Clinical Proteomics:Clinical Proteomics:A Technology to shape the future?A Technology to shape the future?

Cyprus, Nov, 2010

Protein ChemistryProtein Chemistry//Proteomics/PeptideProteomics/PeptideSynthesisSynthesis and and ArrayArray UnitUnit

Institute of Biomedicine/AnatomyInstitute of Biomedicine/Anatomy

BiomedicumBiomedicum HelsinkiHelsinki, 00014 University of , 00014 University of Helsinki, FinlandHelsinki, Finland

E-Mail: marc.baumann@helsinki.fi

(http://research.med.helsinki.fi/corefacilities/proteinchem)

Over 1200 Researchers in only Medical Research(Cancer, Genetics, Developmental Medicine, Neuroscience etc.)

The Medical FacultyThe Medical Faculty

ProteomicsProteomics

What is it all about??What is it all about??

Proteomics in prostate cancer.

Banez LL, Srivastava S, Moul JW.

Center for Prostate Disease Research, Department of Surgery, Uniformed Services University of the Health Sciences, Rockville, Maryland, USA bDivision of Urology, Department of Surgery, Duke University Medical Center, Durham, North

Carolina, USA.

Curr Opin Urol. 2005 May;15(3):151-6.

Mol Cell Proteomics. 2005 Apr;4(4):345.

Proteomics of disease.

Celis JE, Korc M.

We know whatWe know what GenomicsGenomics is!is!

But, wBut, what hat is is ProteomicsProteomics??

2001: Human Genome Project Reveals

3,000,000,000 base pair nucleotides

= only 25,000 genes

=

Only 0.1% of each’s persons DNA differs from any other person

=

And that …And that …

The study of the Proteins expressed by a Genome

= Proteome

= Proteins

So… what makes the Difference?So… what makes the Difference?

same Genome - different Proteome

How can we study Proteomics?How can we study Proteomics?

The technology...The technology...

2D gel electrophoresis2D gel electrophoresis2D liquid chromatography2D liquid chromatography

Micro arraysMicro arrays

An An exampleexample

A technology to find changes in A technology to find changes in ProteomeProteome

Administration of a drug known to bind to an orphan receptor

• Changes in expression level of 23 proteins

A protein Array

TwoTwo--dimensional gel dimensional gel electrophoresis (2D)electrophoresis (2D)

1st dimension, IEF, Proteins are separatedaccording to their isoelectric point (IP) 2nd dimension, SDS-PAGE, Proteins are separated according to their molecular mass Efficient: More than a thousand proteins resolved in E-Coli cell lysates and ~8000 in brain lysates

pH 10

SDS-

PAG

E

pH 3

1st Dimension - Isoelectric Focusing

pH4 pH7

+ -+

pH5.5neutral

pI - isoelectric point

ready made Gel-strips

gelsurface

2DEMw

2nd Dimension - Isoelectric Focusing

Protein Fingerprint: 2Protein Fingerprint: 2--DEDE

pI 6.94.1

Mr(kDa)

200

15

AboutAbout 20002000proteinsproteins

2D-PAGE Direct In-situ Digest

To identify the separated proteins

Basic (+) Acidic (-)

#7

100 fmolrunning the gel- staining each spot of interest ( )- excise- in-gel digestion

PEPTIDE ANALYSIS

Trypsin digest

MALDI TOF/TOF

MALDI-TOF-MS

High Sample Throughput for thePost-Genomic Era

MALDI TOF

Schematic diagram of a Reflector MALDI-TOF mass spectrometer.

MALDI-TOF = Matrix Assisted Laser Desorption/Ionization -Time Of Flight

MALDI TOF/TOF

Quadrupole TOF/TOF

Quadrupole TOF/TOF

Quadrupole TOF/TOF

Typical mass spectrum from a protein band trypsin digest

1 RALRRAPALA AVPGGKPILC PRRTTAQLGP RRNPAWSLQA GRLFSTQTAE51 DKEEPLHSII SSTESVQGST SKHEFQAETK KLLDIVARSL YSEKEVFIRE101 LISNASDALE KLRHKLVSDG QALPEMEIHL QTNAEKGTIT IQDTGIGMTQ151 EELVSNLGTI ARSGSKAFLD ALQNQAEASS KIIGQFGVGF YSAFMVADRV201 EVYSRSAAPG SLGYQWLSDG SGVFEIAEAS GVRTGTKIII HLKSDCKEFS 251 SEARVRDVVT KYSNFVSFPL YLNGRRMNTL QAIWMMDPKD VGEWQHEEFY 301 RYVAQAHDKP RYTLHYKTDA PLNIRSIFYV PDMKPSMFDV SRELGSSVAL351 YSRKVLIQTK ATDILPKWLR FIRGVVDSED IPLNLSRELL QESALIRKLR401 DVLQQRLIKF FIDQSKKDAE KYAKFFEDYG LFMREGIVTA TEQEVKEDIA451 KLLRYESSAL PSGQLTSLSE YASRMRAGTR NIYYLCAPNR HLAEHSPYYE501 AMKKKDTEVL FCFEQFDELT LLHLREFDKK KLISVETDIV VDHYKEEKFE551 DRSPAAECLS EKETEELMAW MRNVLGSRVT NVKVTLRLDT HPAMVTVLEM601 GAARHFLRMQ QLAKTQEERA QLLQPTLEIN PRHALIKKLN HCAQASLAWL651 SCWWIRYTRT P

Number 1 match: tumor necrosis factor type 1 receptor associated protein TRAP-1 (Mr): 76030.27

Automated Database Search (MASCOT)Automated Database Search (MASCOT)

Total coverage: 33.4%

Even a single peptide is enough Even a single peptide is enough for Protein identification!for Protein identification!

We can do a peptide amino acid We can do a peptide amino acid sequence analysis by mass sequence analysis by mass

spectrometry!spectrometry!

2D-PAGE Direct In-situ Digest

(Unknown protein identification)

Basic (+) Acidic (-)

#7

100 fmolrunning the gel- staining each spot of interest ( )- excise- in-gel digestion

PEPTIDE ANALYSIS

Trypsin digest but take a single peptide to the MS!

MALDI MS TOF/TOF or Quadrupole MS TOF/TOF

MS-MS

Fragmentation of a single peptide inthe Mass Spectrometer

>>>> Database search

>>>> Identification of the whole protein

Number 1 match: tumor necrosis factor type 1 receptor associated protein TRAP-1 (Mr): 76030.27

Automated Database Search (MASCOT)Automated Database Search (MASCOT)

Sequence successfully identified

1 RALRRAPALA AVPGGKPILC PRRTTAQLGP RRNPAWSLQA GRLFSTQTAE51 DKEEPLHSII SSTESVQGST SKHEFQAETK KLLDIVARSL YSEKEVFIRE101 LISNASDALE KLRHKLVSDG QALPEMEIHL QTNAEKGTIT IQDTGIGMTQ151 EELVSNLGTI ARSGSKAFLD ALQNQAEASS KIIGQFGVGF YSAFMVADRV201 EVYSRSAAPG SLGYQWLSDG SGVFEIAEAS GVRTGTKIII HLKSDCKEFS 251 SEARVRDVVT KYSNFVSFPL YLNGRRMNTL QAIWMMDPKD VGEWQHEEFY 301 RYVAQAHDKP RYTLHYKTDA PLNIRSIFYV PDMKPSMFDV SRELGSSVAL351 YSRKVLIQTK ATDILPKWLR FIRGVVDSED IPLNLSRELL QESALIRKLR401 DVLQQRLIKF FIDQSKKDAE KYAKFFEDYG LFMREGIVTA TEQEVKEDIA451 KLLRYESSAL PSGQLTSLSE YASRMRAGTR NIYYLCAPNR HLAEHSPYYE501 AMKKKDTEVL FCFEQFDELT LLHLREFDKK KLISVETDIV VDHYKEEKFE551 DRSPAAECLS EKETEELMAW MRNVLGSRVT NVKVTLRLDT HPAMVTVLEM601 GAARHFLRMQ QLAKTQEERA QLLQPTLEIN PRHALIKKLN HCAQASLAWL651 SCWWIRYTRT P

What can Proteomics give us?What can Proteomics give us?

A typical case...A typical case...

ColorectalColorectal CryptCrypt -- PhysiologyPhysiology andand TransformationTransformation

Stem cells are located in the lower crypt poles, colonocytes are migrating to the surface of the crypt

Continuelly replacement in the toxic and mechanically stressing environment of the gut

Hyperproliferation is believed to precede adenoma formation

Loss of APC function leads to deregulated crypt homeostasis and is thought to be the initiating event in adenoma formation

Correlation of Histological Progression and MolecularChanges in Colorectal Cancer

Normal mucosa

Aberrant crypt foci

Early adenoma

Late adenoma

Invasive carcinoma

Metastasis

APC, -catenin mutation

K-ras mutation

18q loss, p53 mutation

Nm23 mut.

Samples

Normal

Polyp

Carcinoma

Liver metastasis

Tissue microdissection

Why Tissue Microdissection?

Normal Tissue

Tumor Tissue

You would like to isolate only the targeted diseased tissue

Laser-based microdissection:

Laser Capture Microdissection Laser Capture Microdissection (Arcturus Autopix)(Arcturus Autopix)

Laser Capture MicrodissectionLaser Capture Microdissection

Emmert-Buck et al (1996) Laser Capture Microdissection Science, Vol 274, Nro 8, p998-1001

Capturing of the vessels in the control Capturing of the vessels in the control brain slidebrain slide

Slide before capture

Slide after capture Cap with the selected tissue

Cap

*

Why Tissue Microdissection?

Cryostat section

Normal Tissue

Tumor Tissue

MALDI MS MALDI MS analysisanalysis directlydirectly fromfrom the the tissuetissuecapturedcaptured on the on the capcap membranemembrane

Electron micrograph, dermal artery with Electron micrograph, dermal artery with some granular osmiophilic materialsome granular osmiophilic material (GOM)(GOM)

CADASILdisease

Scanning/ Gel-Matching /Imageanalysis

Normal

Polyp

Tumor

Mass spectrometrical identification Statistics

NP

T M1st Dimension

2nd Dimension PC based matchingSample enrichment/preparation

2-DE Algorithm

PrincipalComponent

Analysis(t1,t2,t3)

normalnormalmetastasismetastasis

tumortumor

polyppolyp

N P T M N P T M

N=26 N=46

Deviating Proteins (n=112)Identification of 72

Controls: normal liver tissueHCT116 and Lovo cell lines

benign benignmaligne maligne

The left gel segment (a) is zoomed from the normal mucosa,

gel b represents the patients polyp and

gel c is the corresponding segment of the same patients adenocarcinoma

a b c

CR174N CR173P CR172T

Intra-individual expression differences of Cytokeratin 20 in patient 14.

EttanEttanTMTM DiGEDiGEthe quantitative approach to the quantitative approach to

do Proteomicsdo Proteomics

CyDye DIGE Fluor dyesMinimal labelling dyes

Label 50 µg of protein

3 colors: Cy™2, Cy3, Cy5

MW matched (~450Da)

Charge matched (positive)

Label -amino group of lysine

Sensitivity - 0.025 ng

Linear dynamic range over 4 orders of magnitude

Ettan™ DIGE systemAchieving accurate quantitative data

Protein extract 1label with Cy3

Pooled internalstandard label with Cy™2

Protein extract 2label with Cy5

Mix labelled extracts

2-DEseparation

Cy5

Typhoon™Variable Mode Imager

Cy3

Cy2

DeCyder™ Differential AnalysisSoftware

Overlay of normal and patient Overlay of normal and patient protein samplesprotein samples

Normal control = CyTM3 labelled - Blue

Patient A sample = Cy5 Labelled - Red

Increased abundance

Equal abundance

Reduced abundance

Also 2D Databases exist!

Protein Fingerprint: 2Protein Fingerprint: 2--DEDE

pI 6.94.1

Mr(kDa)

200

15

AboutAbout 20002000proteinsproteins

An Analytical Challenge = What about having thousands of proteins in the sample and the need

for automation?

Yeast database search reveals:

6,188 potential proteins

tryptic digestion

344,855 peptides

2D2D--LC/MS/MSLC/MS/MS(2(2--dimensional liquid chromatography mass spectrometry)dimensional liquid chromatography mass spectrometry)

Faster Protein Identification by MS/MSFaster Protein Identification by MS/MS

2D2D--LC/MS/MSLC/MS/MSInstrumental SetInstrumental Set--up SCX/RP/RPup SCX/RP/RP

Loading20 µl/min

Sample & Salt Plugs

µ-Precolumn300 µm i.d. x 1 mm, C8

Nano column75 µm i.d. x 15 cm, C18

Nano LCgradient

Z

SCX µ-Column300 µm i.d. x 5 mm Waste

z

Waters 2D-nano UPLC aquity LC

Loading20 µl/min

Sample & Salt Plugs

µ-Precolumn300 µm i.d. x 1 mm, C8

Nano column75 µm i.d. x 15 cm, C18

Nano LCgradient

Z

SCX µ-Column300 µm i.d. x 5 mm Waste

z

Waters nano-Aquity Nano LC system with

2D

2D2D--LC/MS/MSLC/MS/MSInstrumental SetInstrumental Set--up SCX/RP/RPup SCX/RP/RP

Loading20 µl/min

Sample

Nano LCgradient

Z

Waste

z

Each protein/peptide is separated accordingEach protein/peptide is separated accordingto their physicoto their physico--chemical identitychemical identity

We use the property of proteins/peptides to We use the property of proteins/peptides to behave differently in different pHbehave differently in different pH

The time for one analysis is The time for one analysis is critical?critical?

You can analyze thousands of compounds in a few hoursYou can analyze thousands of compounds in a few hours

You can analyze thousands of compounds in a few hoursYou can analyze thousands of compounds in a few hours

The whole 2D LC can be done in a chipThe whole 2D LC can be done in a chip

The whole 2D LC can be done in a chipThe whole 2D LC can be done in a chip

2D2D--CE/MS/MSCE/MS/MS

High throughput CE

10000 Capillaries

Capillary windows

Autosampler

LED detection (MIT, Forest et al, 2006)

DiagnoDiagno--ProteomicsProteomics

Printing proteins as microarrays for high-throughput function determination

Science. 2000 Sep 8;289(5485):1760-3.

Detecting protein-protein interactions on glass slides

BODIPY

CY3

CY5 and rapamycin

CY5 only

Everything together

Prot G P50 FRB

Printing proteins as microarrays for high-throughput function determination

Science. 2000 Sep 8;289(5485):1760-3.

A single slide holding 10,800 spots. Protein G was printed 10,799 times

A single spot of FRB was printed in row 27, column 109. The slide was probed with BODIPY,CY3, CY5 and rapamycin

Automated MicroAutomated Micro--Chip Robot loaderChip Robot loader

What else can we do?What else can we do?

DE C

2 DE

• Performance

Native IEF and native PAGE

5 variants of hemoglobin

pH 6.7 -7.7

Native IEF and SDS-PAGE

standard IEF proteins

pH 3-10

Denatured IEF and SDS-PAGE

GFAP protein variants expression differences

in control and Alzheimer diseased patients

pH 4-6

HbA1c

HbA

HbF

HbS

HbA2

control AD

SELDI = Chip based surface enchanced laser desorption analysis

The Chip

Current Developments In SELDI Affinity TechnologyMass Spectrometry Reviews, 23, 34-44, (2004)

High Throughput

Micro-Array technology in Proteomics

Protein arrays

Ab-micro array

Protein Array

Micro-Array technology in Proteomics

Microfluidics in a Chip for Proteomics

Biochip-IMB, Ltd.

Biochip-IMB, Ltd.

Express diagnostics tools CUSTOMIZED

IMSImaging mass spectrometry

PrinciplesPrinciplesTissue section (mouse brain)

2,000 15,000 30,000

Ion

inte

nsity

Acquisition x

Acq

uisi

tion

ym/z

12mm

• A laser is rastered over a defined area while acquiring a complete mass spectrum from each position, resulting in molecular images for multiple analytesCornett, et al., Nature Methods 2007

IMS workflowIMS workflow

Stoeckli, M, et al., Analytical Biochemistry 2002

Tissue slide for IMS

Benefits of MALDIBenefits of MALDI--IMSIMS

Analysis of entire sample in one reading

Previous knowledge of molecular composition is not necessary

Allows for investigation of disease formation, progression, and treatment

www.maldi-msi.org

MS imaging advantages

•No labeling requiredBiomolecules are functionally unmodified

•Image biomolecular modificationsPTM’s, Metabolites

•Detailed information on molecular identityBy 2D MALDI

•Large scope of different elements and molecules

Targeted labeling

Label free imaging

IMS vs. histochemical stainIMS vs. histochemical stain

Imaging MS applications……

Drug administration and detection

New Phytologist (2007) 173 : 438 –444m/z 381

m/z 543

m/z 705

Reconstruction of the Carbohydrate moietyOf WHEAT (Triticum aestivum)

Special Applications

Imaging of regions immunoreactive with anti-synaptophysin Ab in healthy human pancreas.

(A) Localization of synaptophysin positive cells by TAMSIM. The monoclonal rabbit anti-synaptophysin is conjugated with the tag El 307 (498 m/z). The false color green points in the section show the presence of the tag El 307 and thus synaptophysin positive cells.

(B) Classical IHC image with the anti-insulin Ab. The dark pink spots correspond to Langerhans islets and so the synaptophysin-positive cells. The distribution of synaptophysin positive cells in (A) is very similar to that in (B).

Immuno-MS-IMS

Thiery, G., et al., Proteomics 2008