .


2.4.3.2 Epitope identification of α-synuclein by mass spectrometric epitope excision and epitope extraction

For the epitope elucidation and structural characterization of αSyn, mass

spectrometric epitope mapping was performed using the anti-αSyn pASY-1 and

anti-αSyn pC20 antibodies. In the epitope excision experiments, the protein was

bound to the antibody column, using 2 h incubation, and trypsin was then

added. After 2 h at 37°C, the unbound peptide fragments were collected in the

supernatant) and after washing (wash fraction), and the immune complex

dissociated by addition of 0.1% TFA (elution fraction). The MALDI-TOF mass

spectra of the supernatant, washing and elution fraction are shown in Figure 43.

Upon epitope excision with the anti-αSyn pASY-1 antibody, the MALDI-TOF

mass spectrum of the supernatant fraction showed almost all tryptic fragments

of αSyn. In the elution fraction two most intensive peaks of the fragments αSyn

(1-23) and αSyn (59-80) were identified which proved that the epitope

recognized by the antibody is discontinuous, located within the N-terminal and

central domains of the protein.


1000 2000 3000 4000 5000 m/z

(1-6)

(35-43)

(22-32) / (24-34)

(33-43) / (35-45)

(46-58)

(44-58) / (46-60)(81-97)

(97-140)

(98-140)

(44-80)

(61-80)

(59-80)

(24-60) 2+

(81-96)

(1-32) 2+

(11-43)

1000 2000 3000 4000 5000 m/z

b

(1-23)A

(1-21)

(59-80)B

[M+H] + exp A : 2440.0 [M+H] + calc A : 2439.9

[M+H] + exp B : 2157.1[M+H] + calc B : 2157.4

1000 2000 3000 4000 5000 m/z

aMDVFMKGLSK AKEGVVAAAE 21KT23KQGVAEAA G32KTKEGVLYV GS43KT45KEGVVH

GVATVAE58KTK EQVTNVGGAV VTGVTAVAQ80K TVEGAGSIAA ATGFVKKDQL


1

51

101 140

c

Figure 43: MALDI-TOF mass spectra of epitope excision experiment using anti-αSyn

pASY-1 antibody: (a), Supernatant excision containing the non-epitope peptide fragments of αSyn using trypsin; (b), Wash excision; (c), Elution fraction after trypsin digestion reveals two fragments of αSyn (1-23) and αSyn (59-80) as antigenic determinants for anti-αSyn pASY-1 antibody. The insert in the upper part contains a schematic representation of the primary structure of αSyn showing in red the possible cleavage sites using trypsin. The cleavage sites determined upon identification of the tryptic fragments present in the elution fraction are indicated in red and numbered. The identified peptides are highlighted on grey background.


A further epitope excision experiment was performed using Glu-C

protease which cleaves peptides after glutamyl and aspartyl residues. The αSyn

was bound to the anti-αSyn pASY-1 antibody for 2 hours, and Glu-C

endoprotease added onto the antigen-antibody column for 2 h at 37°C. The

unbound peptide fragments (supernatant) were removed by extensive washing,

and the epitope- peptide fragments released with 0.1% TFA. Prior to MALDI-MS

analysis, supernatant and elution fractions were desalted with the C18 ZipTip

procedure. The MALDI-TOF mass spectrum of the elution fraction contained

four peptides with the most intensive peak corresponding to the N-terminal

fragment αSyn (1-28).

MDVFMKGLSK AKEGVVAAA20E KTKQGVA28EAA GKTKEGVLYV GSKTK46EGVVH

GVATVA57EKTK EQVTNVGGAV VTGVTAVAQK TV83EGAGSIAA ATGFVKKDQL


1

51

101 140

[M+H] + exp : 2924.3 [M+H] + calc : 2924.4

2000 2500 3000 3500 m/z

(1-20) (58-83)

(1-28)

(47-83)

1500 2000 2500 3000 3500 m/z

(1-20) (58-83)

(1-28)

(47-83)

1500 Figure 44: Mass spectrometric epitope excision with Glu-C using anti-αSyn pASY-1

antibody. MALDI-TOF mass spectrum of the elution fraction shows as the most intensive binding fragment the N-terminal (1-28), which is indicated on grey background in the insert in the upper part of the figure. The schematic representation of the primary structure of αSyn is shown in red the possible cleavage sites using Glu-C.

For epitope extraction, αSyn was first digested for 2 h with the chosen

protease (trypsin or Glu-C) and then applied onto the antibody column. Binding

of the peptide mixture to the antibody was performed for 2 h at 25°C and

washing, dissociation and epitope elution performed in the same manner as in


the epitope excision experiment. The released epitope and non-epitope

peptides were lyophilized, desalted and analyzed by MALDI-TOF-MS. Epitope

extraction of αSyn using trypsin led to the identification of αSyn (59-80), while in

the elution fraction the epitope peptide αSyn (1-23) was not observed. This can

be explained by cleavage of this peptide during the proteolytic digestion. In the

MALDI-MS analysis of the elution fraction using Glu-C protease, the N-terminal

fragment αSyn (1-28) was observed, which confirmed that the epitope is

discontinuous and located within the N-terminal (1-23) and central (59-80)

domains. Thus, the identification of the αSyn interaction sites with the anti-αSyn

specific antibody provided a basis for the design of new potential PD

immunotherapeutic approaches.

An identical epitope excision/ extraction- MS approach was employed to

identify the epitope of αSyn recognized by the anti-αSyn pC20 polyclonal

antibody. As previously described, an anti-αSyn pC20 antibody micro-column

was used to bind 20 µg of αSyn. After 2 h incubation at 25°C, the supernatant

fraction containing unbound protein was removed and 1 µg trypsin was added

and incubated for 2 h at 37°C. In the second step the matrix was washed

extensively and the final volume of the washing buffer collected and analyzed

by MS. The peptides remaining bound to the antibody were eluted, and the

supernatant, washing and elution fractions analyzed by LC-ESI-MS and

tandem- MS. In the spectrum of the elution fraction, αSyn (59-80) was identified

by the [M+2H]2+ and [M+3H]3+ ions. This result was in complete agreement with

Dot blot immuno-analysis (Figure 52) and with epitope identification using

combined bioaffinity- MS (see 2.6.3).


200 300 400 500 600 700 800 900 m/z200 300 400 500 600 700 800 900 m/z

aMDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH



1

51

101 140

386.1

437.3476.4

561.2

648.3

740.3

964.9

643.8

720.1(1-6)

(13-21)(35-43) (46-58)

(61-80) (81-96)

(59-80)(59-80)

200 300 400 500 600 700 800 900 m/z

b

720.0

1080.0

400 500 600 700 800 900 1000 1100 m/z

[M + 3H] 3+

[M + 2H] 2+

c

d

V A T V G T V V A G GAQ

200 400 600 800 1000 1200 m/z

y4 y5

y3y1

y6

y7

y8 y9

y10

59TKEQVTNVGGAVVTGVTAVAQK80

y8y7y6 y5 y4 y3 y1y9y10

MS/MS, m/z 720.1 (3+)

Figure 45: LC/ESI-ion trap-MS/MS mass spectra of fractions of epitope excision

experiment using anti-αSyn pC20 antibody: (a), Supernatant excision containing the non-epitope peptide fragments of αSyn using trypsin; (b), Wash excision; (c), Elution fraction after trypsin digestion reveals αSyn (59-80) as antigenic determinant for anti-αSyn pC20 antibody; (d), MS/MS fragmentation mass spectrum of ion 720.1 (3+) (with y ions assigned), which led to the identification of peptide (59-80) is also displayed. The insert in the upper part of the figure contains a schematic representation of the primary structure of αSyn showing in red the possible cleavage sites using trypsin. The identified peptide is highlighted on gray background.

To identify the epitopes of the mutant αSyn (A30P) from mouse brain

homogenate, a similar epitope excision- mass spectrometry approach was

employed. In this study, tris buffer saline soluble mouse brain homogenate was


extracted as described in 2.2.1, and the polyclonal anti-αSyn pC20 antibody

immobilized on a Sepharose micro-column (see 3.6.2). In the first step, 200 µg

of mouse brain homogenate was bound to the antibody column. After 2 h,

unbound proteins were removed by washing, and trypsin digestion was

performed on the antibody-antigen column for 2 h at 37°C. The proteolytic

peptides in the supernatant and the elution fraction upon dissociation of the

immune complex were collected and analyzed by high resolution mass

spectrometry (LTQ-Orbitrap-MS), using tandem-MS followed by database

search procedures. Table 10 summarizes the peptides obtained with this

approach. These results revealed a discontinuous epitope located in the C-

terminal (103-140) and central (59-97) domains.

Table 10: High resolution mass spectrometric analysis of the elution fraction of a mouse brain homogenate using trypsin in epitope extraction:

Identified peptide [M+H]+ calc [M+H]+ exp ∆m (ppm) 59T – K80 2157.1874 2157.1853 0.9 61E - K80 1928.0447 1928.0463 -0.8 81T – K96 1478.7849 1478.7867 -1.2 81T – K97 1606.8799 1606.8805 -0.4

103N – A140

(116Met, 127Met oxid.)

4318.7235 4318.7257 -0.5

2.5 Synthesis and structural characterization of synuclein polypeptides

2.5.1 Synthesis and structural analysis of α-synuclein peptides and peptide models

The αSyn epitope peptides αSyn (1-23), αSyn (59-80) and control peptides for

epitopes were synthesized by solid phase peptide synthesis (SPPS) [296] on a

semi-automated synthesizer using a NovaSyn-TGR resin and

fluorenylmethyloxycarbonyl (Fmoc)/tert-Butyl (tBu) protection chemistry [297].

Double coupling was used to provide near-complete formation of peptide bonds,

and each synthesis carried out in steps of 10 ÷ 12 amino acid residues to


ensure the use of fresh solvents. The general protocol for synthesis consisted of

(i), removal of Fmoc with a solution containing 2% DBU [298], 2% piperidine in

dimethylformamide (DMF) for 8 min, and (ii), coupling of PyBOP/NMM-activated

amino acid for 20 min followed by washing. DMF was employed as a solvent for

all reactions and washing steps. After completion of synthesis, cleavage of the

peptide from the resin was carried out with TFA, triethylsilane and water for 3 h;

the crude peptides were precipitated with t-butylmethylether, filtered, re-

dissolved in 5% acetic acid, lyophilized, and purified by semi- preparative

HPLC. Structural characterization of the synthetic peptides was performed by

MALDI-MS, ESI-ion trap-MS and MALDI-FTICR-MS. Amino acid sequences,

HPLC data and MALDI-FTICR mass spectra of the purified αSyn peptides are

shown in Figure 46. The amino acid sequences, the HPLC chromatograms and

the [M+H]+ from mass spectra of αSyn peptides obtained by ESI-ion trap-MS

are summarized in Table 11.

0 10 20 30 40 500

50

100

150

29.6

Abs

orba

nce

(mA

u)

Time (min)

H-1MDVFMKGLSKAKEGVVAAAEKTK23-NH2

a

2000 2200 2400 2600 2800 3000 m/z

2437.3171

2438.3216

2439.3247

2440.3232

2437 2438 2439 2440 2441 2442 m/z

2437.3171[M + H] +

[M+H] + exp : 2437.3171 [M+H] + calc : 2437.3305 ∆m = 5.5 ppm


H-59TKEQVTNVGGAVVTGVTAVAQK80-NH2

b

Abs

orba

nce

(mA

u)

0 10 20 30 40 50 600

100

200

300

400

500

600

700

Time (min)

24.03

2156.2009

2000 2100 2200 2300 2400 2500 m/z

[M + H] +2156.2009

2000 2100 2200 2300 2400 2500 m/z

[M + H] +

[M+H] + exp : 2156.2009 [M+H] + calc : 2156.2034 ∆m = 1.16 ppm

Figure 46: Analytical RP-HPLC profiles and MALDI-FTICR mass spectra of purified:

(a), αSyn (1-23); (b), αSyn (59-80).

Table 11: Chemical characteristics of αSyn peptides: αSyn

peptidea

Sequence HPLC

Rt (min)b

[M+H]+calc c [M+H]+exp

d

11 (1-23) H-MDVFMKGLSKAKEGVVAAAEKTK-NH2 29.6 2437.3 2437.5

12 (59-80) H-TKEQVTNVGGAVVTGVTAVAQK-NH2 24.03 2156.4 2156.2

13 (59-80 T72A) H-TKEQVTNVGGAVVAGVTAVAQK-NH2 26.13 2127.4 2127.3

14 (59-80 T64A) H-TKEQVANVGGAVVTGVTAVAQK-NH2 25.36 2127.4 2127.3

15 (59-69) H-TKEQVTNVGGA-NH2 22.4 1102.58 1102.5

16 (70-80) H-VVTGVTAVAQK-NH2 24.6 1071.64 1071.6

17 (81-91) H-TVEGAGSIAAA-NH2 24.5 945.49 945.5

a Fmoc/tBu solid phase peptide synthesis (EPS 221, Abimed) b RP-HPLC purification (UltiMate 3000, Dionex) using a Vydac C4 column c GPMAW software 5.0 (Lighthouse Data, Denmark) d Mass spectrometric analysis by ESI-ion trap-MS using an Esquire 3000 +

In addition to the identification of epitopes of the anti-αSyn pASY-1

specific antibody, a further goal was the characterization and comparison of the

antigenic binding properties of the αSyn epitope peptides and the binding of

different αSyn antibodies to N-terminal and C-terminal αSyn sequences using


immuno-analytical techniques (Dot blot and ELISA). Therefore, the

conformational preferences of the αSyn epitope peptides were analyzed by CD

spectroscopy in the far-UV region. CD spectra of the αSyn epitope peptides are

presented in Figure 47. All CD spectra in water showed a strongly negative

band around 198 nm, characteristic of an unordered structure. The

solubilization of epitope peptides in PBS did not lead to significant

conformational changes, corresponding to a random coil conformation.

180 200 220 240 260-400000

-300000

-200000

-100000

0

100000

water

PBS

H-1MDVFMKGLSKAKEGVVAAAEKTK23-NH2

180 200 220 240 260-600000

-400000

-200000

0

200000

400000

600000

H-59TKEQVTNVGGAVVTGVTAVAQK80-NH2

Mol

. Elli

ptic

ityΘ

(deg

cm2

dmol

-1

Wavelength (nm)

Mol

. Elli

ptic

ityΘ

(deg

cm2

dmol

-1

Wavelength (nm)

water

PBS

Figure 47: CD spectra of αSyn (1-23) and αSyn (59-80) epitope peptides recorded in

water and PBS.

In addition, structural information about the αSyn epitopes was obtained

by molecular dynamic simulation. In this study, HyperChem 6.0 and BallView

programs for molecular modelling were used to design the structure of the

peptides as shown in Figure 48.

NH2

COOH

a αSyn (1-23)


NH2 COOH

b αSyn (59-80)

Figure 48: Molecular modelling of the structure of αSyn epitope peptides using

HyperChem and BallView programs: (a), αSyn (1-23) and (b), αSyn (59-80).

The binding of the αSyn (1-23) epitope peptide to the anti-αSyn pASY-1

antibody was further characterized by affinity- mass spectrometry using MALDI-

TOF-MS. The peptide was dissolved in PBS, pH 7.5 and exposed to the

antibody affinity column. After 2 h incubation at 25°C, the column was washed

with 40 ml buffer and the complex dissociated under acidic conditions. The

results of the experiments are presented in Figure 49. In the MALDI-TOF mass

spectrum of the elution fraction, αSyn (1-23) was identified by the [M+H]+ ions,

thus confirming that αSyn (1-23) specifically bound to the antibody column.


Mw exp : 2438.2 DaMw calc : 2438.9 Da∆m = 0.7 Da

b

a

c

1500 2000 2500 3000 3500 m/z1500 2000 2500 3000 3500 m/z1500 2000 2500 3000 3500 m/z

[M + H] +

1500 2000 2500 3000 3500 m/z1500 2000 2500 3000 3500 m/z

[M + H] +

1500 2000 2500 3000 3500 m/z1500 2000 2500 3000 3500 m/z Figure 49: Binding of αSyn (1-23) to anti-αSyn pASY-1 antibody column by affinity-

mass spectrometry using MALDI-TOF-MS: (a), Supernatant; (b), Wash; (c), Elution fraction showing [M+H]+ of αSyn (1-23) epitope peptide.

The αSyn (1-23) and αSyn (59-80) epitope peptides were further

compared by ELISA for their affinity to the anti-αSyn pASY-1 antibody, using wt-

αSyn as a positive control. ELISA experiments were performed using antigen

dilutions (quantitative comparison of binding intensities). After coating the plates

with the αSyn epitope peptides, anti-αSyn pASY-1 antibody, peroxidase

conjugated goat anti-rabbit IgG and OPD/H2O2 substrate were added and the

absorbance determined at 450 nm. Figure 50 showed that the synthetic

peptides had specific affinity to the anti-αSyn antibody. Wt-αSyn revealed the

highest affinity (0.0027 µM at OD450 of 1.0). Significant differences in the

antibody binding were found for the epitope peptides, with αSyn (59-80) having

a high antigenicity than N-terminal αSyn (1-23) (0.049 µM compared to 0.0797

µM at OD450 of 1.0).


1, αSyn11, αSyn (1-23) 12, αSyn (59-80)

Antigen concentration (µM)

OD

(450

nm

)

0

0.5

1

1.5

2

2.5

0.00001 0.00003 0.0001 0.0003 0.001 0.003 0.01 0.03 0.09 0.27 0.83 2.5

0.0797 µM

0.0027 µM 0.0494 µM

Figure 50: Indirect ELISA of pASY-1 antibody to αSyn (rhomb), αSyn (1-23) (squares)

and αSyn (59-80) (triangles). The pASY-1 antibody shows affinity to the protein and synthetic epitope peptides (1-23) and (59-80). The peptide concentrations needed to obtain an optical density (450 nm) of 1.0 are expressed in µM. Background signals from wells without antigen have been subtracted.

The αSyn control peptides synthesized by SPPS were also characterized

by CD spectroscopy in the far-UV region, and showed a strongly negative band

around 198 nm, characteristic of an unordered structure (Figure 51). CD spectra

of the shorter peptides αSyn (59-69), αSyn (70-80), and αSyn (81-91) revealed

the lowest ellipticities and a less intensive positive band at 210 - 220 nm.

180 200 220 240 260-1000000

-800000

-600000

-400000

-200000

0

16, α-Syn (70-80)15, α-Syn (59-69)

17, α-Syn (81-91)Mol

. Elli

ptic

ityΘ

(deg

cm2

dmol

-1

Wavelength (nm)

13, α-Syn (59-80 T72A)14, α-Syn (59-80 T64A)

Figure 51: CD spectra of αSyn peptides: 13, αSyn (59-80 T72A); 14, αSyn (59-80

T64A); 15, αSyn (59-69); 16, αSyn (70-80); 17, αSyn (81-91) recorded in water at a concentration of 30 μM.


Further characterization of the binding of different αSyn antibodies to wt-

αSyn and αSyn peptides was carried out using Dot blot analysis. The samples

were first spotted on nitrocellulose membranes and unspecific binding sites

were blocked. After washing, the membranes were the incubated with the anti-

αSyn specific antibodies (see Table 20: Primary and secondary antibodies in

the Experimental Part) for 2 h at 25°C. After further washing and addition of the

detection antibodies, membranes were developed and exposed on a film

(exposure times 10 - 60 sec), and the exposed films developed by using an

automated Canon camera.

The Dot blot binding responses of anti-αSyn antibodies to different αSyn

peptides are shown in Figure 52. The anti-αSyn antibodies gave a positive

response for the wt-αSyn and αSyn (59-80), αSyn (59-80 T72A), and αSyn (59-

80 T64A) peptides, while the short peptides αSyn (59-69), αSyn (70-80) and

αSyn (81-91) showed no affinity binding. The anti-αSyn pASY-1 antibody

recognized specifically the N-terminal αSyn (1-23) and central αSyn (59-80)

epitope peptides, in complete agreement with the affinity- mass spectrometry

and ELISA results.


Anti-α/ßSyn Ab

1 11 12 13 14

15 16 17

a

1 11 12 13 14

15 16 17

b

c 1 11 12 13 14

15 16 17

1 11 12 13 14

15 16 17

Anti-αSyn m4B12 Ab

Anti-αSyn mBD Ab

1 11 12 13 14

15 16 17

dAnti-αSyn pC20 Ab

1 11 12 13 14

15 16 17

eAnti-αSyn pASY-1 Ab

1, αSyn11, αSyn (1-23)12, αSyn (59-80)13, αSyn (59-80 T72A)

14, αSyn (59-80 T64A)15, αSyn (59-69)16, αSyn (70-80) 17, αSyn (81-91)

Figure 52: Dot blot analysis of (a), anti-α/βSyn; (b), anti-αSyn m4B12; (c), anti-αSyn

mBD; (d), anti-αSyn pC20; (e), anti-αSyn pASY-1 to wt-αSyn and αSyn peptides.

2.5.2 Structural characterization of synthetic α-synuclein polypeptides

In order to elucidate the aggregation properties of αSyn truncation and

degradation products, syntheses and structural characterization of several αSyn

peptide fragments were carried out. The proteolytic fragments αSyn (71-140),

αSyn (72-140) were prepared by chemical solid phase peptide synthesis

(SPPS) [296] on a semiautomated peptide synthesizer using Fmoc-strategy [297]


on NovaSyn TGR resin using side chain protected amino acids derivates. The

protocol of the synthesis is described in 3.3. The crude products were

precipitated with t-butylmethylether, filtered, re-dissolved in 100% acetic acid

and lyophilized. The crude products were purified by reversed phase high

performance liquid chromatography (RP-HPLC) and analyzed by ESI-MS. The

data showed that two heterogeneous products were obtained; therefore the

proteolytic fragments αSyn (71-140), αSyn (72-140) can be qualified as highly

“difficult” peptide sequences for SPPS, particularly since they contain a part of

the hydrophobic NAC-region and the highly aggregation-prone region Val71–

Val82 of αSyn [299]. Hence, their SPPS synthesis will require special

methodology such as double coupling and capping procedures, which are

pursued in most recent work [300].

Additionally, several polypeptide fragments αSyn (72-140) and αSyn (1-

120) were prepared by recombinant bacterial expression, using E.coli BL21

(DE3) [Lys] as prokaryotic expression system1. Cells were grown and

harvested, then the proteins extracted and resuspended in PBS, and

subsequently purified by RP-HPLC. The ESI mass spectrum of αSyn (72-140)

(Figure 53), showed the formation of the 6+ to 10+ charged molecular ions,

which provided the exact molecular mass characterization with mass accuracy

of 55 ppm.

1 Recombinant polypeptides prepared in collaboration with Prof. M. Leist, at the Dept. of

Biology, University of Konstanz


b

a

Mw exp : 7274.9 DaMw calc : 7274.4 Da∆m = 55 ppm

c

809.3

910.3

1040.2

600 800 1000 1200 1400 m/z

1213.4728.6

8+

7+

6+

9+

10+

809.3

910.3

1040.2

600 800 1000 1200 1400 m/z

1213.4728.6

8+

7+

6+

9+

10+

30.9

20 30 40 500

50

100

150

200

250

300

30.9

20 30 40 500

50

100

150

200

250

300

20 30 40 500

50

100

150

200

250

300

Abs

orba

nce

(220

nm

)

Time (min)

TGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE

GILEDMPVDP DNEAYEMPSE EGYQDYEPEA

72

140

Figure 53: (a), Schematic representation of the primary structure of 10, αSyn (72-140);

(b), Analytical RP-HPLC profile of 10 with retention time (Rt) of 30.9 min; (c), ESI mass spectrum of 10 in 1% aqueous formic acid showing the 6+ to 10+ charged molecular ions.

Further commercially available peptide fragments, αSyn (61-140) and αSyn (96-

140) containing Met residue in position 1, were purified by RP-HPLC and

characterized by CD spectroscopy and mass spectrometry. The amino acid

sequences, the HPLC characteristics and ESI mass spectrometric data of the

αSyn polypeptides are summarized in Table 12.

Table 12: Chemical characteristics of αSyn peptide fragments:

Code Peptidea HPLC

Rt (min)b

[M+H]+calc [M+H]+exp c

5 αSyn (1-120) 32.5 12112.8 12112.1

6 αSyn (61-140) 31.1 8461.2 8460.4

7 αSyn (96-140) 29.8 5218.5 5218.3

8 αSyn (71-140) 31.1 7373.9 7373.8

9 αSyn (72-140) 30.8 7274.8 7274.3

10 αSyn (72-140) 30.9 7274.8 7274.4 a Fmoc/tBu solid phase peptide synthesis (EPS 221, Abimed)


b RP-HPLC purification (UltiMate 3000, Dionex) c ESI ion trap mass spectrometric analysis using an Esquire 3000 + (Bruker Daltonics)

The secondary structure characterization of the αSyn peptides was performed

by CD spectroscopy in far-UV region (260 - 180 nm) (Figure 54). The CD

spectra of all αSyn peptides in water showed a negative band around 198 nm

(π-π* transitions), characteristic of an unordered structure.

Mol

. Elli

ptic

ityΘ

(deg

cm2

dmol

-1

Wavelength (nm)

180 200 220 240 260-1600000

-1400000

-1200000

-1000000

-800000

-600000

-400000

-200000

0

200000

5,6, 7, 8, 9, 10,

α-Syn (1-120)α-Syn (61-140)α-Syn (96-140)α-Syn (71-140)α-Syn (72-140)synthetic

α-Syn (72-140)recombinant

Figure 54: CD spectra of αSyn peptide fragments recorded at a final concentration of

30 µM in water.

The binding affinities of different αSyn antibodies to αSyn polypeptides were

analyzed using ELISA, Dot blot, Western blot and SAW biosensor

determination. Figure 55 illustrates the Western blot analysis of synucleins

using an anti-αSyn mBD antibody, which did not recognize βSyn and αSyn (96-

140). Therefore it can be concluded that the epitope for this antibody is located

in the central highly aggregation-prone domain (Val71–Val82) of wt-αSyn.


M 1 2 3 4 5 6 7 8a 9a 10

1, αSyn2, βSyn3, αSyn (A30P)4, αSyn (A53T)5, αSyn (1-120)6, αSyn (61-140)7, αSyn (96-140)8, αSyn (71-140)a

9, αSyn (72-140)a

10, αSyn (72-140)

a polypeptide synthesized by SPPS Figure 55: Western blot analysis of synucleins using anti-αSyn mBD antibody. Lack of

detection for βSyn and carboxyl terminal fragment αSyn (96-140) is indicated by arrows.

In Table 13, the binding affinity determinations of intact, full-length

synucleins (1, 2, 3, 4) and several αSyn fragments and partial sequences (5, 6,

7, 8, 9) to anti-synuclein antibodies are summarized, using ELISA, Western blot

and Dot blot detection analysis. For ELISA experiments suitable dilutions of the

primary anti-αSyn antibodies were first evaluated by incubating 0.5 μM wt-αSyn

in PBS, 100 μL per well, in triplicate, for 2 h at 25°C and then adding 12 three

fold serial dilutions of antibody. The binding of the antibodies to wt-αSyn was

assessed by incubating HRP-conjugated goat anti-mouse or anti-rabbit

antibodies (1 h at 25°C) and absorbance determination with an ELISA reader.

Subsequently, synucleins were compared for binding to anti-αSyn antibodies by

ELISA, using antigen dilutions from 1.4×10-5 µM to 2.5 µM. The results revealed

that both the mBD and 4B12 antibodies had no affinity to βSyn (2), which lacks

an 11 amino acid- residue sequence within the central amyloidogenic domain

(Figure 2). These antibodies, together with the anti-αSyn α/β antibody did not

bind to 7, αSyn (96-140). All antibodies studied, except anti-αSyn α/β antibody,

recognized specifically the fragment αSyn (72-140).


Table 13: Affinity binding of synuclein proteins (1, 2, 3, 4) and peptide fragments (5, 6, 7, 8, 9) to anti-synuclein antibodies determined by immunoanalytical methods:

Antibody ELISA Western blot Dot blot

Response a Antigenicity b

(nM)

1 Anti-α/βSyn 1/+

2/+

1, 1.5

2, 1.89

1/+ ; 2/+ 1/+ ; 2/+ ; 3/+ ;

4/+ ; 5/– ; 6/+ ;

7/– ; 8/– ; 9/–

2 Anti-αSyn m4B12 1/+

2/–

1, 2.05

2, –

1/+ ; 2/– 1/+ ; 2/– ; 3/+ ;

4/+ ; 5/– ; 6/+ ;

7/– ; 8/+ ; 9/+

3 Anti-αSyn mBD 1/+

2/–

1, 2.5

3, 7.2

4, 7.29

1/+ ; 2/– ; 3/+ ;

4/+ ; 5/+ ; 6/+ ;

7/– ; 8/+ ; 9/+

1/+ ; 2/– ; 3/+ ;

4/+ ; 5/– ; 6/+ ;

7/– ; 8/+ ; 9/+

4 Anti-αSyn pC20 1/+

2/+

3/+

4/+

1, 0.4

2, 1.02

3, 0.78

4, 1.28

1/+ ; 2/+ ; 8/+ ;

9/+

1/+ ; 2/+ ; 3/+ ;

4/+ ; 8/+ ; 9/+

5 Anti-αSyn pASY-1 1/+

2/+

3/+

4/+

1, 2.7

2, 2.3

3, 34.3

4, 18.8

1/+ ; 2/+ ; 8/+ ;

9/+

1/+ ; 2/+ ; 8/+ ;

9/+

a + positive detection response; – negative detection response obtained in indirect

ELISA b The protein/ peptide concentrations in nM needed to obtain an OD450 nm of 1.0 in

indirect ELISA

2.5.3 Preparation and characterization of chemically modified α-synuclein

Chemical modification of proteins with succinic anhydride has been

successfully employed in several structure – function studies of proteins [229, 301,

302]. Among others, succinylation can be efficiently used to characterize the role

of charge on the stability of a folded native protein conformation. Succinic

anhydride reacts with the ε-amino groups of lysine and the N-terminal α-amino

group of proteins, converting them from basic to acidic groups (Figure 92).


Succinylation of αSyn was carried out in 0.3 M sodium phosphate buffer, pH

7.5, with increasing amounts of succinic anhydride ranging from 0.5, 1, to 100-

fold molar excess, which was added to the continuously stirred protein solution

(1 mg mL-1), over 90 min maintaining the pH of 6 the reaction mixture [303]. After

quenching the reaction, proteins were subjected to Tris-tricine gel

electrophoresis, CD spectroscopy and mass spectrometric analysis.

Molar ratio succinic anhydride/ αSyn

0 0.5 1 2 10 50 100 MkDa200 -150 -120 -85 -70 -60 -

40 -50 -

30 -25 -20 -15 -

10 -

Figure 56: Tris-tricine gel electrophoresis of succinylated αSyn: molecular weight marker M (lane 1), αSyn with increasing amounts of succinic anhydride solution dissolved in DMSO: 0 (lane 2), 0.5 (lane 3), 1 (lane 4), 2 (lane 5), 10 (lane 6), 50 (lane 7), 100 (lane 8)-fold molar excess with respect to protein.

Figure 56 shows the Tris-tricine gel of αSyn modified with increased molar

amounts of succinic anhydride. The ESI mass spectrum (Figure 57a) together

with the deconvoluted spectrum (Figure 57b) of succinylated αSyn at an αSyn:

succinic anhydride ratio of 1:1 revealed the presence of five modified proteins.

The most abundant protein derivative (denoted with A) had a molecular mass of

14961.0 Da, corresponding to an αSyn derivative containing five succinylation

groups.


788.419+

832.218+ 881.2

17+936.116+

998.315+

1069.614+

1151.813+

1247.712+

0

1

2

3

Intens.

600 700 800 900 1000 1100 1200 1300 1400 1500 1600 m/z

985.115+

991.815+

998.315+

1005.115+

1011.715+

980 990 1000 1010 1020 1030 1040 m/z

x104

Mw calc C4H4O3 : 100 Da

A

14761.01+ (M+300)

14861.41+ (M+400)

14961.01+ (M+500)

15061.11+ (M+600)

15161.81+ (M+700)

14600 14700 14800 14900 15000 15100 15200 15300 15400 m/z

C

E

B

D

D

BA

C

E

Mw exp A: 14961.0 DaMw calc A: 14961.3 Da∆m = 20 ppm

a

b

Figure 57: (a), ESI mass spectrum of succinylated αSyn (αSyn: succinic anhydride ratio

of 1:1); (b), Deconvoluted ESI mass spectrum of succinylated αSyn shows the presence of five modified proteins. The most abundant protein is A with Mw exp of 14961.0 Da that contains five modified amino groups.

As illustrated in Figure 58b, the extent of modification was increasing with

increasing molar excess of succinic anhydride, and revealed complete

succinylation of all 16 amino groups and the single His50 residue at a molar

ratio of anhydride to protein of 100. This result was confirmed by LC-ESI-

MS/MS fragmentation of the modified protein (Figure 59) digested with Glu-C.


400 600 800 1000 1200 1400 1600 1800 m/z

17+16+

15+

14+

13+

18+

19+

20+

17+

16+

14+

18+

15+

19+

Mw calc C4H4O3 : 100 Da

Mw exp A: 16060.2 DaMw calc A: 16061.3 Da

Mw exp B: 16160.4 DaMw calc B: 16161.3 Da

H2N-MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVHGVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA-COOH

Figure 58: ESI mass spectrum of succinylated αSyn (αSyn: succinic anhydride ratio of

1:100) shows the presence of two modified proteins: in blue αSyn succinylation of 16 amino groups (lysine and N-terminal residues) with Mwcalc A of 16061.3 Da and in red with the additionally modified histidine residue with Mwcalc B of 16160.4 Da. The insert in the upper part of the figure contains a schematic representation of the primary structure of succinylated αSyn. It shows in blue αSyn succinylation of 16 amino groups (lysine and N-terminal residues) and in red the modified histidine.

y2

y3

y4

y5b6

b7

b8

b9

b10

H + 100

218.9

318.0

419.0

490.2649.1

720.1

821.1

920.2

991.2

V T A V - 0.1 G 237.4

200 400 600 800 1000 1200 1400 m/z

-

200 400 600 800 1000 1200 1400 m/z

b10

y2y3

b9

y4

b8

y5

b7

y6

b6

y7y8

47 57G V VHN CH C

CH2

O

N

N

G V A T V A E

OH

O

O

G V VHN CH C

CH2

O

N

N

G V A T V A E

OH

O

O

Figure 59: LC-ESI-MS/MS fragmentation of the double charged ion at m/z 569.6 of

αSyn (47-57) obtained by in solution digestion of succinylated αSyn with Glu-C shows that histidine (H) is succinylated at a molar ratio of the anhydride to the protein of 100:1.


The secondary structure analysis of succinylated αSyn was analyzed in the

succinylation buffer (0.3 M sodium phosphate buffer, pH 7.5 with 1 mg mL-1

succinic anhydride) at 25°C with a scan speed of 1 nm min-1. The CD spectrum

(average of four scans in a wavelength range of 180 and 260 nm) was found to

adopt a random coil conformation with a minimum at 198 - 200 nm (Figure 60).

180 200 220 240 260-2000000

-1500000

-1000000

-500000

0

500000

1000000

Mol

. Elli

ptic

ityΘ

(deg

cm2

dmol

-1

Wavelength (nm) Figure 60: CD spectrum of succinylated αSyn recorded in 0.3 M sodium phosphate

buffer, pH 7.5 in αSyn: succinic anhydride ratio of 1:100. The protein adopts a random coil conformation with a minimum at 198 - 200 nm.

2.6 Identification and quantification of synucleins by combination of bioaffinity and mass spectrometry

Biosensors have been developed as a new and efficient tool of

Bioanalytical Chemistry. A biosensor is an analytical device which converts a

biological response into an electrical signal. Biosensors have applications in

many basic and applied research fields, such as in medical diagnostics,

detection of drugs and explosives, and food quality control. In the study of

biopolymer interactions, surface plasmon resonance (SPR) has been long

established as a “gold standard” for detection and affinity quantification. The

binding of interaction partners increases the total mass and density at the chip

surface, resulting in a local refractive index change that is proportional to the

SPR biosensor signal output [304, 305]. A newly developed type of biosensor


capable of challenging SPR is based on surface acoustic wave (SAW). The

high sensitivity, simultaneous measurement of two types of signals and

resistance to buffer viscosity changes make the SAW biosensor suitable for

broad applications to affinity interaction studies. The principle of the SAW

biosensor is the transformation of an electric signal into a mechanical wave

through piezoelectricity; the wave modifies its amplitude and phase due to

surface mass loading and liquid viscosity changes, and the wave is converted

back into an electrical signal for processing. In more detail, mass loadings

render phase shifts, while viscosity changes induce modifications in both phase

and amplitude.

∆φ

∆A

INPUT SIGNAL OUTPUT SIGNAL

MASS, VISCOSITY

∆φ, ∆A

INPUT SIGNALOUTPUT SIGNAL

a

b

Figure 61: (a), Functioning principle of the SAW sensor: an electrical signal is

transformed in mechanical wave through piezoelectricity; the wave changes its amplitude and phase due to surface mass loading and liquid viscosity changes; the wave is then transformed back into electrical signal for processing; (b) The input and output signals differ in phase and amplitude; mass loadings render phase shifts, while viscosity changes induce modification in both phase and shift.

The S-sens K5 SAW sensor (SAW Instruments) is highly sensitive

towards surface effects and used to detect and quantify mass and viscosity

changes due to biomolecular interactions [306]. This type of biosensor, with five

sensor elements on one sensor chip operated in Lowe-wave geometry, is

working at two fixed frequencies differing by about 0.3 MHz, with φ(f1) - φ(f2) ≈


180° at a frequency range between 130 and 170 MHz [307, 308]. The main

advantage of using two fixed frequencies is that the influences of physical

parameters on the sensor signal such as temperature, salts and viscosity are

largely reduced. SAW biosensor is usable with a number of surface materials,

including gold and noble metals.

A schematic view of phase shift of a single sensor element is shown in

Figure 62. Self assembled monolayer (SAM) formation on the gold sensitive

surface is used as a linker for the covalent immobilization of different molecules.

A first injection of EDC and NHS shows an increase of the signal due to the

conversion of the carboxyl groups of the SAM, present on the surface, into

active N-hydroxysuccinimide ester. The next signal increase is due owed to the

covalent immobilization of the binder (e.g. antibody). This is followed by an

injection of ethanolamine for capping the unreacted active ester groups. The

solvent is changed from water to PBS buffer (needed to ensure near-

physiological conditions) causing a signal shift. The injection of a solution of

protein (αSyn) leads to a further signal increase, showing the affinity binding to

the antibody. Injecting a solution of glycine or HCl 0.1 M (pH 2) abolishes the

bioaffinity and the signal decreases; the bound protein is eluted.

4000 6000 8000 1000045

50

55

60

65

70

75

COOH COOH COOH COOH COOH PBSMQ

2. Immobilization of antibody

3. Blocking1.Activationwith EDC/NHS

4. Buffer exchange 5. Affinity binding of α-Syn

6. Elution with glycine or0.1 M HCl, pH 2

∆φ

Phas

e [d

eg]

Figure 62: Schematic view of phase shift of a single sensor element: 1) activation using

a single injection of EDC and NHS; 2) covalent immobilization of antibody; 3) blocking with ethanolamine; 4) buffer exchange from water (MQ) to PBS buffer; 5) affinity binding of ligand (αSyn); 6) elution using glycine or HCl solution.


2.6.1 Interactions of synuclein proteins and peptides with anti-synuclein specific antibodies

Specific binding of synuclein proteins and peptides with anti-synuclein

specific antibodies was analyzed as a function of time with SAW array

consisting of five sensor elements. A self assembled monolayer (SAM) of 16-

mercaptohexadecanoic acid was formed on the gold surface. The carboxyl

groups were activated using N-hydroxysuccinimide (NHS) and N-(3-

dimethylaminopropyl)-N-ethylcarbodiimide (EDC). Recombinant wt-αSyn and

αSyn epitope peptides 11, αSyn (1-23) and 12, αSyn (1-23), (see 3.3), were

immobilized to the activated SAM prior to the binding of anti-αSyn pASY-1

antibody. Two of the sensor elements were activated, and then the epitope

peptide 11, αSyn (1-23) was immobilized, and the free ester groups were

blocked with ethanolamine (pH 8.5). On the next two sensor elements the

peptide 12, αSyn (59-80) was immobilized. The last sensor element was used

as a reference corresponding to the positive control αSyn. Then, the gold-chip

was placed in the biosensor and the affinity binding of 100 nM anti-αSyn pASY-

1 antibody to the antigens was analyzed at 23°C in PBS binding buffer (pH 7.5).

Bound masses were calculated using the sensitivity of the Love-wave sensor

515° cm2 µg-1 under the assumption that the value is generally applicable.

Figure 63a shows a general scheme of affinity study of an antibody by manual

immobilization of αSyn and αSyn peptides by the SAW. Figure 63b indicates the

phase (° or deg), represented as a black column, and surface mass loading (ng

cm-2) (diagonal pattern column) plotted versus antigens. The highest binding

affinity was shown by wt-αSyn (phase of 3.63° and 7.056 ng cm-2) and was

confirmed by ELISA data (see Figure 50 in 2.5.1).


place Au-chip in Biosensor

add pASY-1 antibody

1. activation with NHS/EDC

2. add αSyn (59-80)

3. ethanolamine

1. activation with EDC/NHS

2. add αSyn (1-23)

3. ethanolamine1. activation with

EDC/NHS2. add αSyn

3. ethanolamine

a

b

0

1

2

3

4

1, αSyn 11, αSyn (1-23) 12, αSyn (59-80)012345678

Phas

e [d

eg]

ng cm-2

Figure 63: Binding affinity study of anti-αSyn pASY-1 antibody using manual

immobilization of wt-αSyn and αSyn epitope peptides and SAW biosensor: (a), General scheme of manual immobilization of antigens; (b), Phase (black column) and ng cm-2 (diagonal pattern column) plotted versus antigens. The highest binding affinity was shown by wt-αSyn (phase of 3.63° and 7.056 ng cm-2) and was confirmed by ELISA data (see Figure 50).

Further experiments were performed to monitor the affinity binding of 10

µM synuclein proteins and peptides on 200 nM anti-αSyn specific antibodies.

Figure 64a shows the real-time sensor curve of the SAW sensor. The injection

of EDC/NHS was performed to prepare the SAM for protein immobilization. The

phase signal during the injection was a result of the viscosity alteration. In

contrast, the remaining signal offset after the injection has to be related to the

association of mass to the sensor surface. It indicates the activation, by forming

an NHS-ester with the carboxyl groups of the SAM. Then, three injections of

200 nM anti-αSyn mBD antibody were performed at time (t) = 1000 s (first

injection) at a flow rate of 20 µL min-1, so that the estimated duration of an

injection wast = 450 s. After the last injection, running buffer was flown over

the sensor, and residual carboxyl groups were deactivated with 1M

ethanolamine. Immobilized anti-αSyn mBD antibody showed for a single sensor


element a phase difference (∆φ) of 13.5°, with the sum of phase difference of all

five sensor elements was calculated to 62.3°. The interactions with αSyn

proteins (1, 2, 3, 4) and peptides (11, 12, 13) were carried out using PBS as

running buffer (pH 7.5), and affinity bound antigens were eluted under acidic

conditions (pH 2). Then, extraction of the data from the sensor signals for all

antigens was employed, and a plot of phase (black column), and surface mass

loading (diagonal pattern column) versus antigens was carried out (Figure 64c).

-1000 0 1000 2000 3000 4000 5000 6000-10

0

10

20

30

Time [s]

EDC/NHS

∆φ

mBD Ab mBD Ab mBD Ab Ethanolamine

Activation

anti-α-Syn mBD Ab (200 nM)

Immobilization Blocking

Phas

e [d

eg] Anti-αSyn mBD antibody

∆φ = 15.1 – 1.6 = 13.5°∑ ∆φ = 62.3°m/A = 62.3° / 515° μg-1 cm2

m/A = 0.121 µg cm-2

n = 0.807 pmol cm-2

a

1, αSyn2, βSyn3, αSyn (A30P)4, αSyn (A53T)11, αSyn (1-23)12, αSyn (59-80)13, αSyn (59-80 T72A)

Phas

e [d

eg]

b

0 200 400 600-1

0

1

2

3

4

Time [s]


c

Phas

e [d

eg] ng cm

-2

0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

1 2 0

1

2

3

4

5

6

7

3 4 11 12 13 Figure 64: (a), Immobilization of 200 nM anti-αSyn mBD antibody on self assembled

monolayer (SAM) gold chip. Phase signals as observed for the injection of EDC/NHS (activation of the SAM), anti-αSyn mBD antibody (immobilization) and ethanolamine (blocking). The signal offsets indicated the association of mass to the sensor surfaces; (b), SAW curves of affinity binding of 10 µM synuclein proteins and peptides on 200 nM anti-αSyn mBD antibody; (c), The phase shift (black column) and surface mass loading (ng cm-2) (diagonal pattern column) plotted versus antigens. The highest binding affinity was shown by 1, αSyn (3.12° and 6.058 ng cm-2).

The affinity binding studies using the SAW biosensor showed that αSyn

proteins bound specifically to the immobilized anti-αSyn mBD antibody. In

contrast, the synthetic N-terminal αSyn (1-23) peptide did not show any binding

affinity. The αSyn (59-80) and αSyn (59-80 T72A) peptides generally showed

binding affinity to the anti-αSyn mBD antibody, consistent with the results

obtained by Dot blot experiments (see 2.5.1).

2.6.2 Determination of dissociation constants of anti-synuclein antibody-ligand complexes

The KD determinations of the anti-αSyn antibodies to synucleins were

done by SAW biosensor, used for binding of increasing concentrations of anti-

αSyn antibody (5 - 200 nM) to αSyn immobilized on the biosensor surface. After

injection, the unbound antibody dissociated and the surface was regenerated

with glycine, pH 2. The obtained KD values were generally in the low nano-

molar range. The bioaffinities were also investigated by Dot blot, ELISA and

SAW biosensor, and the results were found to be in good agreement. For

further evaluation, the resulting curves were exported into Origin and the

integrated FitMaster was applied. The resulting overlay plot and individual fitting

following with a 1:1- binding model are displayed in Figure 65. The pseudo-first


order kinetic constants kobs was determined by the FitMaster and plotted versus

the anti-αSyn pC20 antibody concentration (Figure 65). A linear best fit was

applied using the equation Kobs = koff + kon * C. The KD value was determined

from KD = koff / kon and found in the low nanomolar range.

Moreover, the SAW biosensor was used for real-time measurements to

analyze the interaction of αSyn with an anti-αSyn 4B12 (GeneTex) monoclonal

antibody. The immobilization process was carried out in the fluidic cell of the

system. αSyn at a concentration of 10 µM was immobilized to the sensor chip

coated with SAM (16-mercaptohexadecanoic acid). For the binding

experiments, the running buffer was changed to PBS (pH 7.5). The experiment

was continued with a series of injections at increasing antibody concentrations

(5 - 200 nM). After each injection, the system was given sufficient time to reach

the baseline level by dissociation of the bound antibody, and the surface was

then regenerated with a pulse of 50 mM glycine, pH 2.0. For evaluation, the

resulting curves were exported into OriginPro 7.5 and the integrated FitMaster

was applied. The resulting overlay plot and the individual fits following with a 1:1

binding plus buffer offset are displayed in Figure 65b. A linear best fit was

applied using the equation shown. A KD value of 45.5 ± 2.7 nM was obtained

(Figure 65b).


Anti-αSyn pC20 antibody

ba

0 50 100 150 2000.000

0.002

0.004

0.006

0.008

0.010

Kob

s

C [nM]0 50 100 150 200

0.000

0.002

0.004

0.006

0.008

0.010

Kob

s

C [nM]

-200 0 200 400 600 800

0

5

10

15

20

c1 5 nMc2 10 nMc3 25 nM

c4 50 nM

c5 75 nM

c6 100 nM

c7 150 nMc8 200 nM

Time [s]

Phas

e [d

eg]

KD = 39 ± 11.4 nM

R2 = 0.96

0 250 500 750 1000 1250

0

2

4

6

8

Phas

e [d

eg]

Time [s]

0 50 100 150 2000.000

0.001

0.002

0.003

0.004

0.005

0.006

0.007

0.008

Kob

s

C [nM]

c1 5 nMc2 10 nM

c3 25 nM

c4 50 nMc5 75 nMc6 100 nMc7 150 nMc8 200 nM

Anti-αSyn 4B12 antibody

KD = 45.5 ± 2.7 nM

R2 = 0.99

Figure 65: KD determinations of (a), anti-αSyn polyclonal pC20 (Santa Cruz) and (b),

anti-αSyn monoclonal 4B12 (GeneTex) antibodies on αSyn using SAW biosensor.

Examples of KD determinations of the αSyn-specific antibodies for the

αSyn and βSyn obtained from SAW biosensor are shown in Table 14. The anti-

α/βSyn monoclonal antibody exhibits the highest affinity for 2, βSyn with KD

value of ~12 nM. The affinity of this antibody for 1, αSyn is distinctly lower (KD

50.9 nM), primarily due to a slower kon rate. Further, the anti-αSyn monoclonal

BD antibody exhibits a lower affinity to 1 than the anti-αSyn monoclonal 4B12

antibody. The lowest affinity to 1 is provided by the anti-αSyn pASY-1 antibody

with a KD of approximately 438 nM, due to a slower kon rate of 2.74 10-6 nM-1

sec-1.


Table 14: Kinetic rate and equilibrium dissociation constants of the αSyn-specific antibodies for the αSyn and βSyn:

No. Antibody Ligand kon

(nM-1 sec-1)

koff

(sec-1)

KD

(nM)

1 Anti-α/βSyn 1, αSyn

2, βSyn

1.2978 10-4

1.1159 10-4

0.0066

0.0013

50.93 ± 6.8

11.91 ± 4.45

2 Anti-αSyn m 4B12 1, αSyn 2.8350 10-5 0.0013 45.5 ± 2.7

3 Anti-αSyn mBD 1, αSyn 2.3848 10-5 0.0022 92.25 ± 8.45

4 Anti-αSyn pC20 1, αSyn 3.4794 10-5 0.0014 39 ± 11.4

5 Anti-αSyn pASY-1 1, αSyn 2.7402 10-6 0.0012 437.9 ± 31

Figure 66a shows the comparison of the affinities binding of 50 nM anti-

synuclein antibodies to αSyn using the SAW biosensor. After each affinity

binding, the gold chip was regenerated using 2 injections of glycine, pH 2.

Figure 66b indicates the phase (° or deg), represented as black column, and

surface mass loading (ng cm-2) (diagonal pattern column) plotted versus

antibodies. The highest binding affinity to αSyn was found by the anti-αSyn

pC20 antibody and the lowest affinity by the anti-αSyn pASY-1 antibody. These

data confirmed the KD values shown in Table 14 and additionally are in

agreement with the ELISA data summarized in Table 13 (2.5.2).

0

10

20

30

40

50

60

70

α/β 4B12 BD pC20 pASY-10

20

40

60

80

100

120

140

Phas

e [d

eg]

ng cm-2

Figure 66: Affinity binding of 50 nM anti-αSyn antibodies on immobilized αSyn using

SAW biosensor. Phase (black column) and ng cm-2 (diagonal pattern column) plotted versus anti-αSyn antibodies.


Furthermore, the SAW biosensor was employed for KD determination of

the anti-αSyn mBD antibody to synthetic αSyn (72-140). The binding of

increasing concentrations of anti-αSyn antibody (10 - 200 nM) to polypeptide

immobilized on the biosensor surface was performed. After injection, unbound

antibody was dissociated and the surface regenerated with glycine, pH 2, which

yielded a KD value of 464 nM (Figure 67).

Value Error

Koff 0.03108 5.4455 10-4 sec-1

Kon 6.69885 10-5 4.84723 10-6 nM-1 sec-1

KD = koff/ kon = 463.96 ± 34.54 nM

R2 = 0.96

0 50 100 150 200 2500.00

0.01

0.02

0.03

0.04

0.05

Kob

s

C [nM]0 50 100 150 200 250

0.00

0.01

0.02

0.03

0.04

0.05

Kob

s

C [nM]-200 0 200 400 600 800 1000

0

2

4

6

8

10

c1 10 nMc2 25 nMc3 50 nMc4 100 nMc5 150 nMc6 200 nM

Phas

e [d

eg]

Time [s]

a b

Figure 67: KD determination of anti-αSyn (BD Transduction Laboratories) to αSyn (72-

140) using SAW biosensor; (a), The fitted curves of the increased binding; (b), Pseudo first-order kinetic constant plotted versus concentration. The linear regression was applied for KD of 463.96 ± 34.54 nM.

Determinations of the affinity binding constant of the αSyn

oligomerization-aggregation mixture (type S5) to the anti-oligomer A11 antibody

as performed after immobilization of the antibody to activated Au-SAM using

150 μL of different concentrations of ligand (20 nM - 20 μM) at a flow rate of 20

μL min-1. Affinity binding was performed in PBS buffer pH 7.5, followed by

regeneration with 150 μL glycine buffer. Determination of antibody/oligomer

association kinetics was performed by extracting the data from the sensor

signals, and determinations of dissociation constants obtained by plotting the

pseudo first-order kinetic constant versus concentration, yielding a KD of

approximately 17.5 µM (Figure 68). Thus, the SAW biosensor was shown to be

a highly efficient tool for sensitive kinetic determinations of interactions and

precise evaluation of kinetic constants.


baLinear Regression

Kobs = koff + kon * C

Value Error

Koff 0.01191 8.22943 10-5 sec-1

Kon 6.78468 10-7 7.09118 10-9 nM-1 sec-1

KD = koff/ kon = 17.5 ± 0.2 µM

R2 = 0.99

0

1

2

3

4

Phas

e [d

eg]

-1-200 0 200 400 500

Time [s]300100-100

c1 20 nMc2 200 nMc3 2000 nMc4 20000 nM

Figure 68: KD determination of anti-oligomer A11 antibody to αSyn oligomerization-

aggregation products type S5 using SAW biosensor: (a), The fitted curves of the increased binding; (b), Pseudo first-order kinetic constant plotted versus concentration. The linear regression was applied for KD of 17.6 ± 0.2 µM.

2.6.3 Interaction studies of synucleins and anti-synucleins antibodies by online bioaffinity- MS

While the surface acoustic wave (SAW) biosensor is a sensitive tool

capable to detect bioaffinity interactions, the combination of a SAW with ESI

mass spectrometry enables the simultaneous detection, identification, and

quantification of affinity-bound ligands from biopolymer complexes on a

biosensor chip (see 3.12). The synuclein- anti-synuclein antibody complexes

were analyzed because αSyn antibodies have been extensively employed in

studies of this protein. The anti-αSyn specific antibodies were immobilized on

the chip surface for interaction studies with αSyn proteins and peptides. The

affinity bound antigens were eluted under acidic conditions and analyzed by ESI

mass spectrometry using newly developed interface for intermediate sample

desalting and concentration [309].

Figure 69a shows the affinity binding of αSyn (1) to anti-αSyn monoclonal

4B12 (GeneTex) antibody using the direct coupling of SAW biosensor – ESI-

MS. The antibody immobilization was performed using a single injection of 150

µL and the quantity of protein 1 bound to the antibody was approximately 20.83

ng cm-2. The mass spectrum of the eluted protein 1 provides direct identification

of αSyn by the characteristic pattern of multiply charged ions.


4000 6000 8000 1000045

50

55

60

65

70

75

4000 6000 8000 1000045

50

55

60

65

70

75

PBSMQ

Ethanolamine1M pH=8.5EDC/NHS αSyn

HCl 0.1 Melution of αSyn

Time [s]

0

2

4

6

8

Intens

600 650 700 750 800 850 900 950 m/z

19+ 18+

17+

16+

15+

21+

22+

23+

20+

Anti-αSyn 4B12


Phas

e [d

eg]

x105b

a

Figure 69: (a), SAW binding curve of the affinity interaction between 10 µM wt-αSyn

and 200 nM covalently immobilized anti-αSyn m4B12 antibody. Before studying the affinity interaction, the change of the system buffer from water (MQ) to PBS is carried out. The black lines represent the injections of EDC/NHS, antibody, ethanolamine, αSyn, HCl 0.1 M; (b), ESI mass spectrum of the eluted antigen shows that the protein was bound to the αSyn specific antibody.

A further example of the online SAW-ESI-MS is the interaction αSyn (1)

and βSyn (2) with a polyclonal anti-αSyn pC20 antibody. Figure 70a illustrates

the SAW binding curve of the affinity interaction of αSyn and the immobilized

anti-αSyn pC20 antibody. Before studying the affinity interaction, a change of

the system buffer from water to PBS was carried out. The mass spectrometric

characterization of the eluted protein 1 in the online SAW-MS is shown in Figure

70a. The ESI mass spectrum of the eluted antigen revealed that the protein was

bound to the αSyn specific antibody. When, the analogous experiment was

performed by online SAW-MS to analyze the interaction of βSyn with the anti-

αSyn pC20 antibody, a considerably lower affinity was found (Figure 70b).


0 2500 5000 7500 10000

0

10

20

30

40

50

Time [s]

αSynGlycine, pH 2

elution of αSynEthanolamine

1M pH=8.5

Anti-αSyn pC20 antibody

PBSWater

0.0

0.5

1.0

1.5

500 600 700 800 900 1000 1100 1200 1300 m/z

19+

18+

17+16+

15+

14+

13+ 12+

20+21+

22+

0 2500 5000 7500

0

10

20

30

40

50

Time [s]

PBSWater

βSynGlycine, pH 2

elution of βSynEthanolamine

1M pH=8.5

0.5

1.0

1.5

.

500 600 700 800 900 1000 1100 1200 1300 m/z

17+

19+

18+

16+

15+

14+

20+

.

Intensx105

Intensx104

Mw exp : 14460.1 DaMw calc : 14460.1 Da

Mw exp : 14287.8 DaMw calc : 14287.9 Da

a

Phas

e [d

eg]

Phas

e [d

eg]

b

Figure 70: Affinity binding and mass spectrometric identification by comparison of αSyn

and βSyn: (a), SAW-ESI-MS binding curve of the affinity interaction study between 10 µM wt-αSyn and 200 nM covalently immobilized anti-αSyn pC20 antibody. The quantity of αSyn bound to the antibody is 39.2 ng cm-2. The ESI mass spectrum of the eluted αSyn shows the 12+ to 22+ charged molecular ions; (b), Online SAW-MS of 10 µM wt-βSyn to 200 nM covalently immobilized anti-αSyn pC20 antibody. The quantity of βSyn bound to the antibody is 25.9 ng cm-2. The ESI mass spectrum of the eluted βSyn shows the 14+ to 20+ charged molecular ions.

The SAW biosensor was also employed as an affinity detection method

in conjunction with MALDI-MS analysis for epitope determinations, similar to the

procedures described in 2.4.3. Both epitope excision/ extraction approaches

were carried out. Figure 71 shows the epitope identification of αSyn (1) to anti-

αSyn monoclonal 4B12 (GeneTex) antibody by offline coupling of SAW

biosensor – MALDI-TOF-MS. The antibody immobilization was performed using

a single injection of 150 µL solution at a concentration of 200 nM. For epitope

extraction the antigen 1 was first digested with trypsin and Glu-C for 2 h and

then presented to the antibody on the sensor chip. The quantity of peptide

mixtures of protein 1 bound to the antibody was 4.5 ng cm-2 after trypsin and

10.8 ng cm-2 after Glu-C digestion, using two injections of 150 µL of a solution


of 10 µM. The affinity bound epitope peptides were eluted under acidic

conditions, lyophilized and analyzed by MALDI-TOF-MS using the Bruker

UltrafleXtreme MALDI-TOF/TOF [310]. The mass spectrum of the eluted epitope

peptides of 1 using trypsin is shown in Figure 71b and provided the

identification of three peptides, αSyn (59-80), (61-80), (81-96). MS/MS

fragmentation analysis of the ion 1478.808 (1+) also revealed the identification

of peptide (81-96) (Figure 71c).

0 2500 5000 7500 10000 12500 15000-30

-20

-10

0

10

20

30

40

Phas

e [d

eg]

Time [s]

Immobilizationanti-αSyn 4B12 Ab

αSyn peptide mixture(trypsin)

αSyn peptide mixture(Glu-C)

Antibody Elution (pH 2) Elution (pH 2) Elution (pH 2)

a

(81-96)(61-80)

(59-80)2157.200

1478.8021928.056

0.0

0.5

1.0

1.5

2.0

4x10

Inte

ns. [

a.u.

]

500 1000 1500 2000 2500 3000 3500 m/z

bEDC/NHS


81T V E G A G S I A A A T G F V K96y16 y14 y13 y11 y9 y8 y7 y6 y5 y4

b7b8 b9 b10 b13

81T V E G A G S I A A A T G F V K96y16 y14 y13 y11 y9 y8 y7 y6 y5 y4

b7b8 b9 b10 b13

y4

y5

y6

y7

y8

y9

y11

b13

y13 y14

y161478.808

1279.139

622.266

764.271551.244

693.282

450.258

1021.453

1149.380

877.345

0

200

400

600

800

1000

Inte

ns. [

a.u.

]

200 400 600 800 1000 1200 1400 1600 m/z

b7b9

c

Figure 71: Epitope identification of αSyn (1) to anti-αSyn monoclonal 4B12 (GeneTex)

antibody by offline coupling of SAW biosensor – MALDI-TOF/TOF MS. (a), SAW binding curve of the affinity interaction study between the peptide mixture of 10 µM αSyn digested in solution with trypsin and Glu-C for 2 h at 37°C, and 200 nM covalently immobilized anti-αSyn m4B12 antibody; (b), MALDI-TOF mass spectrum of the elution fraction, after trypsin digestion, reveals three fragments αSyn (59-80), (61-80), (81-96); (c), MS/MS fragmentation mass spectrum of ion 1478.808 (1+) (with y and b ions assigned), which led to the identification of peptide (81-96) is also displayed.

For the epitope extraction, αSyn was digested for 2 h with the chosen

protease (trypsin or Glu-C), and then applied onto the anti-α/βSyn, anti-αSyn

mBD and anti-αSyn pC20 antibodies immobilized on the sensor chip. Binding of

the antigen to the antibody was performed in two injections of 150 µL at a flow

rate of 20 µL min-1, and washing and epitope elution, performed as previously

described. The released epitope and non-epitope peptides were lyophilized,

desalted and analyzed by MALDI-TOF mass spectrometry. Table 15

summarizes the epitope peptides obtained. Most of the identified peptides were

within the central part of the αSyn sequence, leading to the conclusion that this

region is highly exposed to interactions with other molecules. This agrees with

numerous other studies showing the tendency of the N-terminal region to adopt

an α-helix structure, shielding some sites of interaction. On the other hand, the

highly unstructured conformation of the C-terminal region may be too flexible for

establishing stable non-covalent interactions.


Table 15: Proteolytic peptides present in the elution fractions of αSyn by epitope extraction-offline SAW-MS:

No. Antibody Enzyme Epitope sequence

1 Anti-α/βSyn trypsin 81-96

61-80

59-80

Glu-C 47-83

2 Anti-αSyn m4B12 trypsin 81-96

61-80

59-80

Glu-C 47-83

3 Anti-αSyn mBD trypsin 81-96

61-80

59-80

Glu-C 62-83

58-83

47-83

4 Anti-αSyn pC20 trypsin 81-96

61-80

7-34

Glu-C 47-83

Online SAW-MS was employed for a comparative study on the binding

specificity of αSyn epitope peptides. Affinity binding of an equimolar mixture (10

μM) of αSyn (1-23), αSyn (59-80) and αSyn (59-80 T72A) peptides and mass

spectrometric analyses of the eluted peptides in online SAW-MS from the

immobilized anti-αSyn pC20 antibody are shown in Figure 72. The mass

spectrum of the mixture of the eluted peptides showed that the [M+3H]3+ ions of

the αSyn (59-80) and αSyn (59-80 T72A) peptides 12, 13 had the highest

abundance, suggesting highest affinity to the anti-αSyn-antibody (Figure 72b),

which is consistent with the specific epitope peptide identified by SAW-MS.


+3

+4+5+6407.5

+2

+3

+4

+6360.5

+3709.8

+21064.1

+4532.5

488.6

540.6

610.5

720.4

813.51080.1

0.0

0.5

1.0

1.5

2.0

2.5

200 300 400 500 600 700 800 900 1000 1100 1200 m/z

+21080.1

+3720.4

+4540.6

+3

+4532.7

709.8

1064.10

2

4

6

8

200 300 400 500 600 700 800 900 1000 1100 1200 m/z

+2

Intensx106

Intensx106

Intensx105

Intensx105

11, H-1MDVFMKGLSKAKEGVVAAAEKTK23-NH2

12, H-59TKEQVTNVGGAVVTGVTAVAQK80-NH2

13, H-59TKEQVTNVGGAVVAGVTAVAQK80-NH2

b

aMixture

Elution

12, αSyn (59-80)13, αSyn (59-80 T72A)

Figure 72: Affinity binding of an equimolar mixture (10 µM) of αSyn (1-23), αSyn (59-

80) and αSyn (59-80 T72A) to anti-αSyn pC20 antibody by SAW-ESI-MS: (a), ESI mass spectrum of an equimolar mixture (10 µM) of αSyn (1-23), αSyn (59-80) and αSyn (59-80 T72A); (b), ESI mass spectrum of the eluted antigen shows the 2+ to 4+ charged molecular ions of αSyn (59-80) and αSyn (59-80 T72A) peptides.

Furthermore, affinity binding of an equimolar mixture (10 µM) of αSyn (61-140)

and αSyn (96-140) to the anti-αSyn pC20 antibody was performed by SAW-ESI-

MS. Figure 73a shows the ESI mass spectrum of the equimolar mixture, while

in Figure 73b the ESI-MS of the eluted antigen showed that the αSyn (61-140)

peptide fragment bound to anti-αSyn pC20 antibody.


+6

+5

+7746.3

847.1

870.5

941.0

1044.5

500 600 700 800 900 1000 1100 1200 1300 1400 1500 m/z

+9+10

1058.3+8

0

1

2

3

4

5

846.9

941.1

1058.5

0

1

2

3

500 600 700 800 900 1000 1100 1200 1300 1400 1500 m/z

+10

+9

+8

Intensx105

Intensx105

Intensx105

Intensx105

6, αSyn (61-140)Mw exp : 8460.9 DaMw calc : 8460.2 Da

b

aMixture

Elution

αSyn (61-140): αSyn (96-140)

Figure 73: Affinity binding of αSyn (61-140), αSyn (96-140) to anti-αSyn pC20 antibody

by SAW-ESI-MS: (a), ESI mass spectrum of an equimolar mixture (10 µM); (b), ESI-MS of the eluted antigen reveals the αSyn (61-140) fragment bound to the αSyn specific antibody.

Further experiments were performed to monitor the affinity synuclein

fragments to the anti-αSyn monoclonal antibody. The interactions with αSyn (1) synthetic polypeptides 5 αSyn (1-120), 6 αSyn (61-140), 7 αSyn (96-140), and

10 αSyn (72-140) were carried out using PBS as running buffer, and the affinity

bound antigens eluted under acidic conditions. After extracting the data from the

sensor signals for all antigens employed, a plot of phase (black column), and

surface mass loading (diagonal pattern column) versus antigens concentrations

was performed (Figure 74). These affinity binding studies showed that the αSyn

polypeptides bound to the immobilized anti-αSyn BD monoclonal antibody,

while the C-terminal αSyn (96-140) fragment show no binding affinity. The high

sensitivity of the SAW biosensor provided convenient conditions for the

determination of affinities of synuclein- anti-synuclein complexes using the

online SAW-MS system. The mass spectrometric identification of these

complexes is summarized in Table 16.


0 200 400 600-1

0

1

2

3

4

Time [s]

1, αSyn

10, αSyn (72-140)5, αSyn (1-120)

6, αSyn (61-140)7, αSyn (96-140)

0 200 400 600-1

0

1

2

3

4

Time [s]

1, αSyn

10, αSyn (72-140)5, αSyn (1-120)

6, αSyn (61-140)7, αSyn (96-140)

0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

0

1

2

3

4

5

6

7

Phas

e [d

eg]

a

b

Phas

e [d

eg] ng cm

-2

1, αSyn 5, αSyn (1-120) 6, αSyn (61-140) 7, αSyn (96-140) 10, αSyn (72-140)

Figure 74: Affinity binding of 10 µM αSyn peptide fragments on 200 nM anti-αSyn mBD

antibody. (a), SAW binding curves; (b), The phase (black column) and ng cm-2 (diagonal pattern column) were plotted versus antigens. The highest binding affinity is shown by 1, αSyn.

Table 16: Mass spectrometric identification of antibody- ligand complexes by online SAW-ESI-MS:

No. Antibody Supplier Ligand m/za zb

1 Anti-α/βSyn Lifespan Biosciences 1, αSyn

2, βSyn

904.8

953.5

16+

15+

2 Anti-αSyn m4B12 GeneTex 1, αSyn 761.8 19+

3 Anti-αSyn mBD

BD Transduction Laboratories

1, αSyn

3, αSyn (A30P)

4, αSyn (A53T)

965.0

906.4

906.5

15+

16+

16+

4 Anti-αSyn pC20 Santa Cruz 1, αSyn

2, βSyn

6, αSyn (61-140)

12, αSyn (59-80)

13, αSyn (59-80 T72A)

851.8

894.0

941.1

720.4

709.8

17+

16+

9+

3+

3+


No. Antibody Supplier Ligand m/za zb

5 Anti-αSyn pASY-1

Boehringer Ingelheim 1, αSyn

2, βSyn

3, αSyn (A30P)

4, αSyn (A53T)

904.8

953.5

906.4

906.5

16+

15+

16+

16+ a Most abundant ligand ion identified by SAW-ESI-MS; b Charge of the most abundant ligand ion identified by SAW-ESI-MS

In summary, the direct combination of affinity SAW biosensor and mass

spectrometry provides information of biopolymer interactions at the molecular

level, by (i) structure identification of affinity interactions and ligand; (ii)

quantification of antigen-antibody interactions; (iii) epitope determination.

2.6.4 Isolation and structure determination of in vivo oligomerization-aggregation products

2.6.4.1 Structure characterization of in vivo oligomers by affinity mass spectrometry

The immunoblotting approach in combination with high resolution mass

spectrometry enabled the identification of mutant αSyn (A30P) (3) from soluble

mouse brain homogenate proteins. Four protein spots (spot 1, 2, 3 and 4)

(Figure 75a) detected as immunoreactive by the anti-αSyn mBD antibody, were

excised from the Coomassie gel of the TBS soluble mouse brain homogenates

(Figure 75), digested with trypsin, and analyzed by high resolution mass

spectrometry (see 3.9.5). Each protein spot provided an identification hit for

human αSyn.


m/z

Rel

ativ

e Ab

unda

nce

d MS/MS, m/z 1080.943 (4+)

MDVFMKGLSK AKEGVVAAAE KTKQGVAEAP GKTKEGVLYV GSKTKEGVVH



1

51

101 140

c

**

SDS

IEF60 kDa

5 kDapI 3 10 pI 3 10

60 kDa

5 kDa

ba

SDS

IEF

(3)1

1 2 3 4 1 2 3 4

Figure 75: (a), 2D gel electrophoretic separation of elution 1 (E1) containing TBS

soluble mouse (Syn-m-131) brain homogenate proteins within pH 3–10; (b). Western blot using anti-αSyn mBD at 1:500 dilution in PBS-T. The antibody recognizes the monomeric (3)1 of αSyn (A30P) protein; (c), Mass spectrometric identification of Homo sapiens αSyn from spot 2 (see Figure 75). After excision from 2D gel, the protein spot was digested with trypsin, and analyzed by LC-MS/MS. Database search provided identification of αSyn with sequence coverage of > 81%. (c), The fragmentation mass spectrum of ion 1080.943 (4+) (with y and b ions assigned), which led to the identification of peptide (103-140) and to the localization of oxidation of two methionine residues (116Met and 127Met) highlighted with a black star (*) is also displayed.


Affinity mass spectrometric identification of αSyn (A30P) from soluble mouse

brain homogenates was obtained from 200 µg mouse brain homogenate by

addition to the anti-αSyn pC20 antibody micro-column for 2 h. The supernatant

containing unbound protein was then collected, the column washed with 50 mL

PBS followed by 20 mL water, and affinity-bound protein eluted by incubation

with 500 µL 0.1% TFA for 30 min. The obtained fractions were lyophilized and

analyzed by gel electrophoresis and mass spectrometry. Figure 76a shows the

Coomassie stained 1D gel of the supernatant, wash and elution fractions. The

wash fraction revealed no bands indicating that the washing step was

successfully completed. The band from the elution fraction at ~17 kDa was

subjected to in gel digestion using trypsin. Subsequently, LC-ESI-MS/MS

followed by Mascot-Database search was carried out enabling the identification

of αSyn (NACP140, Homo sapiens) (Figure 76b). These data were confirmed

by analysis using direct infusion ESI-ion trap-MS of the elution fraction

solubilized in 1% formic acid and by in solution digestion and analysis by LC-

MS/MS. Table 17 summarizes the peptides (highlighted in red in Figure 76c)

obtained with this approach. In Figure 76d the MS/ MS fragmentation spectrum

of the peptide (81-96) is shown as an example.


supenatant

M washelution

40 -50 -

30 -25 -20 -15 -

kDa

10 -

60 -85 -

200 -

MDVFMKGLSK AKEGVVAAAE KTKQGVAEAP GKTKEGVLYV GSKTKEGVVH



1

51

101 140

a bMatch to: gi4507109 Score 88αSyn [Homo sapiens]Mass: 14451; pI: 4.67

MS/MS, m/z 740.4 (2+)d

c

Figure 76: Affinity mass spectrometric identification of αSyn (A30P) from soluble mouse

brain homogenates. (a), Tris-tricine gel separation of supernatant, wash and elution fractions after affinity binding of 200 µg of soluble mouse brain homogenates to the anti-αSyn pC20 antibody micro-column; (b), MASCOT browser output depicting the sequence coverage for the human αSyn (gi 4507109) with score of 88; (c), In the amino acids sequence of the protein, the identified peptides are shown in red; (d), LC-MS/MS fragmentation mass spectrum of ion 740.4 (2+) (with y and b ions assigned), which led to the identification of peptide (81-96) is also displayed. Peptide (81-96) is in red and underlined in the amino acids sequence.

Table 17: Tryptic peptides of the elution fraction of mouse Thy-1-human (A30P) αSyn brain homogenates identified by LC/ESI-MS:

Nr. Sequence Molecular ion observed M exp M calc

1 (11-21) (536.7)2+ 1071.4 1071.6

2 (59-80) (720.1)3+ 2157.2 2157.4

3 (81-96) (740.2)2+ 1478.5 1478.6

4 (61-80) (643.5)3+ 1927.9 1928.1


Further affinity binding experiments using 200 µg of TBS-soluble mouse

brain homogenate proteins were performed with the immobilized anti-αSyn

pC20 and anti-αSyn mBD antibodies in combination with immunoblotting. Prior

to Western blot experiments, the supernatant, wash and elution fractions were

separated on 10% Tris-tricine separation gel. The separated proteins were then

transferred onto PVDF membranes and probed with: (a), rabbit polyclonal anti-

αSyn pC20 and (b), mouse anti-αSyn mBD antibody. After washing, the

membranes were incubated with secondary antibodies in PBS-T for 1 h at 25°C.

Following five washes, enhanced chemiluminescence (ECL) substrate solution

was used to generate the chemiluminescent signal (Figure 77). The anti-αSyn

pC20 antibody bound specifically to monomeric (3)1 of αSyn (A30P) protein,

whereas the anti-αSyn mBD antibody bound to monomeric (3)1, oligomeric (3)n

and additionally the ∆N (3) truncation products.

supenatant

washelution

supenatant

washelution

a b

(3)2

(3)1

∆N (3)

(3)1

Anti-α-Syn mBD AbAnti-α-Syn pC20 Ab

Figure 77: Western blot analysis of supernatant, wash and elution fractions

corresponding to the 3, αSyn (A30P), from TBS soluble mouse brain homogenate, affinity experiment with the immobilized (a), anti-αSyn pC20 antibody and (b), anti-αSyn mBD antibody. Anti-αSyn pC20 diluted 1:500 specifically recognizes the monomer 3 and anti-αSyn mBD diluted 1:500 specifically recognizes 3, monomer, oligomer and related ∆N (3) truncation products.

Figure 78 illustrates the affinity binding of 100 µg human neuroblastoma

SH-SY5Y αSyn (A53T) cell extracts with the immobilized anti-αSyn pC20


antibody. The polyclonal antibody specifically recognized the 4, αSyn (A53T)

monomer and high molecular weight aggregates (> 200 kDa).

40 -50 -

30 -25 -20 -15 -

kDa

10 -

60 -85 -

200 -(4)n

(4)1

supenatant

washelution

M

Figure 78: 10% Tris-tricine gel separation stained with silver of affinity binding

experiment of human neuroblastoma SH-SY5Y αSyn (A53T) cell extracts with the immobilized anti-αSyn pC20.

Furthermore, affinity binding experiments were performed with the TBS soluble

mouse brain homogenate with the immobilized anti-oligomer A11 antibody. The

supernatant, wash and elution fractions were separated using 10% Tris-tricine

gel electrophoresis and then stained with sensitive silver staining. In the elution

fraction two bands at ~85 kDa and 120 kDa were observed, indicating the

separation of high molecular products present in transgenic mouse brain

homogenate.


40 -50 -

30 -25 -20 -15 -

kDa

10 -

60 -85 -

200 -120 -

supenatant

washelution

M

Figure 79: Affinity binding experiment of TBS soluble mouse brain homogenate with the

immobilized anti-oligomer A11 antibody. Silver staining of 10% Tris-tricine gel separation of supernatant, wash and elution fractions (lanes 2, 3, 4) was performed, indicating affinity separation of high molecular weight products as bands at ~85 kDa and 120 kDa.

2.6.4.2 Online SAW-MS identification of in vivo oligomerization products of αSyn from mouse brain homogenate

Online SAW-ESI mass spectrometric analyses of the TBS soluble mouse

brain homogenate proteins were carried out to the immobilized anti-αSyn pC20

antibody. Figure 80a shows the SAW binding curve of the affinity interaction of

200 µg mouse brain homogenate Thy-1-human αSyn (A30P) and 200 nM anti-

αSyn pC20 antibody, and Figure 80b the ESI-MS analysis of the eluted antigen,

which provided multiply charged ions corresponding to a molecular mass of

14592.2 Da, which clearly differed from the expected mass for 3, αSyn (A30P),

14487.2 Da; the mass increase of 106 Da could be due to some

posttranslational modification during the preparation procedure of the brain

homogenates. Furthermore, identifications of methionine (M) oxidations in the

C-terminal domain of 3 (116Met, 127Met) were obtained (2.4.3.2 and 2.6.4.1).

SAW-ESI-MS applied to the direct analysis of αSyn (A30P) from mouse brain

extracts suggested this method as a powerful new tool to the molecular

characterization of intermediates of protein aggregation.


0 2500 5000 7500

0

10

20

30

40

50

Phas

e [d

eg]

Time [s]

654.4 695.8730.8769.1

811.7

859.3 913.0

973.7

1043.1 1123.5

0

1

2

3

4

600 700 800 900 1000 1100 1200 m/z

23+ 21+ 20+19+

18+

17+

13+14+

15+

16+

Glycine, pH 2Mouse brain

extractsEDC/NHS

149.0145°0.2893 µg cm-2

23.2°0.045 µg cm-2

PBSWater

pC20 Ab

Intensx105

Intensx105

Mw exp : 14592.2 DaMw calc : 14486.2 Da∆m = 106 Da

a

b

Figure 80: Affinity binding of mouse brain homogenates to anti-αSyn pC20 antibody by

SAW-ESI-MS: (a), SAW binding curve of the affinity interaction of 200 µg soluble mouse brain homogenate Thy-1-human αSyn (A30P) and 200 nM anti-αSyn pC20 antibody; (b), ESI mass spectrum of the eluted antigen shows the 13+ to 23+ charged molecular ions.

In a following SAW-ESI-MS experiment, the anti-αSyn monoclonal BD

antibody was immobilized on the chip surface. Upon injection of 150 µL (200

µg) of TBS soluble mouse brain homogenate, a mass loading of 36.5 ng cm-2

was observed. As previously performed, the interaction was investigated in PBS

as running buffer (pH 7.5). The affinity- bound antigen fraction was eluted under

acidic conditions, causing a reduction in surface mass loading of 15.7 ng cm-2

(Figure 81). The flow containing the eluted proteins, after passing through the

guard column, was directed into the ESI source of the mass spectrometer;

however no signal could be detected. Repeating the experiment using identical

conditions provided 28 ng cm-2 proteins bound to the immobilized antibody,

which also provided no MS signal, which might be due to insufficient amount of

sample or by the lack of antigen binding.


MALDIsolvent

200 nMBD antibody

Elution0.1 M HCl

0 5000 10000 15000 20000 25000

0

10

20

30

Phas

e [d

eg]

Time [s]

PBSWater

Mouse brainextracts

Mouse brainextracts

Elution0.1 M HCl

a) b) c)d)

Figure 81: Affinity binding of mouse brain homogenates to anti-αSyn mBD antibody by

SAW biosensor. The antibody was covalently immobilized on the NHS/EDC activated SAM on the chip’s surface by two injections of 200 nM anti-αSyn mBD antibody. After capping with ethanolamine, and buffer exchanging from water to PBS, the affinity interaction with 200 µg soluble mouse brain homogenate Thy-1-human αSyn (A30P) was investigated. The affinity bound proteins [a) 36.56 ng cm-2] were eluted with two HCl injections [b) -15.7 ng cm-2] and directed to the mass spectrometer. Since no signal could be detected, the experiment was repeated [c) 28.14 ng cm-2 bound protein and d) -7.25 ng cm-2 eluted proteins]. No MS signal could be detected.


2.7 Characterization of oligomerization-aggregation of synucleins and synuclein peptides

2.7.1 Oligomerization-aggregation analysis by gel electrophoresis and immunoanalytical methods

Many studies demonstrated that the mechanism of αSyn aggregation is a

nucleation-dependent process that can be induced by seeding with aggregated

αSyn as nuclei [166, 256]. Under these conditions, βSyn, which lacks 11 central

hydrophobic residues, failed to assemble into filaments [147]. Accordingly the

addition of either β- or γSyn in an equimolar ratio to αSyn significantly reduced

the fibrillization rate of αSyn and increased the duration of the lag-time. A

further increase in the relative concentration of either β- or γSyn (2:1 molar

ratio) led to additional inhibition of αSyn fibrillization; finally, the fibrillization was

completely inhibited at a fourfold molar excess over αSyn [311].

It has been reported that βSyn inhibited αSyn aggregation in an animal

model [312], suggesting that a possible factor in the etiology of PD could be a

decrease in the levels of β- or γSyn [311]. The stability of αSyn oligomerization-

aggregation products (S2) incubated with βSyn, in an equimolar ratio in 20 mM

PBS for 2 d at 37°C was investigated by gel electrophoresis and Western blot

analysis. Figure 82b shows the silver staining of the gel separation of αSyn

(lane 2), βSyn (lane 3) and αSyn incubated with βSyn in an equimolar ratio

(lane 4). αSyn formed dimers, oligomers and truncation products, while βSyn

(2) showed no oligomerization. Likewise, αSyn (1) incubated with 2 led to a

slightly inhibition of the αSyn fibrillization.


M 1 2 1:2

40 -50 -

30 -

25 -20 -15 -

kDa

10 -

60 -

85 -

200 -

b c1 2 1:2




1

51

101 140

a

Figure 82: (a), Schematic representation of the primary structure of 1, αSyn showing the central region on grey background, while the eleven central hydrophobic residues that are lacking in 2, βSyn are highlighted in a blue box. The cleavage site of the proteolytic fragment at V71-T72 identified by IMS-MS is indicated; (b), Silver staining of 15% Tris-tricine gel separation of 1, αSyn oligomerization-aggregation products (S2) – lane 2; 2 βSyn – lane 3; and αSyn oligomerization-aggregation products (S2) incubated with 2 in a molar ratio 1:1 – lane 4, after incubation in 20 mM PBS for 2 d at 37°C at a final concentration of 30 µM; (c), Western blot using anti-α/βSyn antibody diluted 1:3000 in PBS-T.

The identification of the proteolytic fragment αSyn (72-140) by IMS-MS

suggested that threonine at position 72 might play an important role in the

aggregation as a fragmentation- sensitive site. The interaction between αSyn

(59-80 T72A) (13), and αSyn (1) was first investigated by incubating the protein

(30 µM) with 13 at molar ratios of 1:1, 1:2 and 1:3 in 20 mM PBS for 2 days at

37°C. The mixtures were separated on a 10% Tris-tricine gel. The gel presented

in Figure 83a indicated separation of monomeric, oligomeric and degradation

products of αSyn. Simultaneously an identical gel was run and used for transfer

on a nitrocellulose membrane and immunoblotting with anti-αSyn pASY-1


antibody. In Figure 83b, lane 5 represents protein 1 incubated with peptide 13 in

1:3 molar ratio that showed an increased in oligomerization of wt-αSyn. In

Figure 83c and d the Dot blot results using anti-αSyn pASY-1 and anti-oligomer

A11 antibodies are shown.

1:13M S2 (1:1) (1:2) (1:3)

40 -50 -

30 -25 -20 -

15 -

kDa

10 -

80 -200 -

a1:13

M S2 (1:1) (1:2) (1:3)

40 -50 -

30 -25 -20 -

15 -

kDa

10 -

80 -200 -

b

c d1:13

S2 (1:1) (1:2) (1:3) 1:13

S2 (1:1) (1:2) (1:3)

Figure 83: Stability of αSyn oligomerization-aggregation products (S2) incubated with

peptide 13, αSyn (59-80 T72A) in a molar ratio 1:1, 1:2, 1:3 in 20 mM PBS for 2 days at 37°C: (a), 10% Tris-tricine separation of 10 µg oligomer-aggregate mixture; (b), Western blot using anti-αSyn pASY-1 antibody diluted 1:2500 in PBS-T; (c), Dot blot using anti-αSyn pASY-1 antibody; (d), Dot blot using anti-oligomer A11 antibody.

Subsequently, the stability of αSyn oligomerization-aggregation products (S3)

incubated with peptide 13, αSyn (59-80 T72A), in molar ratios of 1:10 and 1:100

was carried out in 20 mM NH4(Ac) for 2 days at 37°C. Figure 84a and b

suggested an increased oligomerization-aggregation rate of wt-αSyn, by

formation of large aggregates recognized specifically by the anti-αSyn pASY-1

antibody.


1:13M S3 (1:10) (1:100)

40 -50 -

30 -

25 -20 -

15 -

kDa

10 -

80 -200 -

a b

c d1:13

S3 (1:10) (1:100)

1:13M S3 (1:10) (1:100)

40 -50 -

30 -

20 -

15 -

kDa

1:13S3 (1:10) (1:100)


peptide 13, αSyn (59-80 T72A) in a molar ratio 1:10 and 1:100 in 20 mM NH4(Ac) for 2 days at 37°C: (a), 10% Tris-tricine separation of 10 µg oligomer-aggregate mixture; (b), Western blot using anti-αSyn pASY-1 antibody diluted 1:2500 in PBS-T; (c), Dot blot using anti-αSyn pASY-1 antibody; (d), Dot blot using anti-oligomer A11 antibody.

The stability of αSyn oligomerization-aggregation products incubated with

αSyn (59-80 T72A) in molar ratios of 1:10 and 1:100 in 50 mM PBS for 7 days

at 25°C was further investigated by gel electrophoresis, Western blot and Dot

blot. The lane 4 in Figure 85b revealed only a small band of monomer

suggesting that at increased incubation time and with excess of peptide 13, the

protein forms insoluble aggregates that are not entering in the stacking gel. Dot

blot analyses of anti-αSyn specific antibody and anti-oligomer A11 antibody

(Figure 85c and d) showed the presence of αSyn oligomers, and these data are

supported by data obtained by IMS-MS (Figure 87; 2.7.2).


1:13M S5 (1:10) (1:100)

40 -50 -

30 -25 -20 -

15 -

kDa

10 -

70 -

a b1:13

M S5 (1:10) (1:100)

50 -

20 -

15 -

kDa

c d1:13

S5 (1:10) (1:100)1:13

S5 (1:10) (1:100)


peptide 13, αSyn (59-80 T72A) in a molar ratio 1:10 and 1:100 in 50 mM PBS for 7 days at 25°C: (a), 10% Tris-tricine separation of 10 µg oligomer-aggregate mixture; (b), Western blot using anti-αSyn pASY-1 antibody diluted 1:2500 in PBS-T; (c), Dot blot using anti-αSyn pASY-1 antibody; (d), Dot blot using anti-oligomer A11 antibody.

2.7.2 Oligomerization-aggregation analysis by ion-mobility mass spectrometry

Ion-mobility mass spectrometry studies were conducted to identify in vitro

αSyn aggregation products, distinguishing them by size, charge, and shape, as

well as mass. All analyses were performed on a Synapt HDMS Q-TOF-MS

system as described in detail in 2.3.3.1. Ions were generated by nano-

electrospray ionization, guided through the desolvation and quadrupole regions

of the instrument, and trapped by the T-wave device. Figure 86 shows the plot

of drift time vs. m/z for RP-HPLC purified αSyn (59-80 T72A), illustrating the

multidimensional nature of the data produced by IMS-MS. Ions within the

spectrum can be assigned to 2+ and 3+ charge states of 13 monomer.

Additionally, ions having higher charge (3+) experienced greater separation

field strengths and traversed the chamber more quickly. The mass spectrum of


13 is presented in Figure 86b, showing the formation of [M+3H]3+ and [M+2H]2+

ions, with a most intensive peak at 709.3 (3+). The mass accuracy between the

calculated and experimental average mass was determined to ca. 84 ppm. This

result ascertained the primary structure and homogeneity of αSyn (59-80 T72A)

(13).

[M + 2H] 2+

[M + 3H] 3+

1063.5

709.3

a

b

Mw exp : 2125.0 DaMw calc : 2125.18 Da∆m = 84 ppm

2000

1500

1000

500

100

%

0700 800 900 1000 1100 1200 m/z

4.51 9.03 13.54Drift Time (milli secs)

m/z

13, α-Syn (59-80 T72A)

[M + 2H] 2+

[M + 3H] 3+

Figure 86: (a), Ion-mobility drift time (driftscope data view) of the prepared 13, αSyn

(59-80 T72A); (b), The mass spectrum extracted from driftscope of peptide is showing the formation of [M+3H]3+ and [M+2H]2+ ions. The mass accuracy between the calculated and experimental average mass was 84 ppm.

Finally, the αSyn oligomerization-aggregation products (S5) incubated with

peptide 13, αSyn (59-80 T72A) in molar ratios of 1:10 and 1:100 for 7 days

were analyzed by IMS-MS. The ion-mobility drift times and the extracted mass

spectra of αSyn oligomerization-aggregation products (S5) incubated with

peptide 13, are shown in Figure 87. Signals corresponding to [M+3H]3+ and

[M+2H]2+ ions of peptide 13 were observed, while the monomer 1 was not

detected. The nano-spray capillary became blocked after approximately 2 min,

presumably due to the formation of large aggregates. The comparison of the

ion-mobility plots of the freshly prepared 13 solution (Figure 86) and those of


the incubated mixtures of 1 and 13 (Figure 87) revealed a shorter drift time for

the ions after incubation that exhibit a compact structure, more prone to form

aggregation products, as previously observed in literature [281]. Furthermore,

these results are in agreement with the oligomerization-aggregation stability

studies by Tris-tricine gel electrophoresis and immunoblotting illustrated in

Figure 85.

1400

1200

1000

800

600

m/z

1400

1200

1000

800

600



1:13, 1:10 1:13, 1:100

600 800 1000 1200 1400 600 800 1000 1200 1400 m/z

100

%

0

1:13, 1:10 1:13, 1:100

709.3

1063.5

709.3

1063.5[M + 2H] 2+

[M + 3H] 3+

[M + 2H] 2+

[M + 3H] 3+

a

b100

%

0

Figure 87: (a), Comparison of ion-mobility drift times (drift scope data view) of αSyn

oligomerization-aggregation products (S5) incubated with peptide 13, αSyn (59-80 T72A) in a molar ratio 1:10 and 1:100 in 50 mM PBS for 7 days at 25°C; (b), The mass spectra indicate the presence of [M+3H]3+ and [M+2H]2+ ions of peptide 13. The protein 1 was not observed in either 1:10 or 1:100 incubations because the sprayer was blocked after approximately 1 to 2 min, due to formation of insoluble products.

2.7.3 Oligomerization-aggregation analysis by Thioflavin-T assay

Thioflavin-T is a fluorescent dye that interacts with protein fibrils with a

characteristic increase in fluorescence intensity around 480 nm [313, 314]. αSyn

forms in vitro amyloid fibrils that are similar to those extracted from human brain

LBs [165]. The rate of formation of these fibrils is accelerated by the αSyn (A53T)


mutation [116, 166, 315], while the A30P mutation causes an increase in fibrillization

rate [116, 166]. The lack of a correlation between the PD-causing mutations and

the acceleration of fibril formation suggests that the fibril is not a direct

pathogenic entity in PD.

For thioflavin assays, purified wt-αSyn and αSyn (A53T), αSyn (A30P)

proteins were dissolved in 20 mM PBS up to concentrations of 30 µM and

incubated at 37°C with agitation in a microcentrifuge tube. As a control, a blank

was provided with 25 µM ThT in water. Determinations were carried out in 96-

well plates (black; black bottom) using a fluorescence plate reader (Victor2;

Perkin Elmer), and fluorescence was measured at an excitation of 450 nm and

emission of 486 nm. Fibrillization patterns of wt-αSyn, αSyn (A53T) and αSyn

(A30P) proteins monitored by increase in ThT fluorescence have been

previously reported [316], and it was shown that aggregation rates increase in the

order αSyn (A30P) < wt-αSyn < αSyn (A53T) [316]. These data were confirmed

by the results depicted in Figure 88.

0

25

50

75

100

1, (0 d) 1, (2 d) 3, (0 d) 3, (2 d) 4, (0 d) 4, (2 d)

450 F

486

Figure 88: Thioflavin-T assay of αSyn proteins. The aggregation rate increases in the

following order αSyn (A30P) < wt-αSyn < αSyn (A53T).

Figure 88 compares the fibrillization patterns of αSyn proteins monitored

by the increase in ThT fluorescence. The mutant (A53T) (4) showed the fastest

rate of fibril formation, whereas αSyn (A30P) (3) formed fibrils with a

considerably slower rate than A53T, and even slower compared to wt-αSyn.


M 1 3 4 1 3 4(2 d) (2 d) (2 d) (7 d) (7 d) (7 d)

40 -50 -

30 -25 -20 -15 -

kDa

10 -

60 -

85 -

200 -

M 1 3 4 1 3 4(2 d) (2 d) (2 d) (7 d) (7 d) (7 d)

40 -50 -

30 -25 -20 -15 -

kDa

10 -

60 -

85 -

200 -(αSyn)n

(αSyn)1

(αSyn)2

∆N-αSyn

ba

dcM 1 3 4 1 3 4

(2 d) (2 d) (2 d) (7 d) (7 d) (7 d)

40 -

20 -15 -

kDa

(αSyn)n

(αSyn)1

(αSyn)2

∆N-αSyn

M 1 3 4 1 3 4(2 d) (2 d) (2 d) (7 d) (7 d) (7 d)

40 -

20 -15 -

kDa

(αSyn)n

(αSyn)1

(αSyn)2

Figure 89: 10% Tris-tricine separation of 1, αSyn, 3, αSyn (A30P) and 4, αSyn (A53T)

proteins incubated in 20 mM PBS at 37°C, with shaking at a final concentration of 30 µM: (a), Gel reader for native fluorescence detection showing the monomeric bands of proteins; (b), Coomassie staining; (c), Western blot using anti-αSyn mBD antibody diluted 1:500 in PBS-T; (d), Western blot using anti-αSyn pC20 antibody diluted 1:500 in PBS-T.

To analyze the aggregation process of αSyn truncation and degradation

in detail, a time-dependent aggregation study of, wt-αSyn (1) and 10, αSyn (72-

140) was performed using the ThT fluorescence assay [300]. For this purpose

purified protein 1 and peptide 10 were dissolved in 20 mM PBS pH 7.5 and

incubated at 37°C for several days, respectively, and both 1 and 10 analyzed by

gel electrophoresis (Figure 90a). The monomer (1a) and dimer (1b) of αSyn (1)

were separated; furthermore truncations of 1 were observed which correspond

to the proteolytic truncation fragment 10 (10a). The oligomers verified were not

oligomerization products of the full-length protein 1 but can be assigned as


oligomerization products of peptide 10 (10b; 10d), consistent with the IMS-MS

data of αSyn. The ThT assay was measured with an excitation at 450 nm and

emission at 486 nm, and data analyzed with Graph Pad Prism wherein a blank

was subtracted from each time point and subsequently the sigmoidal increase

of the ThT fluorescence upon fibril formation analyzed (Figure 90b). Under

these conditions the proteolytic peptide 10 takes approximately 48 h incubation

time to reach a steady-state of aggregation as observed by Thioflavin-T

fluorescence, indicating that truncated peptide 10 ( ) mature into amyloid-fibrils

much faster than the full-length protein 1 (•).

M αSyn αSyn 72-1401 10

10d

10a

10b

10c

10d

10a

10b

1b

1a

akDa200 -150 -120 -85 -70 -60 -

40 -

50 -

30 -

25 -

20 -

15 -

10 -


10

1

b

0 20 40 60 80 100Incubation time [h]

56000

48000

4000032000

24000

16000

8000

0

450 F

486

Figure 90: (a), 10% Tris-tricine gel electrophoresis of in vitro aggregation products of 1,

wt-αSyn and 10, αSyn (72-140) stained with silver nitrate. In lane 2 is shown the monomer 1a, dimer 1b as well as the truncation product 10a formed by protein 1. The bands at ~ 27 kDa and 60 kDa are aggregation products 10b and 10c of peptide 10; lane 2: monomer 10a, dimer 10b, oligomer 10c and 10d were formed by peptide 10; (b), Fibrillization kinetics of wt-αSyn protein 1 (•) and αSyn peptide 10 ( ). Each sample at a concentration of 30 µM was incubated in 20 mM PBS at 37°C with agitation. The fluorescence of ThT was measured with an excitation at 450 nm and emission at 486 nm.

2.7.4 Characterization of oligomerization-aggregation of chemically modified α-synuclein

In order to investigate the role of charge on the stability of the folded

native protein conformation via succinylation, comparative stability studies of

αSyn and αSynsuccin. were performed. The proteins were incubated for 7 days at

37°C as previously described (see 2.2.1.1) and then separated by Tris-tricine

gel electrophoresis and transferred to a nitrocellulose membrane. These studies

showed that succinylated αSyn does not form any oligomers or truncation

products within 7 days as shown in Figure 91. By Western blot using anti-αSyn

pASY-1 antibody, a loss of immunological reactivity caused by the chemical

modification was observed, which indicated a significant conformational change

of the succinylated αSyn.


M S5 S5succin S5 S5succin(7d) (7d) (7d) (7d)

M S5 S5succin S5 S5succin(7d) (7d) (7d) (7d)kDa

40 -50 -

30 -25 -20 -

15 -

10 -

ba

30 µM 7 µM 30 µM 7 µM

(αSyn)n

(αSyn)1

(αSyn)2

∆N-αSyn

(αSyn)1

(αSynsuccin)1

(αSyn)2

(αSynsuccin)2

∆N-αSyn

Figure 91: Effects of chemical modification of αSyn on the oligomerization-aggregation

pathway: (a), Tris-tricine separation of αSyn and αSynsuccin. incubated for 7 d at 37°C. αSynsuccin. incubated for 7 d shows no oligomer or truncation products formation; (b) Western blot analysis using anti-αSyn pASY-1 antibody.


3 EXPERIMENTAL PART

3.1 Materials and reagents

In this work the following commercially available materials were used:

PROTRAN nitrocellulose membrane: Whatman (Germany); 96 well Optiplate

black: Perkin Elmer; Low binding test tubes: Sigma Aldrich; Omix Pipette Tips

C18: Varian, Inc.; Fuji Super RX: Fujifilm.

In this work the following commercially available reagents were used:

NHS-activated Sepharose, Coomassie Brilliant Blue G250, Ponceau S (3-

hydroxy-4-[2-sulfo-4-(4-sulfophenylazo) phanylazo]-2,7-naphtalindisulphone

acid), Tween 20 (Polyoxyethylensorbitanmonolaureat), diethanolamine (min

98%), α-cyano-4-hydroxycinamic acid (HCCA): Sigma; deionized water:

Millipore; Hydrochloric acid (37% p.a.), sodium hydroxide (99%): Merck,

endoprotease Glu-C from Staphylococcus aureus: Sigma; Trypsin: Promega, T-

buthylmethyleter (99%), dimethylformamide (DMF, 99.8%), N-Methylmorpholine

(NMM 99.5% p.a.), piperidine (99%), trifluoroacetic acid (TFA, 99% p.a.),

triethylsilane (97%), DHB (di-hydroxy benzoic acid): Fluka. All amino acids (N-α-

protected amino acids), resins and activators (PyBOP) used for peptide

synthesis: NovaBiochem.

The reagents were of analytical grade or highest available purity.

3.1.1 Buffers and stock solutions

The following solutions were prepared in deionized water (MilliQ) and

summarized in the next table:


Table 18: Composition of buffer and stock solutions

Buffer / Solution Composition

1xCathode Buffer 100 mM Tris

100 mM Tricine

0.1% SDS

10xAnode Buffer 2 M Tris

pH 8.96

Tris-HCl/ SDS 3 M Tris

0.3% SDS

pH 8.45

2xTricine sample buffer 100 mM Tris pH 6.8

24% Glycerol

8% SDS

0.02% Coomassie brilliant blue G-250

SDS-PAGE sample buffer 4% SDS

25% Glycerin

50 mM Tris

0.02% Coomassie

6M Urea

pH 6.8.

SDS-PAGE separation gel 0.5 M Tris

0.4% SDS

pH 6.8

SDS-PAGE stacking gel 1.5 M Tris

0.4% SDS

pH 8.8

SDS-PAGE running buffer 25 mM Tris

192 mM glycine

0.1% SDS

Buffer A 10% NH4SO4

2% H3PO4

Coomassie Blue Solution 20% methanol

0.1% Coomassie brilliant blue G-250

in Buffer A

Fixing solution 30% ethanol

10% acetic acid

Incubation solution 11.2% CH3COONa x 3 H2O



30% ethanol

Prior to use: 0.2% Na2S2O3 x 5 H2O

Silvering solution 0.2% AgNO3

Prior to use: 0.02% CH2O

Developing solution 2.5% Na2CO3

Prior to use: 0.01% CH2O

Formaldehyde 37% CH2O

Blot buffer 25 mM Tris

192 mM Glycin

20% methanol

0.1% SDS

pH 8.3

PBS-T 81 mM Na2HPO4 x 2 H2O

21 mM NaH2PO4 x H2O

100 mM NaCl

0.05% Tween 20

pH 7.5

20 mM PBS 20 mM Na2HPO4 x 2 H2O

0.03% NaN3

pH 7.5

50 mM PBS 50 mM Na2HPO4 x 2 H2O

20% ethanol

pH 7

Coupling buffer 500 mM NaCl

200 mM NaHCO3

pH 8.3

Blocking buffer 500 mM NaCl

100 mM Ethanolamine

pH 8.3

Washing buffer 500 mM NaCl

200 mM CH3COONa x 3 H2O

pH 4

PBS ELISA 5 mM Na2HPO4 x 2 H2O

150 mM NaCl

pH 7.5

PBS ELISA-Tween 0.05% Tween-20 in PBS ELISA

Citrate/phosphate 100 mM C6H8O7 x H2O



200 mM Na2HPO4 x 2 H2O

Solvent A 0.1% TFA in H2O

Solvent B 80% ACN

0.1% TFA in H2O

MALDI solvent ACN/ 0.1% TFA in H2O (2:1)

3.1.2 Proteins

The recombinant proteins and peptides employed in this work are

summarized in Table 19.

Table 19: Recombinant proteins/ peptides

Type Source

Recombinant human αSyn Boehringer Ingelheim, Biberach

Recombinant human αSyn Rpeptide

Recombinant human αSyn (A30P) Rpeptide

Recombinant human αSyn (A53T) Rpeptide

Recombinant human αSyn (61-140) Rpeptide

Recombinant human αSyn (96-140) Rpeptide

Recombinant human αSyn (1-120) Prof. M. Leist, Department of Biology, University of Konstanz

Recombinant human αSyn (72-140) Prof. M. Leist, Department of Biology, University of Konstanz

Recombinant human βSyn Rpeptide

3.1.3 Antibodies

The antibodies employed in the affinity studies are presented in Table 20.

Table 20: Primary and secondary antibodies

No Antibody Company Immunogen Host

1 Anti-α/βSyn LifeSpan BioSciences

Rec h* α and βSyn produced in E. coli

mouse


No Antibody Company Immunogen Host

2 Anti-αSyn m4B12 GeneTex Rec h αSyn produced in E. coli

mouse

3 Anti-αSyn mBD BD Transduction Laboratories

Rat αSyn aa. 15-123 mouse

4 Anti-αSyn pC20 Santa Cruz epitope mapping at the C-terminus of αSyn of human origin

rabbit

5 Anti-αSyn pASY-1 Provided by Boehringer Ingelheim

Rec h αSyn produced in E. coli

rabbit

6 Anti-oligomer A11 Invitrogen Synthetic molecular mimic of soluble oligomers

rabbit

7 Goat anti-mouse IgG alkaline phosphatase conjugate

Jackson ImmunoResearch

__ mouse

8 Goat anti-mouse IgG alkaline phosphatase conjugate

Jackson ImmunoResearch

__ rabbit

* human

3.2 Sample preparation for proteome analysis

3.2.1 Expression of recombinant α-synuclein

Expression and purification of αSyn were performed by Dr. Karin Danzer

(Boehringer Ingelheim, Biberach, Germany) as previously described [250]. pET-

5a/αSyn wild type plasmid was used to transform Escherichia coli BL21 (DE3)

pLysS (Novagen, Madison, WI). Expression was induced with isopropyl-β-D-

thiogalactopyranose (Promega, Mannheim, Germany) for 4 h. Cells were

harvested, resuspended in 20 mM Tris and 25 mM NaCl, pH 8.0, and lysed by

freezing in liquid nitrogen followed by thawing. After 30 min of boiling, the lysate

was centrifuged at 17600 x g for 15 min at 4°C. Supernatant was filtered

through a 0.22 µm filter (Millex-GV; Millipore, Bedford, MA) before being loaded

onto a HiTrap QHP column (5 mL; Amersham Biosciences, Munich, Germany)

and eluted with a 25 - 500 mM salt gradient. The pooled αSyn peak was

desalted by Superdex 200 HR 10/30 size exclusion column (Amersham

Biosciences), concentrated using Vivaspin columns (molecular weight cutoff

(MWCO), 5 kDa Vivascience, Stonehouse, UK) and equilibrated to water. The


protein concentration was determined using a BCA protein quantification kit

(Pierce, Rockford, IL). Aliquots were lyophilized and stored at - 80°C.

3.2.2 α-Synuclein oligomers preparation

Preparation of αSyn oligomerization-aggregation products was

performed by long incubation procedure [250] in a variety of conditions using

buffer systems and incubation time summarized in Table 21. αSyn oligomers

type S2 were obtained by dissolving the lyophilized αSyn in 20 mM PBS, 0.03%

NaN3 (pH 7.5) [257] up to 30 µM, corresponding to the range of the reported

concentration of αSyn found in the cytoplasm of neurons [247], followed by

shaking at 37°C. Moreover, αSyn oligomers S3, S4 were prepared by dissolving

recombinant protein in 20 mM ammonium acetate (NH4Ac) and 20 mM

ammonium acid carbonate (NH4HCO3) followed by incubation at 37°C with

shaking for different periods of time. αSyn oligomers type S5 were prepared by

dissolving protein in 50 mM sodium phosphate buffer (pH 7.0) containing 20%

ethanol to a final concentration of 7 µM [250]. In case of oligomers type S6, 10

µM of FeCl3 were additionally added, whereas oligomers type S5 were prepared

without adding FeCl3. After 4 h of shaking, both types of oligomers were re-

lyophilized and resuspended in 50 mM sodium phosphate buffer (pH 7.0)

containing 10% ethanol with a half of starting volume. This was followed by

incubation for 7 days at room temperature. After incubation, αSyn oligomers

were used for characterization studies by gel electrophoresis, immunoanalytical

and mass spectrometric methods.

Table 21: Preparation of αSyn aggregation products:

No Buffer Time (days) Concentration Temperature

S1 water 0 30 37°C

S2 20 mM PBS 2 30 37°C

S3 20 mM NH4Ac 2 30 37°C

S4 20 mM NH4HCO3 2 30 37°C

S5 50 mM PBS 7 7 25°C

S6 50 mM PBS, 10 µM FeCl3 7 7 25°C


3.2.3 Succinylation of amino groups on α-synuclein

Chemical modification of proteins with succinic anhydride has been

successfully used in the study of various aspects of proteins [229]. Succinic

anhydride reacts with the ε-amino groups of lysine and the amino-N-terminal α-

amino group of proteins, in their non-protonated forms, converting them from

basic to acid groups (Figure 92). Succinylation of αSyn was performed in 0.3 M

sodium phosphate buffer, pH 7.5. Increasing amounts of (1 mg mL-1) succinic

anhydride solution dissolved in DMSO (0.5, 1, 2, 10, 50, 100-fold molar excess

with respect to protein) were added to a continuously stirred protein solution (1

mg mL-1), over a period of 90 min with gentle stirring, and the pH of the reaction

mixture was maintained by adding 0.2 M NaOH [303]. The reaction was allowed

to proceed at room temperature (25°C) for 30 min. Then, the protein solution

was thoroughly dialyzed against water for 2 h and subjected to gel

electrophoresis and mass spectrometric analysis.

HO

HO

O

O

H2O

O

O

O

NH

R

H

HN

O

O

OH

R

Succinic acid Succinic anhydride Figure 92: Succinylation of ε-amino groups of lysine and the amino-N-terminal α-amino

group of proteins.

3.2.4 Preparation of mouse brain homogenates

Generation of Thy-1-human (A30P) αSyn transgenic mice has been

described elsewhere [261, 266-268]. Thy1 is a neuron specific promoter that was

introduced to drive over-expression of human, mutated (A30P) αSyn in a

transgenic animal model for Parkinson’s disease. The two mice employed in the

present work, showing a progressive deterioration of locomotors function, were


sacrificed by cervical dislocation. The most important details related to them are

summarized in Table 22.

Table 22: Thy-1-human (A30P) αSyn transgenic mice employed in the study:

Mouse code Sex Birth (y/m/d)* Age (months) Death (y/m/d)

Syn-m-130 m 07/10/17 18.7 09/05/06

Syn-m-131 m 07/10/17 18.7 09/05/06

* (y/m/d) year/ month/ day

After sacrificing, mice brains were harvested and divided into four parts:

forebrain left (FB/L) / right (FB/R), hindbrain left (HB/L)/ right (HB/R) (Figure 93).

αSyn (A30P) protein was extracted from mouse brain tissue using multiple

steps with five different buffers (Table 23). First, 300 µL buffer 1 (TBS plus one

tablet complete protease inhibitor) were added to the brain pieces which were

thereafter crash with a tissue- ruptor for 1 min on ice. The brain mixture was

than rinsed with 200 µL buffer 1 and centrifuged at 120000 x g, for 30 min., at

4°C. The soluble fraction was used as elution 1 (E1: TBS soluble fraction).

Further, the pellet was resuspended in 500 µL buffer 2 (TBS- 1% Triton X-100

plus one tablet of protease inhibitor cocktail), employing a 1 mL syringe and a

needle. After centrifugation (120000 x g, 30 min, 4°C), the soluble fraction was

used as elution 2 (E2) and the pellet was resolubilized in 500 µL buffer 3 (TBS–

1M sucrose plus one tablet of protease inhibitor cocktail) using the same

procedure as in the previous step. Further, the soluble fraction resulted after

centrifugation (120000 x g, 30 min, 4°C) was used as elution 3 (E3), while the

pellet was dissolved in 500 µL buffer 4 (RIPA buffer plus one tablet of protease

inhibitor cocktail). The last centrifugation step provided the elution 4 (E4) and

pellet P5 which, after being brought in solution with a 6 M urea/ 5% SDS buffer,

was also employed as the last αSyn containing fraction. The BCA-assay for

protein quantification of mouse brain fractions containing the TBS, TBS-Triton,

TBS-sucrose, RIPA and urea-soluble proteins was carried out.


E1 (TBS-soluble)P

120000 x g30 min, 4°C

E2 (TBS-Triton soluble)P

E3 (TBS-sucrose soluble)P

E4 (RIPA-soluble)P

P5 (Urea/ 5% SDS-extract)

FB/L

HB/L

FB/L FB/R

HB/L HB/R

FB/L

HB/L

FB/L FB/R

HB/L HB/R

120000 x g30 min, 4°C

120000 x g30 min, 4°C

120000 x g30 min, 4°C

Extracted

brainDivided

brain

Figure 93: Schematic representation of the subcellular fractionation steps in isolation of

αSyn oligomerization-aggregation products from human αSyn (A30P) transgenic mouse brain homogenates. The brains are harvested and divided into four parts: forebrain left (FB/L) / right (FB/R), hindbrain left (HB/L)/ right (HB/R). The soluble fractions (elution 1 ÷ 4) are obtained after centrifugation at 120000 x g for 30 min at 4°C per elution (E). The pellet (P) 5 is mixed with Urea/ 5% SDS; the insoluble proteins are extracted.

Table 23: αSyn oligomerization-aggregation products containing fractions extracted from human αSyn (A30P) transgenic mice brain:

Mouse Brain regions

Elution 1

(TBS)

Elution 2

(TBS-Triton)

Elution 3

(TBS-sucrose)

Elution 4

(RIPA buffer)

Pellet 5

(Urea/SDS)

Syn-m-130 HR/R E1.1 E2.1 E3.1 E4.1 P5.1

HR/L E1.2 E2.2 E3.2 E4.2 P5.2

FB/R E1.3 E2.3 E3.3 E4.3 P5.3

FB/L E1.4 E2.4 E3.4 E4.4 P5.4

Syn-m-131 HR/R E1.5 E2.5 E3.5 E4.5 P5.5

HR/L E1.6 E2.6 E3.6 E4.6 P5.6

FB/R E1.7 E2.7 E3.7 E4.7 P5.7

FB/L E1.8 E2.8 E3.8 E4.8 P5.8


3.2.5 Human SH-SY5Y-A53T neuroblastoma cell culture

The human neuroblastoma cell line SH-SY5Y is widely used as a neural

cellular model system. The cells were prepared into a collagen flask using 500

mL cell growth medium Dulbecco’s modified Eagle’s containing high glucose

(DMEM/F-12+GlutaMax) supplemented with 75 mL of 15% fetal calf serum

(FCS), 4 mM glutamine and incubated at 37°C in the presence of 5% CO2. The

medium was changed every two day three times in a row. Then the medium

was removed and the cells were washed with 5 - 10 mL PBS in order to remove

the serum. Next, 2.5 mL trypsin was added for 3 min; 45 mL medium was

inserted into two new collagen flasks. The cells were washed with 7.5 mL

medium, split in two and incubated at 37°C in the presence of 5% CO2. After 24

h the confluence of the cells was approximately 65%. Then medium was

removed and the cells were washed with 2 x 5 mL PBS. Further, SH-SY5Y cells

were scraped and washed by centrifugation in cold PBS. The cells were

resuspended in 800 µL Triton-lysis buffer (50 mM HEPES, 150 mM NaCl, 1.5

mM MgCl, 1 mM EDTA, 10% glycerol, 1% Triton-X 100, pH 7.5) with one tablet

of protease inhibitor cocktail and incubated on ice for 15 min. After

centrifugation at 1500 x g, 10 min., 4°C protein concentration of supernatant of

3296 µg mL-1 was quantified using BCA assay. Lysates (10 µg of protein) were

resolved by electrophoresis on 4-12% Bis-Tris gradient gel (NuPAGE,

Invitrogen) using NuPAGE 2-(N-morpholino) ethanesulfonic acid (MES) buffer.

After transfer the nitrocellulose membrane is boiled for 5 min in PBS. Then the

membrane was blocked in Odyssey Blocking Buffer for 1 h at 25°C. The blot

was probed with anti-αSyn mBD antibody diluted 1:500 in Odyssey Blocking

Buffer for 1 h, 25°C or overnight at 4°C. Bands were detected using alkaline

phosphatase-conjugated goat anti-mouse 1:15000 (800 nm) secondary

antibody for 1 h at 25°C in dark and imaged with Odyssey Infrared Imager (Li-

COR Biosciences).


3.2.6 BCA assay for protein quantification

BCA Protein Assay is a detergent-compatible formulation based on

bicinchoninic acid (BCA) for the colorimetric detection and quantitation of total

protein. First, a series of seven dilutions of known concentration are prepared

from the 2 mg mL-1 bovine serum albumine (BSA) and assayed alongside the

unknown(s) before the concentration of each unknown is determined based on

the standard curve. Then, the unknown samples are diluted 1:10 in the

corresponding Triton lysis buffer (50 mM HEPES, 150 mM NaCl, 1.5 mM MgCl,

1 mM EDTA, 10% Glycerol, 1% Triton X-100, 100 mM NaF, 10 mM

pyrophosphat, protease inhibitor cocktail, pH 7.5). In the next step, the BCA –

mixture of reagent A (sodium carbonate, sodium bicarbonate, bicinchoninic acid

and sodium tartrate in 0.1 M sodium hydroxide) plus reagent B (4% cupric

sulfate) in a ratio 50:1 is prepared. Further, 25 µL of standards and 25 µL of

diluted samples in duplicates are added to ELISA 96 well plate. Additionally,

175 µL of BCA-Mix are added and incubate 30 min at 37°C (Table 24).

Table 24: Preparation of diluted BSA standards (working range = 125 - 2000 µg mL-1):

Concentration (µg mL-1) BSA stock (µL) Lysis buffer (µL)

2000 300 0

1500 (A) 375 125

1000 (B) 325 325

750 (C) 175 (A) 175

500 (D) 325 (B) 325

250 (E) 325 (D) 325

125 (F) 325 (E) 325

0 (G) 0 400

The optical density is read at 562 nm wavelength in the Farcyte reader (Tecan).

The standard curve is performed by plotting the average Blank-corrected 562

nm measurement for each BSA standard vs. its concentration in μg mL-1. The

standard curve is used to determine the protein concentration of each unknown

sample.


0 300 600 900 1200 1500

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0

BSA concentration (µg mL-1)

OD

(562

nm

)

Figure 94: BSA standard curve obtained by plotting the OD (562 nm) measurement for

each BSA standard vs. its concentration in μg mL-1.

3.2.7 Acetone precipitation for removal of contaminants

For the gel electrophoresis and affinity analysis of mouse brain

homogenate samples, acetone precipitation was used (for removal of salts and

contaminants). The protein content was precipitated at -28°C for 3 h by adding

6 volumes of ice-cold acetone to the sample. After 20 min centrifugation at

14926 × g the residual acetone was removed and the obtained pellet was

allowed to dry.

3.3 Solid phase peptide synthesis

Solid phase peptide synthesis (SPPS) is a fast approach to synthesizing

peptides and small proteins. The C-terminal amino acid is attached to a solid

support via an acid labile bond with a linker molecule shown in Figure 95 [296].

This resin is insoluble in the solvents used for synthesis, making it relatively

simple and fast to wash away excess reagents and by-products.


Polystyren matrix

O CH 3

H 3C O

N H 2

O C O NH C H2C H 2 PEG

Figure 95: Novasyn-TGR-resin for solid phase peptide synthesis. PEG is the

polyethylene glycol spacer between the resin and the polystyrene matrix.

The solid support is a synthetic polymer with reactive groups which react

covalently with the carboxyl group of an N-α-protected amino acid. The amino

protecting groups can then be removed and a second N-α-protected amino acid

can be coupled to the attached amino acid. These steps are repeated until the

desired sequence is obtained. The N-α-protecting group (Fmoc) is then

removed and the amino acid-linker-support thoroughly washed with solvent.

The side chains of more reactive amino acids are blocked or "protected" with

either a tertiary-butyl or a trityl functional group hindering their chemical activity.

αSyn peptide sequences were synthesized with a semi-automated

peptide synthesizer EPS 221 (Abimed, Germany), using the Fmoc strategy [297,

317]. The main steps used for the synthesis cycles of αSyn peptides were: i)

removal of Fmoc group with 20% piperidine in DMF; as side products

dibenzofulvene and carbon dioxide were split of and removed from the sample

by washing with DMF; the reaction time of deprotection was 5 min, ii) washing

with 8 mL DMF followed by coupling of the next amino acid; after the activating

(0.9 M PyBOP, 1.3 M NMM) in DMF solution activated the next Fmoc-amino

acid and it was coupled for 30 min, iii) washing with 8 mL DMF, iiii) after the last

synthesis cycle the N-terminus of the peptide was deprotected by treatment with

a solution containing 20% piperidine in DMF for 5 min. The resin was then

washed with 8 mL DMF and 10 mL ethanol and dried under vacuum. Cleavage

of the peptide from the resin together with the final deprotection of the side

chains was performed with a mixture of trifluoroacetic acid / triethylsilane / water

(95: 2.5: 2.5, v/v/v) for 2 h at room temperature. The peptide was then

precipitated with 40 mL cold tert-butyl-methylether. The resin and the crude


peptide were separated by filtration using 10% acetic acid and then the peptide

collected, frozen with liquid nitrogen and lyophilized.

ONH CH C A

R3

FmocPG

ONH CH C OH

R3

FmocPG

Protected amino acid

Activated amino acid

Activation

Repeat deprotectionand coupling

Protected peptide chain

ONH CH C NH

R2

FmocPG

OCH C OR1PG

Deprotection

ONH2 CH C NH

R2PG

OCH C OR1PG

Deprotected peptide chain

Coupling


ONH CH C NH

R3

FmocPG

OCH C NHR2PG

OCH C OR1PG

Linker Resin

ONH CH C A

R3

FmocPG

ONH CH C OH

R3

FmocPG



Activation

ONH CH C A

R3

FmocPG

ONH CH C OH

R3

FmocPG



Activation

Repeat deprotectionand couplingRepeat deprotectionand couplingRepeat deprotectionand coupling


ONH CH C NH

R2

FmocPG

OCH C OR1PG

Deprotection

ONH2 CH C NH

R2PG

OCH C OR1PG

Deprotected peptide chain

Coupling


ONH CH C NH

R3

FmocPG

OCH C NHR2PG

OCH C OR1PG

Linker Resin Figure 96: General scheme of solid phase peptide synthesis: in the first step

deprotection of the Fmoc group takes place and then the first protected amino acid is coupled onto the resin. After the last synthesis cycle the N-terminus of the peptide is deprotected and the resin is washed and afterwards the peptide is cleaved of the resin. Side chain protecting groups used for the specific amino acids were:

Nα-Fmoc-L-Alanine(Fmoc-Ala-OH), Nα-Fmoc-NG-2,2,3,7,8-pentamethylchroman-6-sulfonyl-L-Arginine (Fmoc-Arg(Pmc)-OH), Nα-Fmoc-Nβ-trityl-L-Asparagine, (Fmoc-Asn(Trt)-OH), Nα-Fmoc-L-Aspartic-acid-β-t-butylester (Fmoc-Asp(OtBu)-OH), Nα-Fmoc-S-trityl-L-Cystein (Fmoc-Cys(Trt)-OH), Nα-Fmoc-Nγ-trityl-L-Glutamine (Fmoc-Gln(Trt)-OH), Nα-Fmoc-L-Glutamic acid-γ-t-butylester (Fmoc-Glu(OtBu)-OH), Nα-Fmoc-Glycine (Fmoc-Gly-OH), Nα-Fmoc-L-Leucine (Fmoc-Leu-OH), Nα-Fmoc-Nε-t-Boc-L-Lysine (Fmoc-Lys(Boc)-OH), Nα-Fmoc-O-t-butyl-L-Serine (Fmoc-Ser(tBu)-OH), Nα-Fmoc-O-t-butyl-L-Threonine (Fmoc-Thr(tBu)-OH), Nα-Fmoc-L-Valine (Fmoc-Val-OH).

3.4 Chromatographic and electrophoretic separation methods

3.4.1 High performance liquid-chromatography (HPLC)

One approach to the study of individual chemical species is to separate

them from each other by analytical or preparative chromatography. As well as

possessing characteristic mass, shape and surface charge, biomolecules may


also differ from each other in terms of their hydrophobicity. Even soluble

proteins will often possess regions of their structure which are rather

hydrophobic. These regions will depend on the primary structure of the protein

and may be used to separate otherwise very similar proteins or peptides. It we

immobilize hydrophobic groups such as hydrocarbon chains on a stationary

phase, sample components may bind to these chains as a result of these

hydrophobic parts of their structure. This is the basis of reversed-phase

chromatography. In this type of partition chromatography, are designed the

stationary phase by the length of hydrocarbon chain (e.g. C-4, C-8, C-18). In

general, shorter-length chains are used for chromatography of proteins while

longer ones are more appropriate for peptides because of their greater relative

hydrophobicity.

Analytical RP-HPLC was performed on a UltiMate 3000 system (Dionex,

Germering, Germany), equipped with LPG-3400A pumps, using a Vydac C4

column (250 × 4.6 mm I.D.) with 5 μm silica (300 Å pore size) (Hesperia CA);

analytical Nucleosil 300-7 C18 column (250 × 4 mm I.D.) with 7 μm silica and

300 Å pore size (Macherey-Nagel, Düren, Germany). Linear gradient elution (0

min 0% B; 5 min 0% B; 50 min 90% B) with solvent A (0.1% TFA in water) and

solvent B (0.1% TFA in acetonitrile-water 80:20) was employed at a flow rate of

1 mL min-1. The solvents were 15 min sonicated in an Ultrasonic (Transsonic

570, Elma) before use and under vacuum degassed. The chromatograms were

recorded by UV detection at 220 nm, employing the VWD-3400 variable

wavelength detector of the UltiMate system, with a flow cell of PEEK, 0.4 mm

long and an internal volume of 0.7 μL. The fractions were collected with the

FOXY Jr ® automated fraction collector.

3.4.2 ZipTip clean up procedure

The ZipTip cleanup procedure was performed using ZipTip® C18 and C4

pipette tips from Omix (Varian). The ZipTip pipette tip was a 10 µL pipette tip

with a bed of chromatography media fixed at its end. It contains C18 reverse-

phase media for desalting and concentrating peptide and protein samples.

ZipTip C18 pipette tips are most applicable for peptides and low molecular


weight proteins, while ZipTip C4 pipette tips are most suitable for low to

intermediate molecular weight proteins. The procedure contains five steps:

wetting and equilibration of the ZipTip pipette tip, binding of the peptides and/or

proteins to ZipTip pipette tip, washing and elution.

Table 25: Solutions required for use with ZipTip pipette tips containing C18 media.

Solution ZipTipC18 Pipette Tips

Wetting solution 50% ACN in MilliQ water

Sample preparation 0.1% TFA in MilliQ water

Equilibration solution 0.1% TFA in MilliQ water

Washing solution 0.1% TFA in MilliQ water

Elution solution 0.1% TFA / 80% ACN

3.4.3 One dimensional gel electrophoresis

Electrophoresis is the movement of positively or negatively charged

molecules in an electric field. It is an analytical tool, indispensable across a

broad range of the biosciences, particularly in analytical studies of proteins.

Biopolymers such as proteins and nucleic acids are folded into compact

structures held together by a variety of non-covalent, ionic interactions such as

hydrogen bonding and salt bridges. It is possible to disrupt these ionic

interactions to denature the biopolymers and then to separate them

electrophoretically by denaturing electrophoresis. Such experiments are

especially useful in the study of proteins since they have a more varied range of

tertiary structure than nucleic acids. SDS-PAGE (polyacylamide-gel

electrophoresis) was performed according to Laemmli. The detergent SDS

consists of a hydrophobic 12-carbon chain and a polar sulphated head. The

hydrophobic chain can intercalate into hydrophobic parts of the protein by

detergent action, disrupting its compact folded structure. Additionally, SDS

coats proteins with a uniform layer of negative charges which causes them to

migrate towards the anode when placed in an electrical field, regardless of the

net intrinsic charge of the uncomplexed proteins. Tris-tricine is another

technique used to separate proteins in the mass range 1 – 100 kDa [229]. It is the

preferred electrophoretic system for the resolution of proteins smaller than 30


kDa. The concentrations of acrylamide used in the gels are lower than in other

electrophoretic systems in order to facilitate electroblotting, which is particularly

crucial for hydrophobic proteins. The different separation characteristics of the

two techniques, SDS-PAGE and Tris-tricine, are directly related to the strongly

differing pK values of the functional groups of glycine and tricine that define the

electrophoretic mobilities of the trailing ions (glycine and tricine) relative to the

electrophoretic mobilities of proteins. The proteins were investigated with gels

depending on their size. The gels were prepared using the mini-gel instrument

MiniProtean from BioRad. In the next table are presented the solutions for SDS-

PAGE separation and stacking gels.

Table 26: SDS-PAGE with 1mm – thick gel.

Solutions Gel concentration

5% 12% 10%

4× Stacking gel buffer 2.5 mL

4×Separation gel buffer - 6 mL 6 mL

MilliQ 5.8 mL 8.4 mL 10 mL

Acrylamide solution 1.7 mL 9.6 mL 8 mL

APS (a) 85 µL 125 µL 125 µL

TEMED (b) 20 µL 20 µL 20 µL

(a) 10% Ammonium peroxydisulfate;

(b) N’, N’, N’, N’- tetramethylethylenediamine.

In Table 27 are presented the corresponding solutions for a Tris-tricine gel

electrophoresis.

Table 27: Tris-tricine-PAGE with 1mm – thick gel.

Solutions Stacking gel Gel concentration

15% 10%

Acrylamide 972 µL 7.5 mL 4.9 mL

Tris-HCl/ SDS 1.86 mL 5 mL 5 mL

MilliQ 4.67 mL 0.9 mL 3.5 mL

Glycerol - 1.58 mL 1.58 mL

APS (a) 40 µL 75 µL 75 µL

TEMED (b) 7.5 µL 10 µL 10 µL


The Power /PAC 1000 instrument from Bio-Rad at constant current in two steps

was used for SDS-PAGE: a) 60 V when the samples were in the stacking gel

and b) 120 V when the sample was in the separating gel; for Tris-tricine

constant 100 V for 1.5 h. The molecular weights of unknown proteins were

estimated by running standard proteins of known molecular weights from 10 to

200 kDa in the same gel (Figure 97). Afterwards the gels were stained by

Coomassie blue or silver nitrate and/or transferred on the nitrocellulose and/or

PVDF membranes to perform immunoblotting or Edman sequencing analysis.

Gel Blot

kDa

10

15

20

3025

200150120100

5040

857060

Figure 97: Molecular weight marker- PageRuler TM Unstained Protein Ladder

(Fermentas) from 10 to 200 kDa.

3.4.4 Two-dimensional gel electrophoresis

The combination of Two-dimensional gel electrophoresis (2-DE) and

mass spectrometry has become a powerful tool in identifying protein structures,

as well as binding and molecular recognition peptide and protein structures [318].

Two-dimensional gel electrophoresis is a method of protein separation, by

which proteins in a mixture are separated according to two independent

properties: the first-dimension is isoelectric focusing (IEF), which separates

proteins according to their isoelectric points (pI); the second-dimension is SDS-

polyacrylamide gel electrophoresis (SDS-PAGE), which separates proteins

according to their molecular weights (MW) (Figure 98).


Protein mixture pH 3.0

pH 10.0

Isoelectric focusing (IEF)

pH 3.0 pH 10.0

SDS electrophoresisSeparation in second dimension

(by size)

Separation in first dimension(by charge)

Figure 98: Schematic representation of the two-dimensional gel electrophoresis. The first dimension separation is done based on isoelectric point (pI) variations, while the second dimension separation occurs due to the size (molecular weight) differences.

For the 2D-gel preparations of samples from mouse brain homogenates and

human neuroblastoma cells, acetone precipitation was used for removal of salts

and contaminants. The protein fractions have been precipitated for 3 h at -28°C

by adding 6 volumes of ice-cold acetone. Then the samples were solubilized in

a solution containing 7 M urea (used to denature and solubilize proteins), 2 M

thiourea (used to improve the solubility of samples), 4% CHAPS (used to

solubilize proteins and minimize protein aggregation), 0.3% DTT (used to

cleave disulfide bonds), 2% Servalyt 3-10 (carrier ampholytes that improve

solubility and ensure uniform conductivity during IEF without altering the pH

gradient of the IPG strip) and a trace of bromopheol blue. The total volume of

sample used for rehydration is specified in the manufacturer data sheet

according to the length of the IPG strip and is a critical parameter for a

successful isoelectric focusing. The samples were applied for 12 h on 17 cm

immobilized pH gradient (IPG) strips (pH range 3-10) using a passive in-gel

rehydration method. Isoelectric focusing (IEF) using immobilized pH gradients

(IPG), was carried out with Multiphor horizontal electrophoresis system


(Amersham Biosciences, München, Germany). The voltage was progressively

increased from 150 V to 3500 during 10 h and is described in Table 28.

Table 28: Voltage gradient for the isoelectric focusing of rehydrated IPG strips Phase Voltage (V) Duration (h:min) Voltage mode

1 150 0:03 gradient

2 150 0:30 constant

3 300 0:15 gradient

4 300 0:30 constant

5 3500 2:30 gradient

6 3500 10* constant * The isoelectric focusing is finished when the current remains constant for 30 min.

After focusing, the IPG strips were equilibrated for 30 min in 6 M urea, 30%

glycerol, 2% (w/v) SDS, 0.05 M Tris-HCl (pH 8.8), 1% (w/v) DTT and traces of

bromophenol blue, then incubated for 30 min in the same solution except that

DTT was replaced by 4.5% (w/v) iodoacetamide. For the second separation

step, the Bio-Rad Protean-II-xi vertical electrophoresis system was used, and

12-15% SDS-PAGE gels of 1.5 mm thickness were prepared. Strips have been

placed on the vertical gels and overlaid with 0.5% agarose in SDS running

buffer. Electrophoresis was performed in two steps: 25 mA/gel for

approximately 30 min, and 40 mA/gel until the dye front reached the anodic end

of the gels. After electrophoresis, the gels were transferred onto a PVDF

membrane for Western blot analysis or immersed in fixing solution (12% TCA in

MilliQ) for 60 min. Then the 2D-gel will be shacked overnight with a mixture of

160 mL buffer A with 40 mL methanol and 4 mL Brilliant Blue G-Colloidal

Concentrate. Further, the gels will be washed with 25% methanol and scanned

with GS-710 Calibrated Imaging Densitometer from BIO-RAD.

3.4.5 Colloidal Coomassie Brilliant Blue Staining

Colloidal Coomassie blue staining has a detection limit of 0.1 to 1 µg

protein/spot [319]. This procedure is based on the binding of the dye Coomassie

Brilliant Blue G-250, which binds non-specifically to virtually all proteins in acid


solution. This binding results in a spectral shift from reddish / brown (λ= 465

nm) to blue (λ= 610 nm). The protein-Coomassie complex absorbs at 595 nm,

and this wavelength is optimal for spectral determination to measure the blue

color from the Coomassie–protein complex. Coomassie Blue binds roughly

stoichiometrically to proteins, so this staining method is well suited for

densitometric determinations. The proteins are detected as blue bands on a

clear background, after fixing the gel with TCA for obtaining maximum

sensitivity.

Protein

Basic and aromatic side chains

H2C N

C2H5

CH3

C

NH

OC2H5

N+H2C

C2H5NaO3S

H3C

SO3-

+

Blue

Amax = 595 nm

Coomassie G-250

Figure 99: Scheme of protein staining by Coomassie blue.

First the gel was fixed for 30 min in fixing solution. Then the gel was shacked

overnight in staining solution, and afterwards the gel was washed with 25%

methanol for 60 seconds and scanned with GS-710 Calibrated Imaging

Densitometer from BIO-RAD.

To overcome the lengthy of Colloidal Coomassie blue staining procedure,

a shorter protocol has been employed, which is based on the binding of the dye

Coomassie Brilliant Blue R-250 (CBB R-250) to proteins. This dye differs from

Coomassie Brilliant Blue G-250 by the lack of two methyl groups. This method

is faster and compatible with mass spectrometry. First the gel was shacked in

Coomassie staining solution (50% methanol, 10% acetic acid, 0.05% CBB R-

250) for 10 min at 25°C and then 30 min at 60°C. Then the gel was shacked in


destaining solution (30% methanol, 10% acetic acid) for 30 min at 25°C. This

last step was repeated until the best contrast was achieved.

3.4.6 Silver Staining

Silver staining was performed according to Heukeshoven and Dernick

(1985) [320]. Silver staining is one of the most sensitive methods for permanent

visible staining of proteins in polyacrylamide gels and has a detection limit of 1

to 10 ng protein/spot [321]. The gel was impregnated with soluble silver ions and

development by treatment with formaldehyde, which reduces silver ions to form

an insoluble brown precipitate of metallic silver. After gel electrophoresis, the

gel was incubated between 30 and 60 min in fixing solution and 30 min or

overnight in sensitizing solution. After 3 washing steps with MilliQ (5 min) the

gel was incubated in staining solution for 20 min. The solution was discarded

and the developing solution was added in 2 steps: 1 min then discarded and 5-

20 min until the desired ratio of spot intensity to background was observed.

3.4.7 Bioanalyzer gel reader for native fluorescence detection

Following gel electrophoretic separations, proteins were visualized using

also the Gel-BioAnalyzer instrument developed by LaVision-BioTec (Bielefeld,

Germany) [322]. This instrument is used in protein detection based on native

fluorescence from individual aromatic residues, mainly tryptophan residues [259].

The instrument has a removable gel tray and is equipped to read unstained

protein gels using LaVision-Biotec scanning software. The experimental setup is

based on a UV excitation source and a detection system within the UV range.

The UV excitation light was generated by a 300 W xenon lamp (265 - 680 nm).

A UV bandpass filter (280 - 400 nm) is incorporated to block the excitation light

from the detection system. From four filter positions (one for UV excitation,

three for visible fluorescence), the UV filter transmitting light at λ = 343 ± 65/2

nm was employed. The large reading area (30 × 35 cm2) provided scanning of

both one- and two-dimensional gels.


3.5 Proteolytic digestion

3.5.1 Digestion in solution

Proteases have widely different, well characterized substrate specificities [323] and cleave preferentially different peptide bonds: trypsin cleaves at the C-

terminal bonds of lysine (Lys) and arginine (Arg) residues (basic amino acids),

but has a preference for Arg particularly at high pH values. In the sequences

Arg-X, Lys-X, cleavage is inhibited if there is a proline (Pro) residue at position

X. Glu-C cleaves mostly at the C-terminus of acidic amino acids, Lys-C at the

C-terminal of lysine. All serine proteases (e.g. trypsin, Lys-C) have the same

general mechanism (Figure 100), which involves into the reaction three specific

amino acids known as the catalytic triad: histidine 57, aspartate 102 and serine

195 residues [324]. The peptide substrate molecule attaches to the catalytic site

of trypsin (or other serine protease) by binding of its hydrophobic group to a

specific nonpolar pocket such as the carbonyl carbon atom of the peptide bond

to be attached which is close to the serine-OH. The hydrogen atom of the serine

transfers to the histidine nitrogen atom and the oxygen atom forms a bond with

the carbonyl atom of the substrate. The proton is transferred to the tetrahedral

intermediate (B) causing the breakage of the first product R’-NH2 and form the

acyl-enzyme intermediate (C). The next step is to hydrolyze (D) the ester bond

of the acyl-enzyme intermediate, and liberate the second product of the peptide

hydrolysis R-COOH and restore the original enzyme state ready for reaction

with the next substrate molecule (E).


Figure 100: Reaction mechanisms at the catalytic centre of the serine protease α-

chymotrypsin: (a) nucleophilic attack of the side chain oxygen of Ser on the carbonyl carbon forming a tetrahedral intermediate; (b) breakage of the peptide bond with assistance from His 57 (proton transfer to the new amino terminus); (c) it is release the first product; (d) nucleophilic attack of water and formation of the tetrahedral intermediate; (e) decomposition of acyl intermediate and release of the second product.

The proteins were digested using trypsin which was applied in an enzyme:

substrate ratio (E: S) of 1:50 and using Glu-C with E: S of 1:20; the substrate

was dissolved in PBS solvent, pH 7.5. Trypsin was first dissolved in a buffer

containing 50 mM acetic acid at a 1 µg µL-1 concentration. The digestion was

performed at 37°C for 30 min until 16 h. The reaction was quenched by freezing

the sample with liquid nitrogen. The Glu-C was dissolved in ultra pure water in a

concentration of 1µg µL-1 and applied for 2 h. Synucleins were proteolytic

degraded with trypsin, Glu-C .Their characteristics are shown in the Table 29.


Table 29: The proteases with their specific cleavage sites.

Protease Type Cleavage

site

Properties References

Trypsin Serine -P1-P1'- P1=Lys, Arg; P1' not Pro

[248]

Endoprotease Glu-C Serine -Glu-P1'- also -Asp-P1'- [325]

3.5.2 Proteolytic in gel digestion

Protein spots were excised manually from the gel and underwent to

digestion with trypsin according to Mortz et al [326] and peptide extraction. The

gel pieces were washed with ultra pure water (MilliQ, Millipore) for 15 min and

shaken with acetonitrile (ACN) to dehydrate the gel. After drying the gel pieces

in a vacuum centrifuge, they were destained with 50 mM NH4HCO3 for about 15

min and dehydrated with 3:2 acetonitrile/ MilliQ (15 min), followed by drying in a

vacuum centrifuge. The gel pieces were swollen in digestion buffer (12 ng µL-1

TPCK-trypsin in 50 mM NH4HCO3) at 4°C (on ice) for 45 min; the gel pieces

were incubated at 37°C overnight (12 h); at last the peptides were extracted

twice with ACN/ 0.1% TFA 3:2. The samples containing the extracted peptides

were lyophilized to dryness and redissolved in ESI solvent (89.5% MilliQ, 9.5%

acetonitrile, 1% formic acid; pH = 2.5) and analyzed using an Esquire 3000 +

hybrid HPLC 1100 Series (Agilent) mass spectrometer.

3.6 Immunological assays

3.6.1 Dot blot and Western blot

Some of the most sensitive procedures for detecting proteins separated

by electrophoresis are based on immunological methods. The term “blotting”

refers to the transfer of biological samples from a gel to a membrane and their

subsequent detection on the surface of the membrane. Western blotting (also

called immunoblotting because an antibody is used to specifically detect its

antigen) was introduced by Towbin, et al. in 1979 [327]. For the Western blot


analysis the proteins were first separated by SDS-PAGE electrophoresis. The

separated proteins were blotted after a semi-dry procedure. 2 pieces of filter

paper having the dimensions of blotted gels have been introduced into Towbin-

buffer (Blot buffer) and than were introduced into the blotting- instrument.

Between these filter papers was introduced a nitrocellulose membrane having

the same dimensions of the blotted gels and also swollen into blot buffer. Then

the gel was introduced also for short time in buffer and than on top of it were

placed another 2 filter papers also swollen in blot buffer. Special care was taken

for removing all air bubbles between the filter papers. The blotting was

performed at 30 mA for each gel for more than 3 h. To see if the proteins were

absorbed onto the nitrocellulose membrane the Ponceau S-Test was carried

out. For this the membrane was immersed for 2 min into a Ponceau S-solution

(0.02% Ponceau S in 0.3% trichloroacetic acid). Then the membrane was

washed with MilliQ water until the paper surface was clean and the protein

spots were slightly red.

Dot blots are used for semi-quantitative or qualitative determination of

membrane-immobilized antigens. For the Dot blot experiments the protein/

peptide samples were directly applied onto a dry nitrocellulose membrane (2 µg,

solved in PBS buffer). After complete dryness, the membrane was immersed

into PBS-0.5% Tween buffer and than treated as in the Western-Blot

experiment.

Enzyme

Substrate

Detectableproduct

Protein binding membrane

Figure 101: Schematic representation of Dot blot experiment.

The Western blot and Dot blot experiments were done as follows: the

membrane containing the sample was first incubated for 1 h in 10% blocking


buffer in water (Rotiblock buffer- represents the trade name for a mixture of a

high molecular weight poly (vinylpyrolidone) with a non-ionic surfactant

dissolved in PBS buffer). Then the solution of the first antibody (anti-synuclein

antibody) was applied and the membrane was incubated 2 h, 25°C. After

washing the membrane 3 times for 10 min, the secondary antibody was applied

and incubated for 1 h at 25°C. Then the membrane was washed for 25 min. For

the development was used a solution of Luminol (ECL-Western-Blotting-

Reagents, Amersham) mixed and applied directly on the membrane. The

exposure was done into a dark room by using a film (Fuji Medical X-Ray Film).

The exposure time was 30 sec to 1 min. The exposed films were developed by

using an automated Canon camera. For detection of the spots after the film

exposure the following Luminol reaction was carried out as described in Figure

102.

NH

NH

NH2

O

O

+ 2H2O2

Peroxidase

- H2O NH

NH

NH2

O •

O •

OH

OH

NH2

O

O

+ N2 + hν

OO

N

N

NH2

OH

OH

OO

Figure 102: Reaction scheme of Luminol detection in Dot/ Western blot experiments.

Moreover, the proteins extracted from mouse brain homogenates and

human neuroblastoma cells were transferred to nitrocellulose membranes and

processed for immunobloting using a high sensitive Odyssey Infrared Imager

system (Li-COR Biosciences) [328]. This imager introduces the direct infrared

fluorescence detection, which gives a quantitative analysis and wide linear

dynamic range that chemiluminescence can not. It allows direct detection

eliminating the need for film, darkroom and the harsh chemicals associated with

film development. After proteins were transferred at 65 mA/ gel for 2 h, the


nitrocellulose membranes were blocked in Odyssey Blocking Buffer for 1 h at

25°C. The blot was probed with anti-αSyn mBD antibody diluted 1:500 (v/v) in

Odyssey Blocking Buffer for 1 h, 25°C or overnight at 4°C. After three 10 min

PBS-T washes the bands were detected using IR-labeled secondary antibody

(IR800 goat anti-mouse) with absorbance – 770 nm and emission – 794 nm

diluted 1:15000 (v/v), for 1 h at 25°C in dark and imaged with Odyssey Infrared

Imager (Li-COR Biosciences).

3.6.2 Preparation of antibody columns

The antibody immobilization on the affinity material was performed using

the dry NHS-activated 6-aminohexanoic acid-coupled Sepharose 4B (Sigma-

Aldrich). Accessible α-anime groups present on the N-terminal of the antibody

react with NHS-esters and form amide bonds. A covalent amide bond is formed

when the NHS-ester cross-linking agent reacts with a primary amine, releasing

N-hydroxysuccinimide (NHS). Approximately 100 µg of anti-synuclein specific

antibody were dissolved in coupling buffer (pH 8.3). The solution was added to

dry NHS-activated 6-aminohexanoic acid-coupled Sepharose (Sigma) and the

coupling reaction was performed for 1 h at 25°C. The principle of the

immobilization of the antibody on the NHS-Sepharose is showed in the Figure

103.

OHN O

N

O

O

O+

N OO

HO

-

Sepharose

H2N R

OH

(CH2)5

OOH

HN

(CH2)5NH

RO

SepharoseO

HN O

N

O

O

O+

N OO

HO

-

Sepharose

H2N R

OH

(CH2)5

OOH

HN

(CH2)5NH

RO

Sepharose

Figure 103: Principle of the immobilization of the antibody to the NHS-Sepharose.


The Sepharose-coupling product was loaded into a ca. 0.8 mL micro-column

(Mobitec, Göttingen, Germany) providing the possibility of extensive washing

without significant loss of material. The flow through was collected and

investigated through SDS-PAGE. The matrix was washed sequentially with 0.5

M amino-ethanol, 0.5 M NaCl (pH 8.3), 0.1 M CH3COONa, and 0.5 M NaCl (pH

4.0). Each solvent was used with a total volume of 18 mL (three times 6 mL).

The micro-column was then stored at 4°C in 5 mM Na2HPO4, 150 mM NaCl,

(pH 7.5). The washing steps are given below: 6 mL buffer A (Block buffer, 0.1 M

Ethanolamine, 0.5 M NaCl, pH 8.3), 6 mL buffer B (Washing buffer, 0.2 M

NaOAc, 0.5 M NaCl, pH 4.0) and repeat three times for each buffer and

afterwards the column was stored 30 min at 25°C and than washed again as

described before. At the end the column was washed with 10 mL PBS buffer

and stored at 4°C.

Luer lock

Membrane

Gel

Membrane

Luer lock

Luer lock

Membrane

Gel

Membrane

Luer lock Figure 104: Schematic representation of the affinity column: the micro column is filled

with NHS Sepharose material and then the antibody solution is added and the column is washed extensively.

3.6.3 Affinity-mass spectrometry methods

Affinity-mass spectrometry was carried out on the antibody micro-column

which contains anti-synuclein specific antibody immobilized on dry NHS-

activated 6-aminohexanoic acid-coupled Sepharose 4B (Figure 105). A mixture

of 20 µg αSyn and 100 µL PBS ELISA buffer was added to the micro-column

followed by shaking for 2 h at the 25°C. After incubation was collected the

supernatant containing the non-bound protein and then the micro-column were

washed with 40 µL PBS and 10 µL MilliQ. The last 1 mL was collected. The

bound protein was eluted by incubation the column with 500 µL 0.1% TFA for


15 minutes. Afterwards the antibody micro-column was washed with 10 mL

0.1% TFA and 20 µL PBS. The supernatant, last wash and the elution were

lyophilized, desalted by ZipTip procedure and analyzed by mass spectrometry

or gel electrophoresis. This control experiment was important to establish the

function of the column.

non-covalent binding of proteins

addition of protein/ peptide mixture

eluted bound protein/ peptides

0.1% TFA

supernatant containingunbound proteins/ peptides

MS analysis

Tris-Tricine

MS analysis

Tris-Tricine

antibodymicro-column

Figure 105: Schematic representation of the affinity mass spectrometry principle.

3.6.4 Epitope excision and extraction experiments

The analysis of epitope (also called epitope mapping) is defined as the

identification of the antigenic determinant recognized by the paratope of an

antibody. Analytical procedures for the molecular elucidation of specific binding

sites in proteins and peptides have been developed since the early 1990’s in

our laboratory, by combining the isolation of antibody-bound peptides using

immune-affinity techniques followed by the precise structural identification of

epitope peptides by mass spectrometry (Figure 106) [289-291].


Antigen

Antibody

Antibody

m/z m/z m/z

proteolysisExcisionExtraction

washing dissociation

Mass Spectrometric Peptide Mapping

Non-epitope peptides Epitope peptide Antigen

Figure 106: Analytical scheme of epitope identification analysis using a combination of

epitope excision/extraction and mass spectrometric peptide mapping followed by sequence analysis.

Epitope excision/ extraction experiments were carried out in PBS ELISA (5 mM

Sodium dihydrogen phosphate, 150 mM NaCl, pH 7.5) buffer with a

physiological pH of 7.5, which was found to be especially effective for the

stabilization of the immune complex [329]. For epitope excision experiments 20

µg of αSyn (corresponding to antigen: antibody molar ratio of 2:1 with 100 µg

IgG immobilized on the Sepharose column) were applied onto the anti-synuclein

specific antibody micro-column and allowed to bind for 2 h at 25°C for complete

binding of antigen. The first supernatant which contains the non-bound protein

was collected after 1 h. After washing with 10 mL PBS ELISA buffer, proteolytic

digestion was performed for 2 h at 37°C with 0.4 µg trypsin and 1 µg

endoproteinase Glu-C (Sigma-Aldrich, St. Louis, MO, USA) in 100 µL PBS

buffer. Supernatant non-epitope fragments were removed again by blowing out

the column with a syringe, and the matrix material washed with 40 mL PBS

buffer and 10 mL MilliQ. The last 1 mL was collected. After removal of the


proteolytic non-epitope fragments, the immune complex was dissociated using

500 μL 0.1% TFA; the column was shaken gently for 15 min and the released

peptides collected into a micro-reaction cup. The samples were then lyophilized

and stored until mass spectrometric analysis. The column was regenerated by

washing with 10 mL 0.1% TFA followed by 20 mL PBS buffer. When handled in

this way, affinity micro-columns may be used at least 10 to 20 times without

significant loss of antigen binding capacity. For the epitope extraction

experiments the antigen was first digested with the chosen protease for 2 h at

37°C, it was presented to the antibody column. The binding of the antigen to the

antibody was performed for 2 h at 25°C. Washing, dissociation and epitope

elution steps were then performed in the same manner as in epitope excision.

The released epitope and non-epitope peptides were lyophilized and desalted

using the ZipTip procedure and analyzed by MALDI-TOF mass spectrometry.

Before the epitope excision/ extraction experiments with affinity column, the

column was tested in an affinity chromatography experiment.

3.6.1 Enzyme-linked immunosorbent assay

An ELISA (enzyme-linked immunosorbent assay) is an assay for binding

affinity in which a target molecule is coated onto a plastic plate, a second

molecule (the analyte) is added, and a third molecule is used to detect binding

of the analyte. ELISA is rapid and convenient, can detect less than a nanogram

(10-9 g) of a protein. ELISA can be performed with either polyclonal or

monoclonal antibodies, but the use of monoclonal antibodies yields more

reliable results.

For ELISA experiments suitable dilutions of the primary anti-αSyn

antibodies were first evaluated by incubating 0.5 μM wt-αSyn in PBS, 100 μL

per well, in triplicate, for 2 h at 25°C and then adding, after blocking and

washing, 12 three fold serial dilutions of antibody. The binding of the antibodies

to wt-αSyn was assessed by incubating HRP-conjugated goat anti-mouse or

anti-rabbit antibodies (1 h at 25°C) and measuring the absorbance with an

ELISA reader. The concentration of antibodies necessary for obtaining half of

maximum binding at OD450 nm of 1.0 was calculated. For background


subtraction, triplicate wells containing PBS were incubated in the first step for

each antibody dilution. All incubations were followed by washing steps. The

following values were obtained: anti-α/βSyn Ab 1:10000 v/v, anti-αSyn 4B12 Ab

1:10000 v/v, anti-αSyn mBD Ab 1:5000 v/v, anti-αSyn pC20 Ab 1:3000 v/v, anti-

αSyn pASY-1 Ab 1:2500 v/v (see 7.4)

Indirect ELISA (Figure 108), using antigen dilutions, was carried out for

the immuno-analytical characterization of synuclein proteins and peptides.

Ninety-six-well ELISA plates (BioRad, Hercules, CA) were coated overnight at

25°C with 100 µL/well of antigen (serial dilutions from 2.5 µM to 0.00001 µM

concentration). After coating, the wells were washed with 200 µL/well PBS-T

0.05% Tween-20 v/v in PBS- phosphate buffer saline (Na2HPO4 5 mM, NaCl

150 mM, pH 7.5) and the nonspecific binding sites were blocked with 200 µL

5% BSA (Sigma- Aldrich, Steinheim, Germany) for 2 h at 25°C. After incubation

and washing steps, anti-synuclein specific antibody was added to the plate.

After another 2 h of incubation at 25°C, the unbound first antibody was washed

away and 100 µL of peroxidase labeled goat anti-mouse or rabbit IgG (Jackson

Immuno Research, West Grove, PA) diluted 5000 times in 5% BSA was added

to each well. After an additional incubation for 2 h, the wells were washed three

times with PBS-ELISA-Tween and once with 50 mM sodium phosphate- citrate

buffer, pH 5. 100 µL/well of 0.1% o-phenylenediamine dihydrochloride (OPD)

(Figure 107) (Merck, Darmstadt, Germany) in sodium phosphate- citrate buffer

at c = 1 mg mL-1 and 2 µL of 30% hydrogen- peroxide (Merck, Darmstadt,

Germany) per 10 mL of substrate buffer was added to the plates. The

absorbance was measured at λ = 450 nm on a Wallac 1420 Victor2 ELISA Plate

Counter (Perkin Elmer, Boston, MA). In order to compare the binding intensities

of different peptides, the concentration of protein/ peptides which was

necessary for obtaining half maximum binding at OD450 nm of 1.0 was calculated.


+ 2H2O2

NH2

NH2HRP, pH 5.0

N

N

NH2

NH2

2

2,3-diaminophenazine(OPD)

o-phenylenediamine(OPD)

Figure 107: Schematic representation of the reaction catalyzed by horseradish peroxidase. The oxidation product of o-phenylenediamine produced by horseradish peroxidase is 2,3-diaminophenazine. This product is orange-brown in color and can be read spectrophotometrically at 450 nm.

Figure 108: Schematic representation of indirect ELISA.

3.7 Thioflavin-T assay

Thioflavin-T (ThT) is a fluorescent dye, which interacts with protein fibrils

with a characteristic increase in fluorescence intensity in the vicinity of 480 nm [313, 314]. Purified synuclein proteins were dissolved in 20 mM PBS up to 30 µM

concentration and incubated at 37°C with agitation in a microcentrifuge tube. As

a control, a blank was provided with 25 µM ThT in water. The measurement


was carried out in 96-well plate (black; black bottom) by using fluorescence

plate reader (Victor2; Perkin Elmer). Thioflavin-T assay was performed with

CW-lamp (F450), emission filter (T486), measurement time of 1.0 s and a CW-

lamp energy of 12385.

3.8 Circular dichroism spectroscopy

Circular dichroism spectroscopy (CD) has been extensively applied to the

structural characterization of peptides and proteins. The application of CD for

conformational studies in peptides or proteins can be grouped into: (a)

monitoring conformational changes (e.g., monomer-oligomer, substrate binding,

denaturation, etc.) and (b) prediction of secondary structural content. Some

characteristics for the secondary structure (the local spatial arrangement of a

polypeptide’s backbone atoms without regard to the conformations of its side

chains) elements are given below:

CD of random coil: positive band at 212 nm (π-π*)

negative band at 195 nm (n-π*)

CD of β-sheet: negative band at 218 nm (π-π*)

positive band at 196 nm (n-π*)

CD of α-helix: positive band at 192 nm (π-π*)

negative bands at 208 nm (π-π*) and at 222 nm (n-π*)

For the calculation of molar ellipticity [θ] in deg dmol-1 cm2, the difference

between the absorption between left and right of circular polarized light (AL-AR)

was measured according to the Lambert- Beer law:

θ= 33(AL-AR) where Δε is the molar extinction coefficient;

where (AL-AR)= Δε *c*d c is the molar concentration;

and θ= θ/ (10*c*d) [dmol-1 cm2] d is the diameter of the cuvette in cm;

where θ is measured in mdeg;

c is the concentration in mol L-1.

The CD spectroscopy experiments were carried out with a Jasco J-715

Spectrapolarimeter, equipped with a Xe-lamp, a double monochromator, a


modulator and detector (a photomultiplier tube). The Xe lamp is used as the

light source; the light that passes through the monochromator is not only

monochromated, but also linearly polarized and oscillates in the horizontal

direction; after that the linearly polarized light is modulated into right and left

circularly polarized beams. The spectra were registered at room temperature

(25°C) with a scan speed of 1 nm/min and the cuvettes diameter was 0.1 cm

path length. The spectra were averages of four scans in a wavelength range

between 180 and 260 nm (far-UV). Before each measurement a blank spectrum

was performed in the same solvent as the sample to be measured. Molar

elipticities were calculated with Jasco-700 Software.

3.9 Mass spectrometric methods

Mass spectrometry is the method of choice for the identification of proteins

because of its superior speed, sensitivity and versatility. The “joined forces” of

sophisticated biochemical and mass spectrometry approaches are the driving

forces in the entire field of proteomics today, which enabled researchers to

embark on studies of much larger and more complicated systems than

previously possible [241, 245, 246, 330-333]. A mass spectrometer consists of an ion

source, a mass analyzer that measures the mass to charge ratio (m/z) of the

ionized analytes, and a detector that registers the number of ions at each m/z

value (Figure 109).


Figure 109: General construction of a mass spectrometer. The main components: ion

source, mass analyzer and detector, are controlled by a computer that also processes the signals and delivers the mass spectra. Each element is exemplified with devices used in the biopolymer analysis. MALDI is usually coupled to TOF analyzers, whereas ESI is mostly coupled to ion traps and triple quadrupole instruments and used to generate fragment ion spectra (collision induced dissociation (CDI) spectra).

One key advantage of mass spectrometric analyses over other analytical

techniques is its capability to study extremely small quantities of molecules with

high sensitivity. Ionization techniques such as electrospray (ESI) and matrix

assisted laser desorption and ionization (MALDI) [334] are directly responsible for

these advanced possibilities. ESI, as well as MALDI have demonstrated their

capabilities for mass spectrometric analysis of biopolymers up to several

hundred thousand Daltons [234]. Conventional “soft-ionization” mass

spectrometric methods such as electrospray ionization (ESI), HPLC-MS are not

suitable to direct in situ analysis of conformational states and intermediates.

Ion-mobility mass spectrometry (IMS-MS) is emerging as a new tool to probe

protein structures and interactions due to its potential for separation

polypeptides by conformational states, shape and topology.

3.9.1 MALDI-TOF mass spectrometry

Because of the high sensitivity and the tolerance of many biological

buffer solvents, MALDI-MS is a suitable technique for analysis of biological

samples and complex mixtures [335, 336]. In MALDI-MS the sample with an

organic matrix (usually aromatic acids) (Figure 110) is applied on a target and

desorbed by laser irradiation [337]. The ionization takes place by an effective co-


crystallization of the sample with the matrix molecules, for this reason the

sample preparation is a very important process. Ions are usually generated by

bombarding the sample with short-duration (1 - 10 ns) pulses of UV light from a

nitrogen laser. When the laser strikes the matrix crystals, the energy deposition

is thought to cause rapid heating of the crystals brought about by matrix

molecules emitting absorbed energy in the form of heat. The rapid heating

causes sublimation of the matrix crystals and expansion of the matrix and

analyte into gas phase. Ions might be formed through gas-phase proton-transfer

reactions in the expanding gas phase plume with photoionized matrix

molecules. MALDI matrix plays a key role in MALDI and is a non-volatile solid

material that absorbs the laser radiation resulting in the vaporization of the

matrix and sample embedded in the matrix [338]. The matrix also serves to

minimize sample damage from the laser radiation and to facilitate the ionization

process.

OH

HO

OHO

HO

OH

O

C

N

A. B.a b

Figure 110: Matrices used in MALDI-TOF-MS: (a), α-Cyano-4-hydroxycinnamic acid, (HCCA) and (b), in MALDI-FTICR-MS 2,5-Dihydroxybenzoic acid (DHB).

α-cyano-4-hydroxycinnamic acid (HCCA) is recommended for peptides and

proteins under 10 kDa of mass. Best results are obtained in the 500-5000 Da

range and therefore HCCA is the matrix of choice for peptide mass

fingerprinting, being usually used for MALDI-TOF mass spectrometry; 2, 5-

dihydroxy benzoic acid (DHB): is used for the analysis of small molecules and

peptides (200 to 1000 Da), and DHB is usually employed in MALDI-FTICR

mass spectrometry. A good matrix should fulfill the following requirements:

absorption at the wavelength of the laser radiation (λ = 337 nm) to provide

sufficient energy deposition in the sample; and to be soluble in the same solvent


as the analyte. They have to crystallize upon sample preparation. An illustration

of the MALDI process is shown schematically in Figure 111.

Figure 111: Schematic representation of MALDI mass spectrometry. A mixture of

matrix and sample is applied on the target and desorbed by laser ionization. The desorbed ions are moved in an electric field and separated in a time of flight analyzer.

Time-of-flight analysis is based on accelerating a set of ions to a detector with

the same amount of energy. With MALDI-TOF analyte ions gain additional

activation energy and fragmentation occurs by collisions with matrix molecules

and residual gas molecules during their flight in the field free drift path; this post

source decay creates metastable ions. By stepping the reflector voltage (usually

20 kV), the metastable ions can be brought into the detector and their fragment

masses can be analyzed. Because the ions have the same energy, yet a

different mass, the ions reach the detector at different times. The smaller ions

reach the detector faster because of their greater velocity and the larger ions

take longer, thus the analyzer is called time-of-flight because the m/z is

determine from the ions time of arrival. The arrival time of an ion at the detector

is dependent upon the mass charge, and kinetic energy of the ion. MALDI-TOF

mass spectrometry was performed using a Bruker (Bruker Daltonics, Germany)

Biflex ITM linear TOF mass spectrometer with a SCOUT-26-ionization source

video system, nitrogen UV laser (337 nm), and a dual channel plate detector.

Each spot on the target was prepared using mixture between matrix and

sample. 1 µL of a freshly prepared saturated solution of HCCA (α-cyano-4-

hydroxycinnamic acid) in ACN: 0.1% TFA (2:1, v/v) was applied onto the dried

target, and then 1 µL of the peptide (sample) solution was applied over. Internal

or external mass calibration was performed using a peptide mixture in a


concentration of 1 pmol µL-1. The spectra were registered using an accelerating

voltage of 20 kV and a laser attenuation power of 45%. A number off 40 laser

shots were applied for accumulating one spectrum.

3.9.2 FTICR mass spectrometry

The development of Fourier-Transform-Ion-Cyclotron-Resonance

(FTICR) has enabled a breakthrough for the high resolution mass spectrometry

structure analysis of biomolecules [339, 340]. FTICR is based on the principle of a

charged particle orbiting in the presence of a magnetic field in a stable cyclic

motion. The analyzer cell is an ultra high vacuum (<10-10 mbar) trap in which

ions can be stored for extended periods of time. In a FTICR mass spectrometer,

the mass analysis is performed in a cubic or cylindrical cell placed in a strong

magnetic field. The cell consist of two opposite trapping plates, two opposite

excitation plates and two opposite detection plates (Figure 112). Each ion

moving in a spatially uniform magnetic field will describe a circular so-called

cyclotron motion as a result of the Lorenz force and the centrifugal force

operating on it in opposite directions. The cyclotron frequency is dependent only

on the mass over charge and magnetic field, as described by the general

equation ωc = z/m×B, where ωc is the unperturbed cyclotron frequency, z is the

charge, B is the magnetic field strength and m the mass of the ion. The main

advantage of FTICR is that the cyclotron frequency is independent from the

kinetic energy and the velocity of ions that could be influenced by the ionization

process.


Figure 112: Schematic representation of ICR cell, a cubic trapped ion cell used in

FTMS. Coherent motion of the ions in the cell induces an image current in the receiver plates. The time domain signal is subjected to a Fourier transform algorithm to yield a mass spectrum.

3.9.2.1 MALDI-FTICR mass spectrometry

MALDI-FTICR mass spectrometric analysis was performed with a Bruker

APEX II FTICR instrument (Bruker Daltonics, Bremen, Germany) equipped with

an actively shielded 7T superconducting magnet (Magnex, Oxford, UK), a

cylindrical infinity ICR analyzer cell, and an external Scout 100 fully automated

X-Y target stage MALDI source. A 100 mg mL-1 solution of 2,5-

dihydroxybenzoic acid (DHB) in acetonitrile/0.1% TFA in water (2:1) was used

as the matrix. 0.5 μl of matrix solution and 0.5 μl of sample solution were mixed

on the stainless steel MALDI target and allowed to dry. A pulsed nitrogen laser

of 337 nm was used to desorb the ions from the target. External calibration was

carried out using the monoisotopic masses of singly protonated reference

peptides in the mass range, m/z 100 – 5000. Acquisition and processing of

spectra were performed with the XMASS software (Bruker Daltonics).

3.9.2.2 Nano-ESI-FTICR mass spectrometry

Nano-ESI-FTICR MS analysis was performed with a Bruker APEX II

FTICR mass spectrometer equipped with an actively shielded 7T

superconducting magnet (Magnex, Oxford, UK), a cylindrical infinity ICR

analyzer cell, an APOLLO electrospray ionization source, and an API1600 ESI

control unit. The sample was dissolved in a spraying solution containing 30%

methanol, 69% water and 1% formic acid. 8 µL sample solution were loaded


into the gold coated nano-spray capillary using a GELoader pipette tip. The

nano-spray capillary was than placed into the fitting of the XYZ stage. The

voltage applied between the tip of the nano-spray capillary and the glass

capillary inside the instrument was manually adjusted between -700 and -1200

V.

3.9.3 ESI-ion trap mass spectrometry

Electrospray ionization (ESI) is a soft ionization technique that

accomplishes the transfer of ions from solution to the gas phase. The technique

is extremely useful for the analysis of large, non-volatile, chargeable molecules

such as protein and nucleic acid polymers. For the development of ESI mass

spectrometry of biomolecules, John B. Fenn received the Nobel Prize 2002 in

Chemistry. ESI-MS is an ionization method which produces gaseous ionized

molecules from a solution [234] by creating a fine spray of charged droplets in an

electric field [235, 236]. An illustration of the ESI process is shown in Figure 113.

The repulsion between the charges on the surface causes finally intact ions to

leave the droplet by a process known as a “Taylor cone” [341]. Electrospray

ionization is generally leading to the formation of multiply charged molecules.

This is an important feature since the mass spectrometer measures m/z, thus

making it possible to analyze large molecules with an instrument of a relatively

small mass range. The mass analyzer is responsible for the accuracy, range,

and sensitivity of a mass spectrometer. Two most efficient types of mass

analyzers used in this work are time-of-flight (TOF) and ion trap. Different

analyzers can be used with different desorption methods.


-

--

-

- -

Desolvated macromolecular ion Solvated macromolecular ionAerosol of fine charged droplets (microdroplet)

Electrospray

10 µm 10 nm 1 nm

Taylor-Cone

Figure 113: ESI mass spectrometry. The formation of the charged droplets in form of

highly charged ions takes place in the gas phase. The size of the droplets decreases when reaching the Rayleigh limit. The solution is evaporated into the vacuum system of the ion source. In this example the desolvated ion has a 3+ charge [241].

The ion trap mass analyzer is the three dimensional analogue of the linear

quadrupole mass filter and is based on the motion of ions in an rf electric field

(Figure 114). Ions are subjected to forces applied by an rf field in all three

directions. The ion trap consists of three electrodes with hyperbolic surfaces:

two end-cap electrodes at ground potential and between them a ring electrode

to which a radio frequency voltage is applied. The device is radial symmetrical

and r0 and z0 represent its size.

Figure 114: Geometry of the ion trap mass analyzer, consisting of a ring electrode and

two end caps electrodes with special shape.

The ion trap employs an alternating electric field to stabilize and destabilize the

passing ions. In the case of ion trap the electric field is used to store ions and to

release them in time to be analyzed. The mass separation can be done by

several methods. The most important for analytical purposes are: -Stafford’s


mass-selective instability mode scan the fundamental rf-voltage amplitude

applied to the ring electrode. Thus, ions of increasing mass-to-charge ratio

adopt unstable flight paths and leave the trap towards the detector; -resonance

ejection utilizes again the fundamental rf- voltage amplitude to bring the ions

into resonance with the supplementary rf- voltage at the end cap electrodes.

Thus they absorb sufficient power to leave the ion trap. In this way the mass

range of the ion trap can be extended. The ion trap mass analyzer applied to

peptide sequencing generally are combined with an ESI ion source.

A MS/MS experiment starts with ionization and injection of the generated

ions into the ion trap. Here the parent ion is isolated and collision-induced

dissociation (CID) takes place as a result of collisions in the helium damping

gas due to the resonance excitation of the ions. After fragmentation, the CID

spectrum of the selected precursor ion is recorded by sequentially ejecting the

product ions; only masses higher than 28% of the parent ion can be stabilized

inside the ion trap. Ion trap is about 50 times more sensitive than a triple

quadrupole mass spectrometer and the resolution can be improved using a

special scan mode. Proteins and peptides are oligomers of repeating (-NH-

CHR-CO-) units, differing only in the nature of the side chain, R. The amino acid

residues are held together by peptide bonds, (-NH-CO-) which have lower bond

energies than standard (-N-C-) bonds. When proteins or peptides fragment,

peptide bonds are commonly broken releasing mostly intact amino acid

residues. In MS experiments, however, there may also be fragmentation at

other localization in addition to peptide bonds resulting in a complex pattern of

ions. These are related to each other by incremental m/z differences because

only 20 R-groups of known structure are found in proteins and because

breakage points have fixed structural localizations relative to each other. The

nomenclature proposed by Roepstorff which is widely used to describe

polypeptide fragmentation in MS is illustrated in Figure 115.


Figure 115: Peptide cleavage nomenclature proposed by Roepstorff and Fohlman.

Peptide ions fragment at the peptide backbone to produce major series of fragment ions. Fragment ions from N-terminus are called a, b, c, and fragment ions from the C-terminus are called x, y, z ion.

Ions formed from a polypeptide during MS may retain a positive or negative

charge either on their C-or N-terminus. A horizontal line pointing towards the C-

or N-terminus at the breakage point is used to denote which fragment carries

the charge in that particular ion. For the N-terminal ions there are an, bn, cn and

dn (numbered from the N-terminus) while the C-terminal ions are designated xn,

yn and zn (numbered from the C-terminus). The ion represented by a1

represents the first residue in the sequence (minus the CO group) while a2

represents the first two residues (minus the CO group) and so on. Identical

main-chain breakage points are denoted by the pairs a/x, b/y, c/z (each member

of a pair referring to a positive charge retained either on the N- or C-terminus,

respectively). Breakage points denoted by dn, vn and wn are due to side-chain

fragmentations. There are used to distinguish residue pairs such as Leu/Ile and

Gln/Lys which have identical masses.

αSyn proteins and peptides were solubilized in 1% formic acid (HCOOH)

at a final concentration of 10 ÷ 30 µM and measured through direct infusion with

an Esquire 3000 + ion trap device. All MS results were obtained using

atmospheric pressure chemical ionization (APCI) in the positive ion mode. Mass

spectra were recorded in the full scan mode, scanning from m/z 100 to 3000.


Ion source parameters were 20 psi nebulizer gas and 7 L min-1 of drying gas

with a temperature of 300°C (Table 30).

Table 30: The ESI-ion trap parameters:

Parameter Value

Target Mass 1000

Compound stability 100%

Trap drive 80%

Nebulizer 20 psi

Dry gas 7 L min-1

Dry temp 300°C

Mode positive

Scan 100 - 3000

Average scans 6

3.9.4 LC-ESI-ion trap mass spectrometry

The technique of LC-ESI-MS is one of the most exciting developments of

recent times in analytical methodology. The combination of HPLC with MS is a

tandem between the two most powerful standalone analytical techniques. An

LC-MS instrument consists of three major components: an LC (to resolve a

complex mixture of compounds), an interface (to transport the analyte into the

ion source of a mass spectrometer) and a mass spectrometer (to ionize and

mass analyze the individually resolved components). Due to of the widespread

use of liquid chromatography, the LC-system is the most common form of

sample delivery for the instrument. The electrospray ionization is optimized to

accept flow rates up to 1 mL min-1. The nebulization process for both of these

ion sources is assisted with nebulizing gas and counter current drying gas.

LC-MS experiments were carried out using an Agilent Technologies

(Waldbronn, Germany) HP1100 liquid chromatograph for binary gradient elution

(pump model G1312A), including autosampler (G1313A) and a DAD (G1315 B),

coupled to an Esquire 3000 + ion trap mass spectrometer from Bruker Daltonics

(Bremen, Germany). A 150 mm x 4.6 mm x 3 μm Discovery RP-18 column was

used for the separation of the peptides. The elution system was 0.2% aqueous


formic acid (eluent A) and 0.2% formic acid in acetonitril (eluent B). Mass

spectra were recorded in the full scan mode, scanning from m/z 200 to 1500.

Ion source parameters were 20 psi nebulizer gas and 9 L min-1 of drying gas

with a temperature of 300°C. MS/MS experiments were carried out in the

autofragmentation mode. Six microscans were collected for each full MS scan

and 20 microscans were collected for each MS/MS scan, with a maximum

accumulation time for ions entering the trap of 200 ms.

Table 31: The LC- ESI-MS gradient:

Time %A %B

0 98 2

5 98 2

65 40 60

69 2 98

84 2 98

89 98 2

100 98 2 Eluent A: 0.2% HCOOH in MilliQ

Eluent B: 0.2% HCOOH in ACN

3.9.5 Linear trap quadrupole (LTQ) Orbitrap mass spectrometry

The linear ion trap quadrupole Orbitrap (LTQ Orbitrap) instrument has

become a powerful tool in proteomics research with accurate mass and high

resolution similar to those achievable with FTICR instrumentation [342-344]. The

LTQ accumulates, isolates, and fragments peptide ions. Alternatively, isolated

ions can be fragmented by higher energy collisional dissociation. The ions are

trapped in the orbitrap on a circular trajectory around the central spindle

electrode. The outer counter electrode, with a barrel-like shape, is coaxial,

surrounding the inner spindle electrode. Basically this a modified Kingdon trap [345]. The m/z values are determined from the harmonic oscillations of the ions

orbits along the trap axis. Since this frequency is independent of the energy and

spatial spread of the orbitally trapped ions, the measurement of their m/z values

is very precise.


αSyn (A30P) mouse brain homogenates from two-dimensional gels were

analyzed by reversed phase liquid chromatography nanospray tandem mass

spectrometry (LC-MS/MS) using an LTQ-Orbitrap mass spectrometer (Thermo

Fisher) and an Eksigent nano-HPLC. The dimensions of the reversed-phase LC

column were 5 μm, 200 Å pore size C18 resin in a 75 μm i.d. × 10 cm long

piece of fused silica capillary (Hypersil Gold C18, New Objective). After sample

injection, the column was washed for 5 min with 95% mobile phase A (0.1%

formic acid) and 5% mobile phase B (0.1% formic acid in acetonitrile), and

peptides were eluted using a linear gradient of 5% mobile phase B to 40%

mobile phase B in 65 min, then to 80% B in an additional 5 min, at 300 nL min-1.

The LTQ-Orbitrap mass spectrometer was operated in a data dependent mode

in which each full MS scan (30000 resolving power) was followed by five

MS/MS scans where the five most abundant molecular ions were dynamically

selected and fragmented by collision-induced dissociation (CID) using a

normalized collision energy of 35% in the LTQ ion trap. Dynamic exclusion was

allowed. Table 32 summarizes the identified peptide sequences in all four spots.

The mass spectrometric identification of wt-αSyn in spot 1, 2, 3, 4 is shown in

Figure 75.

Table 32: Summary of high resolution LTQ-Orbitrap MS/MS based protein identification for spots 1, 2, 3 and 4 (see Figure 75). The human αSyn protein was identified in spot 2 with sequence coverage of 81.43%.

Spot no. Identified peptide [M+H]+ calc [M+H]+ exp ∆m (ppm)

2 11A – K21 873.4676 873.4675 0.09

2 13E – K21 1072.5997 1072.6001 -0.42

1, 2, 3 33T – K43 1180.6572 1180.6599 -1.09

1, 2, 3, 4 35E – K43 951.5146 951.5152 -0.68

1, 2, 3, 4 44T – K58 1524.8380 1524.8387 -0.51

1, 2, 3, 4 46E – K58 1295.6954 1295.6978 0.79

1, 2, 3, 4 59T – K80 2157.1874 2157.1882 -0.41

1, 2, 3, 4 61E - K80 1928.0447 1928.0487 -0.20

1, 2, 3, 4 81T – K96 1478.7849 1478.7863 -0.95

1, 2, 3, 4 81T – K97 1606.8799 1606.8814 -0.25

1, 2, 3 103N – A140

(116Met, 127Met oxid.)

4318.7235 4318.7281 -0.74


The measurements were performed by Dr. Andreas Marquardt at the

Proteomics Facility, Department of Biology of the University of Konstanz.

3.9.6 Ion-mobility mass spectrometry

Ion-mobility mass spectrometry (IMS-MS) is most recently emerging as a

powerful analytical tool for analysis of conformation-dependant interactions of

proteins and reaction intermediates, which are not feasible to analyze by

conventional mass spectrometry (MS), such as electrospray ionization MS (ESI-

MS) [237-240]. The system enables the analysis of samples differentiated by size

and shape, as well as mass [286]. An IMS-MS instrument must perform five

basics processes: sample introduction, compound ionization, ion-mobility

separation, mass separation and ion detection. Ion-mobility spectrometry and

mass spectrometry share a similar theoretical background and the historical

development of these was presented recently by Uetrecht and co-workers [278].

Mass spectrometric methods are limited to the separation of ions measured by

mass-to-charge (m/z) ratios and ion intensities, while ion-mobility separates

ions based on their mobility and measures the time that it takes an ion to

migrate through a buffer gas in the presence of low electric field (drift time). The

velocity of the ion, ν (drift velocity) is directly proportional to E with the

proportionality constant K called ion-mobility, and is determined by measuring

the time required, tD to traverse a drift cell of known dimensions, d:

ν =dtD

KE =

Measurements between laboratories can be compared by normalizing values to

standard conditions that produce the reduced mobility (K0) using the relation

K0 =dEtD

273T

P760

x x

where P and T correspond to buffer gas pressure and temperature.

It is also possible to describe the K0 in terms of the characteristics of the ion and

the conditions of the drift cell, namely the ions charge, z e, average collision


cross section, Ω, the number density of the drift gas, N and the reduced mass of

the ion and buffer gas, μ, using the relation:

K0 =3ze16N

1Ω

2πµkBT

x x

1/2

Ion-mobility mass spectrometric measurements were carried out on a

Waters (Manchester, UK) SYNAPT G1 and G2 HDMS (high- definition mass

spectrometer) system described in detail elsewhere (2.3.3.1). This consists of a

hybrid quadrupole, ion-mobility and orthogonal acceleration (oa-TOF) geometry.

The ions passed through a quadrupole and either set to transmit a substantial

mass range or to select a particular m/z before entering the Triwave device.

Trivawe is consisted of three T-Wave devices. The first device, the Trap T-

Wave, accumulates ions. The stored ions are gated into the second device, the

IMS T-Wave, where they are separated according to their mobilities. The final

Transfer T-Wave is used to transport the separated ions into the oa-TOF for MS

analysis [282]. The injection volume was 5 µL. The desalting was performed on

the cartridge 10 min at 20 µL min-1 with gradient of acetonitrile from 10% to

90%. The acquisition characteristics were: 350 to 4000 m/z, sample cone

voltage of 25 V, IMS pressure 0.45 bar, wave height 5 to 15 V. The

measurements were performed by Waters personnel at Waters Corporation,

Manchester, UK. Data acquisition and processing were carried out with

MassLynx (V4.1) and DriftScope (V2.1) software supplied with the instrument.

3.10 Programs for mass spectrometry

3.10.1 GPMAW

For the correct molecular weight determination of peptides and proteins

and the proteolytic fragments obtained after protease digestion, the GPMAW

5.0 (General Protein/ Mass Analysis for Windows) (Lighthouse Data, Denmark)


program was used. This program made the calculation of the monoisotopic and

average masses of the peptides or proteins possible, also the simulation of the

proteolytic digestion of peptides and proteins, followed by searching for different

fragments having a known mass. Using this program is possible to introduce

different modifications at certain amino acid positions. One can obtain the

average and the monoisotopic values for [M+H]+ ions and also the values of

fragment ions having different charges. GPMAW 5.0 program may predict the

secondary structure and the hydrophobicity of a given protein or peptide

sequence.

3.10.2 Data Analysis

The Bruker Daltonics DataAnalysis 3.3 application is used for processing,

advanced data mining, and browsing analyses acquired on Bruker Daltonics

mass spectrometers. Data Analysis can load multiple analyses at a time and

allows for simple and reliable protein/ peptide analysis, structural elucidation,

and compound identification based on MS and MS/MS spectra. All processed

results are stored with an analysis for later retrieval or re-processing.

Automated deconvolution of multiple charged ions for analysis of biomolecules,

such as proteins and, peptides and oligonucleotides can be used.

3.10.3 Mascot

The large-scale and rapid identification and characterization of proteins

represents a central step in the understanding of a proteome. Identification

involves the matching of analytical data against protein databases in order to

assign a protein as already known or to describe it as novel. For protein

identification, Mascot (Matrix Science Ltd., London) with MS/MS ion search

engine and peptide mass fingerprint (PMF) (http://www.matrixscience.com)

search engines was used. The search was carried out within the NCBInr protein

database, which is a compilation of several databases including Swiss-Prot,

PIR, PRF, PDB and GenBank. The National Center for Biotechnology


Information (NCBI) has made strong efforts to cross-reference the sequences in

these databases in order to avoid duplication.

3.10.4 HyperChem 6.0

HyperChem 6.0 software was used for the molecular dynamic simulation

of proteins and peptides. AMBER96 parameters were applied for the geometry

optimization and the calculations were performed taking into account the

presence of water molecules.

3.10.5 BallView 1.1.1

BALLView is a free molecular source software employed in this work for

the visualization and modelling of molecular structures [346]. The program is able

to import structures from the most common molecular structure formats (PDB,

MOL, MOL2 and SD). Structures can be visualized with all standard graphical

models and coloring methods. Different parts of a molecule can be freely

visualized and selected for special tasks.

3.10.6 PDQuest software

For imaging and analyzing 2D gels, PDQuest software was used. After the

gel has been scanned advanced algorithms are available to remove

background noise, gel artifacts, and horizontal or vertical streaking from the

image.

3.11 SAW biosensor

The biosensor system used (S-sens® K5 System, Biosensor GmbH, Bonn,

Germany) employs surface acoustic waves. The waves are produced through

inverse piezoelectric effect on the surface of quartz chip covered with a thin

layer of gold. Viscosity changes and mass loadings on the chip’s surface affect

the phase and amplitude of the acoustic waves, which are transformed back

into electrical signal through direct piezoelectric effect. For achieving high


sensitivity in detecting interactions that take place in solution, the instrument

uses special shear waves of Love type. Their confinement to a very thin layer of

substance, as well as the displacement of matter parallel to the interface solid-

liquid, permits a good conservation of the energy of the Love waves, which

increases their susceptibility to surface effects (i.e. mass loading and viscosity

changes) and lowers the noise level in the signal.

The instrument is composed of the main measuring unit, containing a

quartz chip, electronics and a syringe pump, and an automated auto-sampler.

The central element is the ST-cut quartz sensor chip containing five sensor

elements, where the surface acoustic waves are produced, allowed to travel

along the surface and transformed back into electrical signal for analysis. Mass

loading on the chip’s surface and liquid viscosity changes in the liquid running

on it will induce modifications in the amplitude and phase of the acoustic wave.

In particular, mass loading will cause phase shifts, whereas viscosity changes

produces modifications in both phase and amplitude. The quartz chips are

covered with a thin gold coating, used for immobilization of different compounds

containing sulfur (i.e. thiol groups). In the present work 16-

mercaptohexadecanoic acid was used as a linker. By immersing the chip

overnight in a 10 μM solution of 16-mercaptohexadecanoic acid (5.77 mg acid

in 2 mL chloroform) a so caller self assembled monolayer (SAM) is formed,

whereby the thiol groups bind to the gold surface, the hydrocarbonated chains

align parallel one to another due to hydrophobic effect, and the carboxyl groups

orient themselves at the free surface. Different compounds containing free

amino groups can be covalently immobilized by forming a peptide bond with

these groups. However, the reactivity of the carboxyl groups must be enhanced

for the formation of the peptide bond. This can be achieved by modifying the

carboxyl into an active ester. Once the chip is placed inside the instrument, one

can set a specific flow rate for the buffer as well as the details concerning the

injections of different solutions from autosampler. First injection will contain a

mixture of 200 mM EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) and

50 mM NHS (N-hydroxysuccinimide) solutions, in a volumetric ratio of 1:1

(Figure 116). The resulting hydroxysuccinimide ester reacts with the compound


to be immobilized (peptide, protein or antibody), injections of different

concentrations and volumes being used. The free remaining active ester groups

are capped with 1 M solution of ethanolamine, pH 8.5. Injecting solutions of

different compounds, their affinity to the immobilized partner can be observed.

For eluting the affinity bound compounds from the chip, were employed acidic

conditions, injecting for example glycine 50 mM (adjusted to pH 2.0) or HCl 0.1

M (pH 1.0) [347]. All affinity interactions were studied in PBS buffer. For KD

determinations, the affinity was investigated at different concentrations of

analyte found in solution, in a sequential series of injections, each of them

followed by an acidic elution step (regeneration). The gold coated chips can be

reused after a thorough cleaning step with Piranha-solution (H2O2 30% / H2SO4

98% 1:1) for 45 min, that would each all organic compounds on the surface.

O-

O

NCN

N

NCHN

N

NCHN

NO

O

NO

OHO

H+

H+

NHCHN

NO

O

N

O

O

ONHCHN

NOO

-H+

+

+

Figure 116: Outline of the mechanism of reaction in the EDC/ NHS activation of

carboxyl group.

The quantity of bound compound to the chip can be evaluated from the

measured phase shift (recorded with the K15 software) using the following

sensitivity calibration factor:


515 =φ (°)

m (µg) x A(cm2) The measurements were recorded and the instrument was controlled by the

Biosense K12 software. The series of injections from autosampler was

programmed using the SequenceMaster 6.0 software. The binding curves were

analyzed with Origin Pro 7.5 and its engine was also employed by FitMaster for

fitting the binding curves according to a mathematical model that considered a

remaining residue affinity bound to the surface even after the acidic elution step,

as well as for determining the dissociation and association reaction rates (koff

and kon) from the shape of the fitted curves. A linear best fit was applied using

the equation Kobs = koff + kon * C. The average koff [Unit in sec-1] equals the

intersection with the y-axis. The slope of the fitted straight line is a measure of

the kon rate [Unit in Conc-1 sec-1]. The KD value was calculated with KD = koff /

kon.

3.12 Online of SAW-ESI-MS

The online combination of a surface acoustic wave biosensor with

electrospray ionization mass spectrometry, SAW-ESI-MS, is a highly efficient

technique recently developed in our laboratory, which enables the direct

detection, identification, and quantification of affinity-bound ligands together with

dissociation constant (KD) determination in a wide range of affinities [309]. The

online coupling between the SAW-sensor chip and the ESI-MS source was

achieved by interfacing the two instruments with a guard column, installed on a

Rheodyne six-port valve, to which also an HPLC instrument was connected,

used for obtaining precise composition and constant flow of solvents for the

sample elution from the guard column. The interface provides both ligand

concentration and in situ desalting step for the dissociated complex (Figure

117).


Figure 117: General scheme of the SAW-ESI-MS system: the SAW biosensor

(containing the gold covered quartz chip) is connected to ESI-MS through an interface consisting of a Rheodyne six ports injection valve, with an extra front needle port. A guard column for sample desalting and concentration is inserted in the loop of the injection valve, being the central element of the interface. An HPLC instrument is also connected to the interface, used as solvent delivery system. The blue arrows indicate the path of the analyte from SAW biosensor (where the bioaffinity is investigated) through the guard column (where the desalting occurs) to the ESI-MS (for ligand identification). The flow rates are given on the path arrows.

In a typical coupling experiment, the antibody was immobilized on the

chip surface by injecting a solution of 200 nM. Then, the peptide or protein was

allowed to interact with the antibody at a concentration of 10 µM in PBS buffer.

The affinity bound peptide or protein was eluted with 50 mM glycine at pH 2 at a

flow rate of 20 µL min-1. During the elution injection, the exit capillary of the

biosensor was connected to the inject unit, and the liquid that washed the chip

was allowed to flow through the guard column. The analyte on the column was

then washed at a flow rate of 70 µL min-1 with a mixture containing 5% solvent

B, delivered by the HPLC system. After one minute, the composition of the

mixture was changed to 75% solvent B. The eluted peptide or protein sample

from the column was directed to the ESI source of the mass spectrometer. After


acquiring the MS signal, the guard column was washed thoroughly with solvent

B and then equilibrated with solvent A, for a new experiment.

The SAW biosensor has been employed as an affinity detection method

in conjunction with MALDI-MS for epitope determination similar to the

procedures described in 2.4.3. Both epitope excision/ extraction approaches

have been experimentally carried out, although only epitope extraction was

successful. The antibody immobilization was performed using a single injection

of 150 µL solution at a concentration of 200 nM. For the epitope extraction

experiments the antigen, αSyn (1) was first digested with trypsin and Glu-C for 2

h at 37°C, it was presented to the antibody immobilized on sensor chip coated

with SAM. Further, the affinity bound epitope peptides were eluted under acidic

conditions (pH 2), collected, lyophilized and then analyzed by mass

spectrometry. MALDI-TOF-MS measurements were performed by Bruker

personnel at Bruker Daltonics, Bremen, Germany using UltrafleXtreme MALDI-

TOF/TOF [310]. Table 33 summarizes the epitope peptides obtained with this

approach.

Table 33: Proteolytic peptides present in the elution fractions of αSyn by epitope extraction-offline SAW-MS:

No. Antibody Enzyme Peptide sequence

[M+H]+ exp a [M+H]+ calc

b ∆m (ppm)

1 Anti-α/βSyn trypsin 81-96 1478.839 1478.7849 -36.58

61-80 1928.109 1928.0447 -33.35

59-80 2157.256 2157.1874 -31.80

Glu-C 47-83 3633.872 3633.9810 29.99

2 Anti-αSyn m 4B12 trypsin 81-96 1478.875 1478.7849 -60.93

61-80 1928.134 1928.0447 -46.32

59-80 2157.268 2157.1874 -37.36

Glu-C 47-83 3633.850 3633.9810 36.05

3 Anti-αSyn mBD trypsin 81-96 1478.802 1478.7849 -11.56

61-80 1928.056 1928.0447 -5.86

59-80 2157.200 2157.1874 -5.84

Glu-C 62-83 2128.123 2128.1608 17.76

58-83 2614.392 2614.4410 18.74

47-83 3633.902 3633.9810 21.74


No. Antibody Enzyme Peptide sequence

[M+H]+ exp a [M+H]+ calc

b ∆m (ppm)

4 Anti-αSyn pC20 trypsin 81-96 1478.801 1478.7849 -10.89

61-80 1928.082 1928.0447 -19.35

7-34 2727.448 2727.5363 32.37

Glu-C 47-83 3633.872 3633.9810 29.99 a Mass spectrometric analysis (UltrafleXtreme MALDI-TOF/TOF, Bruker Daltonics) b GPMAW software 5.0 (Lighthouse Data, Denmark)

3.13 Edman sequence determination

In the most useful method of N-terminal residue identification, the Edman

degradation occurs in three stages, each requiring different conditions [270].

Amino acid residues can therefore be sequentially removed from the N-terminus

of a polypeptide in a controlled stepwise fashion. The phenyl isothiocyanate

(PITC) reacts with the N-terminal amino groups of proteins under mildly alkaline

conditions to form their phenylthiocarbamyl (PTC) adduct. This product is

treated with anhydrous hydrofluoric acid, which cleaves the N-terminal residue

as its thiazolinone derivative but does not hydrolyze other peptide bonds. The

thiazolinone- amino acid is selectively extracted into an organic solvent and by

treatment with aqueous acid is converted to the more stable

phenylthiohydantoin (PTH) derivative that can be identified by RP-HPLC with

detection at 270 nm.


Figure 118: The Edman degradation occurs in three stages, each requiring different

conditions: 1) mildly alkaline conditions; 2) anhydrous hydrofluoric acid; 3) aqueous acid. Phenylthiohydantoin (PTH) derivative can be identified by RP-HPLC with detection at 270 nm.

For N-terminal sequence determination, proteins were first separated by

polyacrylamide (15%) gel electrophoresis and transferred onto a PVDF

membrane (0.2 µm) (BioRad, München, Germany). Further, the PVDF

membranes were washed with water for 15 min, then with methanol for 5-10

sec, and incubated in the staining solution (0.1% Coomassie Brilliant Blue R-

250 in 40% methanol in water) until protein spots became visible. The

membranes were then destained in destaining solution (50% methanol in water)

until the bands were visible. The membranes were air-dried. The spots of

interest were excised, destained with 100% methanol and applied into the


sequencing cartridge. Sequence determinations were performed on an Applied

Biosystems Model 494 Procise Sequencer attached to a Model 140C

Microgradient System, a 785A Programmable Absorbance Detector, and a

610A Data Analysis System. All solvents and reagents were of highest available

purity (Applied Biosystems). The sequencing experiments were done by Adrian

Moise, Laboratory of Analytical Chemistry and Biopolymer Structure Analysis,

Department of Chemistry of the University of Konstanz.

4 SUMMARY 202

4 SUMMARY

In recent years, mass spectrometry (MS) has become a major analytical

tool in biochemistry and structural biology. In particular, electrospray mass

spectrometry (ESI-MS) has emerged as a powerful technique for analyzing

intact gas phase ions from large biomolecules and supramolecular complexes.

However, in contrast to the large number of ESI-mass spectrometric studies of

protein structures, structure modifications and proteomics, the molecular

characterization of protein “misfolding” and aggregation species by mass

spectrometry had hitherto little success; possible explanations are (i), the low

concentration of intermediate species, and (ii), the slow rates of aggregate

formation in vitro. Conventional “soft-ionization” mass spectrometric methods

such as ESI-MS and HPLC-MS are not suitable to direct “in-situ” analysis of

conformational states and intermediates at different concentrations. Recently,

ion-mobility mass spectrometry (IMS-MS) is emerging as a new tool to probe

complex biomolecular structures, due to its potential to separate mixtures of

protein complexes by conformation state, spatial shape and topology. Thus,

IMS-MS implements a new mode of separation that allows the differentiation of

protein conformational states.

One of the hallmarks of Parkinson’s disease (PD) and related neurological

disorders is the accumulation in human brain of intracellular high molecular

weight α-synuclein (αSyn) aggregation products as fibrils. Oligomeric

intermediates have been suggested to represent major neurotoxic species;

however, chemical structures of αSyn oligomers and possible intermediates

have not been hitherto identified. The first parts of this thesis deal with the

isolation and structure identification of αSyn oligomerization-aggregation

products in vitro and in vivo.

The first part was focused on the molecular characterization of human

αSyn (wt-αSyn) and two mutants, αSyn (A53T) and αSyn (A30P) using HPLC,

high resolution mass spectrometry, gel electrophoresis and circular dichroism

4 SUMMARY 203

spectroscopy. Additional information about the wt-αSyn structure was obtained

by molecular dynamic simulation, which indicated a flexible, random coil

structure. Using mass spectrometric methods, proteolytic degradation studies of

synucleins were carried out for identifying the cleavage specificity and preferred

cleavage sites of different proteases.

The second part of the thesis was focused on the in vitro and in vivo

characterization of αSyn oligomerization-aggregation products, employing gel

electrophoresis, Dot blot and Western immunoblotting. The in vitro

oligomerization of αSyn was carried out by incubation at 37°C in sodium-

phosphate (pH 7.5) for up to seven days. The formation of oligomers was

monitored by Tris-tricine polyacrylamide gel electrophoresis which revealed

bands corresponding to monomeric and oligomer-like αSyn at approximately 37

and 48 kDa. In addition, three bands of minor abundances with molecular

weights lower than full-length αSyn indicated the formation of truncation or

degradation products. The bands corresponding to αSyn monomer and dimer

were excised from the gel, digested with trypsin and analyzed by HPLC-ESI-

MS. ESI-MS and tandem-MS of the tryptic peptides revealed the presence of

monomeric and dimeric αSyn with full-length sequences, respectively; additional

structural characterization was obtained by Edman sequencing. Direct ESI-MS

of αSyn upon incubation in an aqueous buffer for 3 hours provided the

identification of small amounts of N-terminally truncated αSyn (7-140). In

contrast to these results, attempts to identify the truncation and degradation

products by direct mass spectrometric analysis and HPLC-MS were

unsuccessful, presumably due to their low concentration.

The application of ion-mobility mass spectrometry (IMS-MS) to

oligomerization-aggregation mixtures of αSyn in vitro enabled the first

identification of specific fragments corresponding to truncation and degradation

products that have been previously observed by gel electrophoresis, but not

identified. Most important, a highly aggregating fragment was identified,

resulting from cleavage at the central aggregation domain of αSyn, between

residues Val-71 and Thr-72 (7.2 kDa). These results showed that IMS-MS can

4 SUMMARY 204

be successfully applied to the identification of hitherto undetected truncation

products of neurodegenerative target proteins. Aggregation studies of the

corresponding carboxy-terminal fragment, αSyn (72-140) prepared by both

chemical synthesis and recombinant expression, showed a substantially faster

rate of fibrillization compared to the intact full-length αSyn protein. The chemical

structure elucidation of in vivo αSyn oligomerization products from human αSyn

transgenic mouse brain homogenates and human neuroblastoma cell cultures

was performed using two-dimensional polyacrylamide gel electrophoresis (2D-

PAGE) and high resolution mass spectrometry, as well as affinity binding

studies by online combination of a surface acoustic wave (SAW) biosensor and

ESI-MS. Immunoblotting in combination with high resolution- MS provided the

identification of αSyn (A30P) from soluble mouse brain homogenate proteins.

Spots that were detected as immunoreactive with the anti-αSyn mBD antibody

were analyzed by high resolution- MS, leading to the identification of αSyn and

of two methionine oxidized products (116Met and 127Met).

In a third part of the thesis, the epitope structures recognized by anti-αSyn

specific antibodies were identified. The mass spectrometric analysis was

combined with epitope excision and extraction procedures previously developed

in our laboratory. For the epitope excision and extraction experiments, affinity

columns were prepared by immobilizing the antibodies on Sepharose. For

protein digestion trypsin and Glu-C endoproteases were employed, and for

analysis MALDI-TOF-MS, as well as ESI-ion trap-MS used. The epitope

excision, followed by mass spectrometric analysis provided direct information

that the epitopes recognized by polyclonal antibodies (pASY-1; pC20) were

discontinuous and located in the N-terminal (1-23) and central (59-80) regions.

Comparative binding studies of anti-αSyn specific antibodies with the epitope

peptides αSyn (1-23) and αSyn (59-80), and synthetic mutant model peptides

were performed by ELISA, Dot blot and affinity- mass spectrometry. The results

showed that αSyn (1-23) and αSyn (59-80) bound to the anti-αSyn pASY-1

antibody in a concentration- dependent manner.

4 SUMMARY 205

A further part of the dissertation was focused on applications of an online

combination of a surface acoustic wave (SAW) biosensor with ESI-MS that

enabled the direct detection, identification, and quantification of affinity-bound

ligands from synuclein- anti-synuclein antibodies complexes on a biosensor

chip. The specific antibodies were covalently immobilized on the gold coated

surface of the quartz chip. The interactions with wt-αSyn, mutants αSyn (A53T),

αSyn (A30P), αSyn peptides and βSyn were determined. The obtained KD

values were in the low nano-molar range and in good agreement with

bioaffinities investigated by Dot Blot, ELISA, SAW biosensor and SAW-ESI MS.

Moreover, the SAW biosensor has been employed as an affinity detection

method in conjunction with MALDI-MS for epitope determination of αSyn.

In the last part of the thesis, chemical stability studies of wt-αSyn, αSyn

(A53T), αSyn (A30P) and βSyn proteins were performed. The formation of

oligomers was monitored by Tris-tricine polyacrylamide gel electrophoresis, Dot

blot, Western blot, and Thioflavin-T assay. It was shown that aggregation rates

increased in the order αSyn (A30P) < wt-αSyn < αSyn (A53T). In comparison to

the sequence of αSyn, βSyn which lacks 11 central hydrophobic residues did

not form any aggregates. First interaction studies between αSyn (59-80 T72A)

peptide and αSyn at various molar ratios showed that this peptide comprising

the aggregation domain provided a significant increase of the aggregation rate

of wt-αSyn. In contrast, chemical modification of αSyn by succinylation

stabilized its structure and completely inhibited degradation and

oligomerization-aggregation.


5 ZUSAMMENFASSUNG

Die Massenspektrometrie hat sich in den letzten Jahren als eine wichtige

Methode der Strukturanalyse in der Biochemie und Molekularbiologie erwiesen.

Insbesondere die Elektrospray- Massenspektrometrie (ESI-MS) hat sich als

leistungsfähige Methode der Analyse von Biopolymer- Ionen und von nicht-

kovalenten supramolekularen Komplexen etabliert. Während ESI-

massenspektrometrische Techniken in vielen Untersuchungen von

Proteinstrukturen, Strukturmodifikationen sowie zur Proteomanalytik eingesetzt

wurden, waren Anwendungen zur Analyse von Protein- Fehlfaltung und –

Aggregation bisher nur wenig erfolgreich; mögliche Ursachen hierfür sind (i), die

geringen Konzentrationen von reaktiven Intermediaten, sowie (ii), die

langsamen Bildungsgeschwindigkeiten der enstehenden Aggregate.

Konventionelle „soft-ionization“ Methoden wie ESI-MS und HPLC-MS sind für

die direkte Analyse von Intermediaten der Proteinaggregation nicht geeignet.

Dagegen hat sich die Ionenmobilitäts- Massenspektrometrie (IMS-MS) in

jüngster Zeit als neue, leistungsfähige Methode zur Analyse von komplexen

Biomolekülstrukturen in geringen Konzentrationen entwickelt. Aufgrund der

effizienten Trennung von Protein- Komplexen und –Gemischen mit

unterschiedlicher Konformation und Topographie stellt die IMS-MS eine neue

Methode der Trennung und Differenzierung von Intermediaten von Protein-

Aggregaten dar.

Die Akkumulierung von hochmolekularen Aggregaten und fibrillären

Strukturen des Hirnproteins Alpha-Synuclein (αSyn) ist ein charakteristisches

Merkmal der Parkinson’schen Krankheit und verwandter neurologischer

Erkrankungen. Dabei wird die Bildung von oligomeren Intermediaten hoher

Neurotoxizität generell als Schlüsselreaktion angenommen; jedoch konnten die

Strukturen der Oligomeren und deren möglicher Zwischenprodukte bisher nicht

identifiziert werden. Zielsetzungen der vorliegenden Dissertation waren die

Isolierung, elektrophoretische und chromatographische Trennung,


Strukturaufklärung, sowie die Darstellung und biochemische Charakterisierung

von αSyn- Oligomeren.

In einem ersten Teil der Arbeit wurde die Primär- und Sekundärstruktur-

Charakterisierung von rekombinantem αSyn (wt-αSyn), sowie von zwei

Mutanten [αSyn (A53T); αSyn (A30P)] durchgeführt, vor allem mit

massenspektrometrischen, elektrophoretischen Methoden sowie CD-

Spektroskopie. Zusätzliche Strukturinformationen wurden mittels

Molekülmodell- Simulation erhalten, die eine flexible ungeordnete Struktur der

α-Synucleine zeigten. Durch massenspektrometrische Peptide-mapping-

Analyse wurden proteolytische Abbaureaktionen mit verschiedenen Proteasen

charakterisiert.

In einem zweiten Teil wurden Oligomerisierung- Aggregationsprodukte

von αSyn in vitro mittels Gelelektrophorese sowie immunanalytischer Methoden

(Immunoblot; ELISA) charakterisiert. Oligomerisierungsreaktionen wurden in

Phosphat- Puffer (pH 7.5) mittels Tricin- Gelelektrophorese über verschiedene

Zeiten bis zu 7 Tagen verfolgt. Die gelelektrophoretische Analyse zeigte

Monomere und Oligomere (ca. 37 und 48 kDa) sowie mehrere Banden mit

niedrigeren Molekulargewichten und geringerer Intensität als das monomere

αSyn (15 kDa), die auf Abbauprodukte hinwiesen. Die Gelbanden entsprechend

dem monomeren und dimeren αSyn wurden isoliert und nach Abbau mit

Trypsin durch HPLC-MS charakterisiert. Die massenspektrometrischen

Ergebnisse sowie zusätzliche Edman- Sequenzierung der Banden ergaben

jeweils vollständige αSyn- Sequenzen. Ein erstes Abbauprodukt mit trunkierter

N-terminaler Sequenz αSyn (7-140) konnte nach 3-stündiger Inkubation durch

ESI-Massenspektrometrie nachgewiesen werden; dagegen waren Versuche zur

direkten massenspektrometrischen Analyse der αSyn- Abbauprodukte ohne

Erfolg.

Die Identifizierung von αSyn- Proteinfragmente gelang erstmals mit Hilfe

der Ionenmobilitäts-Massenspektrometrie (IMS-MS), wobei Abbauprodukte


(Trunkierung) sowie proteolytische Fragmente entsprechend den

elektrophoretischen Proteinbanden nachgewiesen wurden. Von besonderem

Interesse war die Identifizierung eines C-terminalen Proteinfragments durch

Spaltung in der zentralen Aggregationsdomäne (Val71-Thr72), das hohe

Reaktivität der Aggregationsbildung zeigte. Diese Ergebnisse zeigen die

erfolgreiche Anwendung der IMS-MS zur direkten Aufklärung der bisher nicht

identifizierbaren Abbauprodukte. Erste Aggregations- Untersuchungen des

durch chemische Festphasensynthese sowie durch rekombinante Expression in

E. Coli dargestellten Fragments αSyn (72-140) zeigten eine erhebliche höhere

Aggregationsgeschwindigkeit im Vergleich zum intakten αSyn. Weitere

Strukturuntersuchungen von αSyn- Oligomerisierungsprodukten in vivo aus

Hirnhomogenat von transgenen Mäusen, sowie aus humanen

Neuroblastomzellen wurden mittels Gelektrophorese, hochauflösender

Massenspektrometrie, sowie durch Kombination eines Bioaffinitäts (Biosensor)-

Systems und ESI-MS durchgeführt. Die elektrophoretischen Banden wurden mit

einem αSyn- spezifischen Antikörper nachgewiesen, und nach Isolierung aus

dem Gel die Mutante αSyn (A30P) sowie Strukturmodifikationen durch Met-

Oxidation identifiziert.

In einem dritten Abschnitt der Arbeit wurden Untersuchungen zur

Aufklärung der Epitopstrukturen von αSyn- spezifischen Antikörpern unter

Anwendung der in unserem Laboratorium entwickelten proteolytischen

Excisions- und Extraktions- Methoden in Kombination mit Massenspektrometrie

durchgeführt. Hierzu wurden Sepharose-immobiliserte Antikörper zum in situ

Abbau mit Trypsin und Glu-C-Endoprotease hergestellt, und MALDI-TOF-MS

sowie ESI-MS angewendet. Die massenspektrometrischen Ergebnisse ergaben

für zwei polyklonale αSyn- Antikörper (ASY-1; pC20) diskontinuierliche

Epitopstrukturen im N-terminalen Bereich (1-23) sowie der zentralen

Aggregationsdomäne (59-80). Die Epitope- Peptide sowie weitere αSyn-

Partialsequenzen wurden durch Festphasensynthese dargestellt und mit

verschiedenen Antikörpern mittels ELISA, Immunoblot und Affinitäts-

Massenspektrometrie charakterisiert.


In einem weiteren Abschnitt wurden Epitopstrukturen von αSyn-

spezifischen Antikörpern, sowie Antikörper- Interaktionen verschiedener

Synucleine und αSyn- Partialpeptide durch direkte (online) Kombination eines

SAW (Surface-acoustic-wave)- Biosensors und ESI-MS charakterisiert.

Interaktionen wurden für wt-αSyn, αSyn- Mutanten, βSyn sowie verschiedene

αSyn- Partialsequenzen bestimmt und ergaben Affinitätskonstanten bzw. KD-

Werte im Bereich von 12- ca. 400 nmol, in Übereinstimmung mit den

Affinitätsuntersuchungen der Antikörperspezifitäten mit anderen Methoden

(ELISA; Affinitäts-Massenspektrometrie).

Im letzten Teil der Arbeit wurden vergleichende Untersuchungen zur

chemischen Stabilität und in vitro- Aggregation von αSyn, Mutanten des

humanen αSyn, sowie von βSyn mittels Gelelektrophorese, sowie mit dem

Thioflavin T-Assay durchgeführt. Mit der Thioflavinmethode konnte gezeigt

werden, dass die Aggregationsgeschwindigkeit der αSyn- Mutante (A30P)

geringer ist im Vergleich zum wt-αSyn, dagegen zeigte die Mutante αSyn

(A53T) eine erhöhte Aggregationsgeschwindigkeit. Im Gegensatz zu den αSyn

zeigte das βSyn, bei dem die zentrale hydrophobe Domäne (73-83) fehlt, weder

Oligomerisierung noch nachweisbare Aggregation. Erste Untersuchungen zur

Interaktion und Aggregation von αSyn- Peptiden mit dem wt-αSyn ergaben eine

erhöhte Aggregationsgeschwindigkeit durch die Peptidmutante αSyn (59-80

T72A). Im Gegensatz dazu zeigte ein durch Succinylierung aller Aminogruppen

modifiziertes αSyn hohe Stabilität und keine Oligomerisierung und Aggregation.

6 BIBLIOGRAPHY 210

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7 APPENDIX 236

7 APPENDIX

7.1 Appendix 1

Abreviations Name

aa amino acid

ACN Acetonitrile

Ab Antibody

APS Ammoniumperoxodisulfat

BSA Bovine serum albumine

CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-

propanesulfonate

CID collision-induced dissociation

CD Circular dichroism

CDR Complementary determining region

Da Dalton

2-DE 2-dimensional gel electrophoresis

DHB Dihydroxybenzoic acid

∆m Mass difference

DMF Dimethylformamide

DTT Dithiothreitol

ELISA Enzyme linked immunosorbent assay

ESI-MS Electrospray ionization- Mass spectrometry

FTICR Fourier transform-ion cyclotron resonance

Fmoc 9-Fluorenylmethoxycarbonyl

HCCA 4-Hydroxy-α-cynamic acid

HPLC High performance liquid chromatography

h hours

IEF Isoelectric focusing

IPG Immobilized pH gradient

IgG γ-Immunglobulins

7 APPENDIX 237

mAb Monoclonal antibody

MALDI-MS Matrix-assisted laser desorption-/ionization-mass

spectrometry

min Minute

m/z Mass over charge ratio

MS Mass spectrometry

NMM N-Methyl-morpholine

OD optical density

pAb Polyclonal antibody

PBS Phosphate buffered saline

PD Parkinson disease

pH Negative logarithm of H3O+-iones concentration

ppm Parts per million

PyBOP Benzotriazol-1-yloxy-tris-pyrrolidinophosphonium-PF6-salt

RP Reversed phase

Rt retention time

SDS-PAGE Sodiumdodecylsulfat-Polyacrylamid-Gel electrophoresis

T Tesla

TBS Tris buffer saline

TCA Trichloroacetic acid

TEMED N,N,N',N'-Tetramethylethylendiamine

TFA Trifluoroacetic acid

TOF Time of flight

Tris Tris-(hydroxymethyl-) aminomethane

Tween Polyoxyethylen Sorbitan Monolaurat

UV Ultraviolet

V Volt

°C Grad Celsius

7 APPENDIX 238

7.2 Appendix 2

Table 34: Amino acids abbreviations:

Name One letter code

Three letter code Monoisotopic mass (Da)

Alanine A Ala 71.037

Arginine R Arg 156.101

Asparagine N Asn 114.042

Aspartic acid D Asp 115.026

Cysteine C Cys 103.009

Glutamine Q Gln 128.058

Glutamic acid E Glu 129.042

Glycine G Gly 57.021

Histidine H His 137.058

Isoleucine I Ile 113.084

Leucine L Leu 113.084

Lysine K Lys 128.094

Methionine M Met 131.040

Phenylalanine F Phe 147.068

Proline P Pro 97.052

Serine S Ser 87.032

Threonine T Thr 101.047

Tryptophan W Trp 186.079

Tyrosine Y Tyr 163.063

Valine V Val 99.068

7 APPENDIX 239

Table 35: Approximate pKa values of ionizable groups of amino acids and peptides:

Residue Functional group

(Proton donor) (Proton acceptor)

pKa

C-terminal -COOH -COO- 3.1

Asp -COOH -COO- 3.9

Glu -COOH -COO- 4.3

His -NH3+ -NH2 6

N-terminal -NH3+ -NH2 8

Cys -SH -S- 8.5

Tyr -OH -O- 10.5

Lys -NH3+ -NH2 10.5

Arg -NH3+ -NH2 12.5

Ser -OH -O- 13

Thr -OH -O- 13

7 APPENDIX 240

7.3 Appendix 3

Table 36: Amino acid sequences of synucleins: the amino acids variations in the sequence compared to the wild type αSyn (wt-αSyn) are highlighted in red.

Code Protein Sequence

1 αSyn MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA

2 βSyn MDVFMKGLSM AKEGVVAAAE KTKQGVTEAA EKTKEGVLYV GSKTREGVVQ GVASVAEKTK EQASHLGGAV FSGAGNIAAA TGLVKREEFP TDLKPEEVAQ EAAEEPLIEP LMEPEGESYE DPPQEEYQEY EPEA

3 αSyn (A30P) MDVFMKGLSK AKEGVVAAAE KTKQGVAEAP GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA

4 αSyn (A53T) MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVTTVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA

5 αSyn (1-120) MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP

6 αSyn (61-140) MEQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA

7 αSyn (96-140) MKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA

8 αSyn (71-140) H-VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA-NH2

9 αSyn (72-140) H-TGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA-NH2

10 αSyn (72-140) TGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA

7 APPENDIX 241

7.4 Appendix 4

0

0.5

1

1.5

2

1:885

7350

0

1:295

2450

0

1:984

1500

1:328

0500

1:109

3500

1:364

500

1:121

500

1:405

00

1:135

00

1:450

0

1:150

01:5

00

Antibody dilutions

OD

(450

nm

)

Anti-α/βSyn

Anti-αSyn m4B12Anti-αSyn mBDAnti-αSyn pC20

Anti-αSyn pASY-1

Figure 119: ELISA binding of the antibodies to wt-αSyn. αSyn was first added (0.5 μM

in PBS, 100 μL per well) in triplicate, for 2 h at 25°C. Next, 12 three fold serial dilutions of antibody were added to the plate. The binding of the antibodies to wt-αSyn was assessed by incubating HRP-conjugated goat anti-mouse or anti-rabbit antibodies (1 h at 25°C) and measuring the absorbance with an ELISA reader.

2126.1916

2000 2100 2200 2300 2400 2500 m/z

[M+H] + exp : 2126.1916 Da[M+H] + calc : 2126.1928 Da∆m = 0.56 ppm

H-59TKEQVTNVGGAVVAGVTAVAQK80-NH2

a

26.13

0 10 20 30 40 50

0

50

100

150

200

250

300

Abs

orba

nce

(mA

u)

Time (min)

[M + H] +

7 APPENDIX 242

709.8

1064.2

2127.3

500 1000 1500 2000 2500 m/z

+1

+2

+3709.8

1064.2

2127.3

500 1000 1500 2000 2500 m/z

+1

+2

+3

[M+H] + exp : 2127.3 Da[M+H] + calc : 2127.4 Da∆m = 47 ppm

H-59TKEQVANVGGAVVTGVTAVAQK80-NH2

b

0 10 20 30 40 500

50

100

150

200

250

300

Abs

orba

nce

(mA

u)

Time (min)

25.36

0 10 20 30 40 500

50

100

150

200

250

300

Abs

orba

nce

(mA

u)

Time (min)

25.36

c

H-59TKEQVTNVGGA69-NH2

0 10 20 30 40 500

50

100

150

20022.4

Abs

orba

nce

(mA

u)

Time (min)

551.8

1102.5

500 600 700 800 900 1000 1100 1200 m/z

1 +

2 +551.8

1102.5

500 600 700 800 900 1000 1100 1200 m/z

1 +

2 +


H-70VVTGVTAVAQK80-NH2

d

24.6

0 10 20 30 40 500

50

100

150

200

250

Abs

orba

nce

(mA

u)

Time (min)


536.4

1071.6

500 600 700 800 900 1000 1100 1200 m/z

1 +

2 +536.4

1071.6

500 600 700 800 900 1000 1100 1200 m/z

1 +

2 +

7 APPENDIX 243

1 +

H-81TVEGAGSIAAA91-NH2

24.5

0 10 20 30 40 50

0

50

100

150

200

25024.5

0 10 20 30 40 50

0

50

100

150

200

250

Abs

orba

nce

(mA

u)

Time (min)

945.4

750 850 950 1050 1150 m/z

945.4

750 850 950 1050 1150 m/z


e

Figure 120: Analytical RP-HPLC and MALDI-FTICR mass spectrum of pure: (a), αSyn

(59-80 T72A) and ESI-MS mass spectra of pure: (b), αSyn (59-80 T64A); (c), αSyn (59-69); (d), αSyn (70-80); (e), αSyn (81-91).

Dissertation Camelia Vlad .pdf Sequence - uni-konstanz.de

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