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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
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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...
14
Hydrophobic Amino Acids
Aliphatic Aromatic
Neutral
Sulphur-containing
15
Hydrophilic Amino Acids
PolarCharged
Semi-charged
16
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