1

Proteomics ABC

• 23,000 genes in the Genome but– ca. 1,000,000 proteins caused by Exon splicing

– 300+ Post-translational modifications

• Dynamic Range– Cell 106, Plasma 1012

• The Dynamic Proteome– Temporal (milliseconds, month)– Spatial (cell, organelle),– Developmental (100+ cell types in the body, years)– All proteins exist in dynamic complexes – This determines their function and is highly dynamic

2

Gene (DNA)

Transcription(gene expression)

~23,000 genes

Post-translationalmodifications ofproteins

P

P B1

B3

P

S B2

B4

phosphorylation

glycosylationhe

tero

geni

tyconfirmation

~500,000 proteins

Translation

protein Cprotein A protein B

mRNA(alternative splicing)

form A form Cform B

~150,000 proteins

Human Proteomics: many proteins, few genes

3 3

Proteomics: The 2-D PAGE-MS Workflow

Sample Prep

Image analysis

Sample labelling

Automated spot picking

Spot digestion

MALDI spotting

Image acquisition

SeparationSpecificdetection

Spot Handling Workstation

Protein IDwithMALDI-ToF

Laboratory workflow system

4

Proteomics: The LC-MS Based Workflow

LC based workflow

Samplepreparation

Protein pre-fractionation, Protein depletionGroup-specific protein enrichment

Protein digestion

RT: 0.00 - 147.01

0 20 40 60 80 100 120 140

Time (min)

0

20

40

60

80

100

0

20

40

60

80

100

Re

lative

Ab

un

da

nce 0

20

40

60

80

10052.6632.77

18.84 46.16 57.14

44.10

12.57

1.57 75.8658.03 87.56 129.57121.87 137.2296.28

41.06

26.8522.22

13.3432.88

50.9312.54 128.3954.17 132.4157.88 75.96 87.63 124.3989.932.55

18.5217.01

42.0727.731.34

1.89 30.91 75.8650.18 57.70

76.9914.82 87.6770.04 134.87129.1497.96 113.09

NL:4.05E9

Base Peak MS yeast_120ming_05

NL:3.24E9

Base Peak MS yeast_120ming_06

NL:1.02E9

Base Peak MS yeast_120ming_07

Proteinidentification & characterization

Multidimensional nanoscalepeptide separation

5

Complementary approaches

•Limited sensitivity in MS, because of in-gel digestion

•Many samples analysed because of parallel runs

•Separation and display of intact proteins

•Information on protein isoforms and PTMs available

•Higher sensitivity because of in-liquid digestion

•Fewer samples analysed (ca.12 hours / sample

•Analysis of peptides

•PTMs and protein isoforms not directly displayed

2DE-MS approach vs. MDLC-MS approach

6

Gene Expression

Central dogma of molecular biology

7

Gene Structure

8

Post-translational modifications

• Proteolytic cleavage– Fragmenting protein

• Addition of chemical groups

– Phosphorylation: activation and inactivation of enzymes– Acetylation: protein stability, used in histones– Methylation: regulation of gene expression– Glycosylation: cell–cell recognition, signaling– GPI anchor: membrane tethering– Hydroxyproline: protein stability, ligand interactions– Sulfation: protein–protein and ligand interactions– Disulfide-bond formation: protein stability– Deamidation: protein–protein and ligand interactions– Ubiquitination: destruction signal– Nitration of tyrosine: inflammation

9

Primary Structure

10

Secondary Structure -Alpha Helices

11

Secondary Structure -Beta sheets

12

Tertiary and Quaternary Structure

Tertiary structure - fold of a given chain Quaternary structure - protein functional unit

13

Protein Structure

• Protein structure can be divided into:- Primary (amino acid sequence)

- Secondary (local folding structure)- Tertiary (overall fold of amino acid chain)- Quaternary (subunits composing functional protein)

mRNA: 5’-AUGGCUUGUUUACGAAUU... - 3’3 letter code: NH2-Met-Ala-Cys-Leu-Arg-Ile-... COOH

1 letter code MACLRI...

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Hydrophobic Amino Acids

Aliphatic Aromatic

Neutral

Sulphur-containing

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Hydrophilic Amino Acids

PolarCharged

Semi-charged

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Acid-Base Properties of Amino Acids

•All amino acids have acidic andbasic functional groups– carboxyl group is acidic– amino group is basic

• Amino acids that lack charged Rgroups are zwitterions at neutral pH

• Aspartic and glutamic acids are negatively charged at neutral pH

•Arginine and lysine are positively charged at neutral pH

17

What it pKa?

• The pKa for a functional group is the pH at which the acidic or basic group on 50% of the molecules in a solution are ionised

•Amino acids can ionise their N-terminal amino group, the C-terminal carboxy group and sometimes the side chains

•At neutral pH 7, the charges are:Asp, Glu -1 (pKa = 3.9, 4.3)His +1/0 (pKa = 6.0)Cys 0/-1 (pKa = 8.3)Arg, Lys +1 (pKa = 12.5, 10.5)Tyr 0 (pKa = 10.1)

18

Principle of Electrophoresis

• v = E• v = migration velocity (cm/s)

• = electrophoretic mobility (cm2/Vs)–charge, size, shape of molecule,–viscosity, pore size, buffer pH and –ionic strength, temperature of medium

• E = electric field strength (V/cm)

• Power = Voltage Current

-

19

Courses of Electric Parameters

vo lta g e [V ] (d isc -e le c tro p h o re sis.)

vo lta g e [V ] (IEF)

m a x. 2 0 0 0 V (IEF)

m a x. 6 0 0 V (e le c tro p h o re sis)

m a x. 5 0 m A

m a x. 3 5 W

c u rre n t [m A ]

3 0 m in 1 h : 3 0 m in1 h 2 h

p o w e r [W ]

tim e

20

Electrophoresis Systems

Vertical Horizontal

21

Electrophoretic methodspH

= c

onst

= A

= B

pI

pI

A

B

m

m

R

R

A

B

pH

=

pH

LT

Zone E lectrophoresis Isotachophoresis

pH

gra

die

nt

3

4 5

6

7

8

9

10

A and B are sam ple com ponents.

22

Isoelectric Focusing

Protein Separation according to Isoelectric Point-(pI) the pH at which the number of + and -charges are the same

23

Gel Additives

•Glycerol, Sucrose

• IPG buffers

•Reducing Reagents (DTT, 2-mercaptoethanol)

•Urea

•Non-Ionic Detergents (TritonX-100, Brij-)

•Zwitterionic Detergents (CHAPS)

•Anionic Detergents (SDS)

•Cationic Detergents (CTAB)

•Organic Solvents (e.g. DMSO)

•Enzymes, Substrates

24 4-May-04

Free carrier ampholytes (CA-IEF)

Pharmalytes®

Ampholine®

before start in the e lectric fie ld

pH

separation d istance

pH

separation d istance

pH

separation d istance

increasing pI

carrier am pholytes

25

Isoelectric Focusing

26 4-May-04

Isoelectric focusing: procedure

• Prefocusing

• Location of sample loading

• Mode of sample loading

• Sample entry

• Separation temperature

• Separation time

• Fixing and staining

27

IEF with carrier ampholytes

gel

PharmalytesAmpholines

decreasing pI

electricfield

long IEFtime

where R = Hor - (CH ) - COOH,x = 2 or 3

28

The Cathodal Drift

•

29 29-Apr-04

Titration Curves

• Cathode

Anode

pH 3 pH 10

30

Limitations of IEF with carrier ampholytes

• Gradient drift causes loss of basic & acidic proteins

• Reproducibility of the first dimension in 2D-PAGE

• Protein loading capacity of gel rods

• Ultranarrow intervals are not stable

31

Immobilized pH gradients (IPG)

Immobiline Gels(0.5 mm gel layerson film supports)

Acrylamido buffers: Immobiline®

CH2=CH-CO-NH-R,

R contains a carboxylic

or a tertiary amino group

32 4-May-04

Features of Immobilized pH Gradients

• Few acrylamide derivatives

• No gradient drift

• Tailor-made gradients

• Very narrow gradients possible: e.g. pH 4.2-4.4

• Stable basic pH gradients

• Gradient profile is stable

• True equilibrium method

• High loading capacity

• IEF in narrow strips

33

Conventional and Universal Strip Holders

Cup-loading stripholders

Regular stripholders

34

2-D Electrophoresis, Immobiline DryStrips and Buffers

35

Agarose IEF of fish sarcoplasm proteins

+

-

pH 3.5

pH 9.5

36

SDS Polyacrylamide Gel Electrophoresis

Separation according to the Molecular Weights

37

Gel structures and compositions

AGAROSE POLYACRYLAMIDEGelation of the polysaccharide sol by chilling Chemical polymerisation of acrylamide monomers and

NN´-methylenbisacrylamide (Bis)

1% agarose (w/v) ca. 150 nm;0.16 % agarose (w/v) ca. 500 nm.

→ ∅→ ∅

→ ∅

Total acrylamide concentrationand Crosslinking:

T = 100 [%]; C = 100 [%]a + ba + b

a:g acrylamide; b:g Bis;V: volume in mL

5 % T / 3 % C 5 nm

× ×V

b

38

SDS sample preparation

•

nonreducing SDS treatment

1 - 2 % (w/v) SDS

native

reducing SDS treatment

1 - 2 % (w/v) SDS+ DTT, DTE or 2-mercaptoethanol

3 min at 95 C

reducing SDS treatment and alkylation

1 - 2 % (w/v) SDS

+ DTT, DTE or 2-mercaptoethanol3 min at 95 C

+ iodoacetamide or vinylpyridine

o o

39

Molecular weight standards

• Peptide Markers (P)

– 2.5 - 17 kDa

• Low Molecular Weight (L)

– 14 - 94 kDa

• High Molecular Weight (H)

– 53 - 212 kDa

• Rainbow Markers (R) 10 - 250 kDa

R

40

Protein polyacrylamide gel electrophoresis PAGE

1 2 3 4 5 6 7 8 9 10

41

Disc electrophoresis

42

Discontinuous electrophoresis

43

Problems with Polyacrylamide Gels

•Toxic Monomers

–Acrylamide, Bis, ammonium persulphate

•Polymerization effectiveness

–temperature, acrylamide concentration, pH value,oxigen, inhibitors (additives, boric acid),quality of chemicals

•Chemicals in the matrix

–monomers, ammonium persulphate, ions

•Shelf life

–pH value must be below pH 7

•Limitation in pore size

44 Add Title Here 8 pt. Arial

Practical hints for vertical PAGE

•NO HCl in cathode buffer!

•glycine in anode buffer not necessary

•overlayering the gel edge

•prerun only with Tris-HCl

•open cassette with WonderWedge

45 Add Title Here 8 pt. Arial

Practical hints for SDS PAGE

•add reducing reagent after boiling

•scavenge excess reducing reagent with iodoacetamide

•treat sample at high pH (8 — 8.8)

•use at least 2 % SDS

•do not boil nonreduced samples

•use DTT or DTE instead of 2-mercaptoethanol

46

Cell-Map Proteomics

47

Systematic Complex pull-downs

48

Final verified complex

49

Zooming the Picture Out

50

The Big Picture

51 29-Apr-04

Preparative IEF on Multiphor

• Flatbed Focusing in Sephadex G-200 superfine

–with standard equipment (Multiphor)

–high loading capacity (1 g)

–many applications

–not sensitive to precipitated protein

–recovery from dextran gel easy