Principles and Methods
of 2-D Electrophoresis
中央研究院
生物化學研究所
曾湘文 博士
Oct. 26, 2006
Proteome Analysis and Proteomics
"The analysis of the entire PROTEin complement
expressed by a genOME, or by a cell or tissue type."
Wasinger VC et al, Electrophoresis 16 (1995)
“Proteomics is the study of quantitative changes of
expression levels and their application to drug discovery,
diagnostics and therapy.”
Two basic technologies:
2-D electrophoresis of complex protein mixtures
Identification and structure analysis of proteins with
mass spectrometry methods
Advantages of 2-D Electrophoresis
tolerant to crude sample loads:
no prepurification (like chromatography) has
to be employed.
highly resolution.
are a very effective fraction collectors
proteins are protected inside the gel matrix
2D Workstation
Analyze 2-D
Gels
Proteomics Technology
Sample preparation
Two-dimensional electrophoresis
Detection of spots
Image analysis
Spot excising
Enzymatic digestion of proteins
Mass spectrometry
Bioinformatics
Sample Preparation
Cell washing
Cell disruption
Protein precipitation
Solubilization
Protection against protease activities
Removal of
nucleic acids
lipids
salts,buffers, ionic small molecules
insoluble material
Cell washing
To remove contaminant material.
Frequent used buffer
PBS (phosphate buffer saline):
sodium chloride, 145 mM (0.85%) in
phosphate buffer, 150 mM, pH7.2
Tris buffer sucrose (10mM Tris, 250 mM
sucrose, pH 7,2)
Enough osmoticum to avoid cell lysis
Cell disruption methods
Gentle lysis method
1. Osmotic lysis (cultured cells)
Suspend cells in hypoosmotic solution.
2. Repeated freezing and thawing (bacteria)
Freeze using liquid nitrogen
3. Detergent lysis (yeast and fungi)
Lysis buffer (containing urea and detergent)
SDS (have to be removed before IEF)
4. Enzymatic lysis (plant, bacteria, fungi)
Lysomzyme (bacteria)
Cellulose and pectinase (plant)
Lyticase (yeast)
Cell disruption (continued)
Vigorous lysis method
1. Sonication probe (cell suspension)
Avoid overheat, cool on ice between burst.
2. French pressure (microorganism with cell wall)
Cells are lysed by shear force.
3. Mortar and pestle (solid tissue, microorganism)
Grind solid tissue to fine powder with liquid nitrogen.
4. Sample grinding kit (for small amount of sample)
For precious sample.
5. Glass bead (cell suspension, microorganism)
Using abrasive vortexed bead to break cell walls.
Cell disruption (continued)
Key variable for successful extraction from
crude material
1. The method of cell lysis
2. The control of pH
3. The control of temperature
4. Avoidance of proteolytic degradation
Removal of contaminants
Major type of contaminants:
1. DNA/RNA
2. Lipids
3. polysaccharides
4. Solid material
5. Salt
DNA/RNA contaminant
DNA/RNA can be stained by silver staining.
They cause horizontal streaking at the acidic part of
the gel.
They precipitate with the proteins when sample
applying at basic end of IEF gel
How to remove:
1. precipitation of proteins
2. DNase/RNase treatment
3. sonication (mechanical breakage)
4. DNA/RNA extraction method (phenol/chroloform)
Removal of other contaminants
Removal of lipids: Removal of solid
>2% detergent material
Precipitation
Centrifugation
Removal of Removal of salts
polysaccharides:
Microdialysis
Enzymatic
Precipitation
precedure
Precipitation
Effect of DNase Treatment
E. coli extract on 7 cm pH 3-10 NL
+ DNase - DNase
Protein precipitation
Ammonium sulfate Not efficient, de-salting
(salting out) necessary
TCA precipitation Can be hard to re-
solubilize
Acetone and/or ethanol Leaves SDS behind, but
many proteins not
precipitated
TCA plus acetone
More effective than either
alone, good for basic
proteins
Effect of sample precipitation
E. coli lysate precipitated
with TCA/acetone and Crude E. coli lysate
resuspended
Protein solubilization
Urea (8-9.8 M) , or 7 M urea / 2 M thiourea
Detergent (CHAPS,…)
Reductant (DTT, 2-mercaptoethanol)
Carrier ampholytes (0.8 % IPG buffer)
Sonication can help solubilization
Sample can be heated only prior to addition of
urea
Extraction:Comparison Urea vs
Urea/Thiourea
8 M urea Rat liver 7 M urea / 2 M thiourea
Reductants
DTT (dithiothreitol) most commonly used
DTE interchangeable with DTT
(dithioerythreitol)
2-mercaptoethanol required at high concentration, contains
impurities, but may have solubilization
benefits (?).
tributylphosphine Poorly soluble, very hazardous
triscarboxyethylpho Good reductant, but negative charge makes
sphine it unsuitable for 1st dimension.
triscyanoethylphosp Uncharged, soluble, but efficacy as reductant
hine is in doubt.
Protease inhibitors
PMSF Most commonly used
(phenylmethyl sulfonyl Inactivates serine and cysteine proteases
fluoride) Is inactivated by DTT and 2-
mercaptoethanol
AEBSF (Pefabloc) More soluble, less toxic than PMSF, but
can cause charge modifications(?).
EDTA Inhibits metalloproteases
Peptide protease May show up in 2-D pattern
inhibitors (leupeptin
aprotinin etc.)
High pH Inhibits most proteases, but avoid Tris
base
Effect of salt
E. coli extract pH 4-7
no salt 30 mM NaCl
De-salting techniques
Dialysis Slow
Spin dialysis Detergents can
concentrate with protein
Gel filtration Protein losses
Precipitation/ Complicated, can cause
resuspension losses
Effect of dialysis
Pre-dialysis sample Dialyzed sample
pH 5 6 7.5 10 pH 5 6 7.5 10
Desalting by Low Voltage IEF
Bovine vitreous proteins
150 V / 30 min 100 V / 5 hrs
Effect of sample prep technique
(Drosophila larva extract)
Homogenate precipitated with
Homogenized in 8 M urea, Homogenized in 2% SDS 80% acetone, 10% TCA.
4% CHAPS Heated at 95 ºC 3 min Resuspended in 8 M urea, 4% CHAPS
First dimension is pH 3-10 L run on IPGphor in 8 M urea, 2% CHAPS, 0.5% IPG
buffer, 65 mM DTT
First Dimension
First Dimension: Denaturing IEF
High molar (8 mol/L) urea, thiourea
one conformation of a protein
for protein solubility
prevents protein aggregates and hydrophobic
interactions
Non-ionic or zwitterionic detergent
for protein solubility
IPG Buffer (carrier ampholyte mixture)
for protein solubility
raises the conductivity of the DryStrips
DTT, DTE (no 2-mercaptoethanol)
prevents different oxidation steps
IEF with Carrier Ampholytes
gel
Pharmalytes
Ampholines
electric long IEF
field time
where R = H
or - (CH ) - COOH,
x = 2 or 3
decreasing pI
Plot of the net charge of a protein versus the
pH of its environment
Immobiline DryStrips: 1st Generation
11 cm strips:
pH 4-7
pH 3 - 10
pH 6 - 11
7 cm, 13 cm
and 18 cm strips:
pH 4-7
pH 3 - 10 L (linear gradient)
pH 3 - 10 NL (non-linear gradient)
pH 6 - 11
Comparison: E. Coli Protein Extract
in IPG pH 6-11
7 cm 11 cm 13 cm 18 cm
Wide and Narrow pH Gradients
pH 33 44 5 56 67 78 89 10
pH 9 10
Wide gradients are applied for:
entire protein spectrum
Narrow gradients are applied for:
increased resolution
44 55 66 7 7 8 8 9
9 increased loading capacity
to detect and analyse more
proteins
Mouse liver proteins From A. Görg et al. (1999)
IPG 4 - 7 Number of spots
1564
IPG 4 - 5 218 IPG 5 - 6 1491 IPG 5,5 - 6,7 1429
Increased Resolution: Blow - Ups of Spots
Mouse liver proteins
IPG 4-7 IPG 4-7
IPG 5-6 IPG 5,5-6,7
From A. Görg et al. (1999)
Guidelines for choosing Immobiline
DryStrip gels
Focusing Time
Under Focusing Over Focusing
FRANCE/oct98:JLL
The IPGphor Platform
IPGphor Strip Holder
Transparent cove
Pressure block
Base
Anode
Sample application wells
Cathode
IPGphor features
Platform accommodates up to 12 strip holders
7, 11 , 13 , 18 and 24 cm strip holders
Cup-loading stripholders for all lengths
Built-in power supply delivering 8000 V, 1.5 mA
Built-in Peltier cooling, 18 - 25 °C
Programmable “delayed start” rehydration period
10 possible programs, 10 phases each (ramp or step)
Safety lid
Effect of Rehydration under Low Voltage
Mouse liver proteins
18 cm IPG strips
A. No voltage
applied
B. 30 V applied
during
rehydration
from A. Görg et al.Life Science News 1 (1998) 4-6
Pipetting the sample into the
stripholder
Placing the IPG DryStrip into the
stripholder
Conventional and Universal Strip Holders
cup-loading stripholders
standard stripholders
Two-dimensional
electrophoresis
Theoretical pI and Mr map of yeast cell proteins
(calculated from MIPS data)
1000
Mr / kDa
100
10
1
2 4 6 8 10 12 14
Theoretical pI
Principle of 2-D Electrophoresis
1. First dimension:
denaturing isoelectric focusing
separation according to the pI
2. Second dimension:
SDS electrophoresis
separation according to the MW
The 2-D electrophoresis gel resolves
thousands of protein spots.
Common reagents of PAGE
Monomer: Acrylamide
Basic unit in PAGE gel
Neurotoxic
Bridge: Bis, [N,N'-methylene-bis(acrylamide)]
Cross-linker
Neurotoxic
Free radical generator: Ammonium persulfate
Generation of free radical
Riboflavin (vitamin B2) can also be used
Catalyst: TEMED (tetramethylethylenediamine)
Assist transfer of electron of free radical
Chemical structures
Acrylamide Bis, [N,N'-methylene-bis(acrylamide)]
Ammonium persulfate TEMED (tetramethylethylenediamine)
Choice of electrophoretic system
GE (Amersham biosciences)
23 x 20 cm
8 x 10 cm
16 x 16 cm
Choice of electrophoretic system
Bio-Rad
Second Dimension on Vertical Equipment
applying the
pipetting the IPG strip
agarose low
overlay
melting
agarose
Staining Methods
Colloidal Coomassie stain (Gel Code Blue
from Pierce)
Fluorescent stain (Molecular Probes)
Sypro Ruby
Difference Gel Electrophoresis (DIGE)
Coomassie stain
Silver stain
Sypro Ruby protein staining
1. Simple protocol. No overstainng.
2. 1-4000 dynamic range.
3. Less protein to protein variation
4. Stains glycoproteins, lipoproteins and Ca2+
binding proteins and other difficult-to-stain proteins
5. Do not stain DNA/RNA
6. MS compatible
7. Expensive
2-D Fluorescence Difference Gel
Electrophoresis (2-D DIGE)
Staining of Polyacrylamide Gels
Silver staining Coomassie blue staining Sypro Ruby staining
Technology
Staining
Silver
Coomassie blue
Fluorescent dyes
Sypro Ruby
Radioisotopic labelling
Comparison of protein staining methods
for proteomics
Methods Sensitivity Charcteri
SYPRO Ruby 1 ng 1. MS compatible
2. High sensitivity
3. Need special image acquiring
instrument.
Silver stain by Merril 1 ng 1. High sensitivity
2. Glycoprotein and other low abundance
proteins can be detected
Silver stain by Gottlieb 1 ng 1. MS compatible
2. High sensitivity
Coomassie Blue G-250 10 ng 1. MS compatible
2. Easy to handle
Coomassie Blue R-250 50-100 ng 1. MS compatible
2. Low cost
Image Analysis
Imaging analysis
Imaging
Desktop scanner
Fluorescence imager
Phosphorimager
Bio-Rad FXPro Plus
“Typhoon” - Variable Mode Imager
Multi-color
fluorescence
Phosphor-imaging
Chemiluminescence
Technology
Image analysis
e.g. Bio-Rad PDQuest
Database storage of many
gel images
Multi-image manipulation
and comparison
Creation of master gel
image (“typical” profile)
Comparison of individual
experimental gels to
master
Identification of variant
spots
Imaging analysis of difference 2-DE
A (Control, hydrophilic) B (LPS-treated, hydrophilic)
97 97
66 3C 66 3
9
45 45
37
30 30
30
42
15 46
36
20.1
45 47 48
20.1
19
14.4 14.4 41
3.2 4.0 5.0 5.5 6.0 7.0 8.0 9.0 10.0 3.2 4.0 5.0 5.5 6.0 7.0 8.0 9.0 10.0
1,650 individual protein spots
Spot 900- 3D Viewer
Control
900
LPS-treated
Protein Identification by MALDI-TOF
1. Cut protein spot 2. Protein digestion
Protease
4. Spot onto MALDI chip 3. Peptide purification
5. MALDI-TOF analysis 6. Peptide fragment fingerprint
Referances
More help:
from the Amersham Pharmacia
Biotech Handbook:
On the Internet:
http://www.apbiotech.com
Angelika Görg’s manual on her
Website:
http://www.weihenstephan.de/blm/
deg
2004
