2D Gel Analysis

David Wishart University of Alberta Edmonton, AB david.wishart@ualberta.ca

Lecture 1.3 1

Separation & Display Tools

1D Slab Gel Electrophoresis
2D Gel Electrophoresis

Capillary Electrophoresis
HPLC (SEC, IEC, RP, Affinity, etc.)

Protein Chips
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2D Gel Electrophoresis
Simultaneous separation and detection of ~2000 proteins on a 20x25 cm gel Up to 10,000 proteins can be seen using optimized protocols
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Why 2D GE?
Oldest method for large scale protein separation (since 1975) Still most popular method for protein display and quantification Permits simultaneous detection, display, purification, identification, quantification Robust, increasingly reproducible, simple, cost effective, scalable & parallelizable Provides pI, MW, quantity
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Steps in 2D GE
Sample preparation
Isoelectric focusing (first dimension) SDS-PAGE (second dimension) Visualization of proteins spots Identification of protein spots Spot pattern evaluation/annotation
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Steps in 2D GE

Lecture 1.3

Sample Preparation
Sample preparation is key to successful 2D gel experiments Must break all non-covelent proteinprotein, protein-DNA, protein-lipid interactions, disrupt S-S bonds Must prevent proteolysis, accidental phosphorylation, oxidation, cleavage, deamidation
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Sample Preparation
Must remove substances that might interfere with separation process such as salts, polar detergents (SDS), lipids, polysaccharides, nucleic acids Must try to keep proteins soluble during both phases of electrophoresis process
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Cell Disruption Methods

Vigorous Methods

Sonication French press Glass bead disruption

Gentle Methods

Lecture 1.3

Enzymatic lysis Detergent lysis Freeze-thaw Osmotic lysis

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Sample Preparation
Proteolysis Protection Contaminant Removal

PMSF Pefabloc EDTA EGTA

Dialysis Filtration Centrifugation Chromatography

leupeptin

Solvent Extraction

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Protein Solubilization
8 M Urea (neutral chaotrope) 4% CHAPS (zwitterionic detergent) 2-20 mM Tris base (for buffering) 5-20 mM DTT (to reduce disulfides Carrier ampholytes or IPG buffer (up to 2% v/v) to enhance protein solubility and reduce charge-charge interactions
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Lecture 1.3

Other Considerations
Further purification or separation?
Subcellular fractionation Chromatographic separation Affinity purification

Optimizing electrophoresis parameters

IEF pH gradient, Acrylamide %, loading

Limits of detection
ng? (Coomasie stain) pg or fg? (Western)
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Detergent Fractionation
Cells Extraction with Digitonin/EDTA
supernatent Cytoplasmic Fraction pellet

Extraction with TritonX100/EDTA

supernatent Organelle Membranes pellet

Extraction with SDS/EDTA

supernatent Nuclear pellet Cytoskeletal (in SDS)
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Lecture 1.3

Subcellular Fractionation

Human mitochondrial proteins

Lecture 1.3

Human nuclear proteins

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Differential Solubilization
Protein sample

Extraction with 40mM Tris Base

supernatent Fraction 1 pellet

Extraction with 8M Urea, 4% CHAPS

supernatent Fraction 2 pellet

Extraction with 5M Urea, 2M Thiourea 2% CHAPS, 2% SB3

supernatent Fraction 3 pellet

Extract with SDS

Fraction 4
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Lecture 1.3

Steps in 2D GE
Sample preparation
Isoelectric focusing (first dimension) SDS-PAGE (second dimension) Visualization of proteins spots Identification of protein spots Spot pattern evaluation/annotation
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2D Gel Principles

SDS PAGE

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Isoelectric Focusing (IEF)

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IEF Principles
Increasing pH + + + + + + + + + _ _ _ _ _ _ _ _ _ C A T H O D E

A N O D E

pI = 5.1

pI = 6.4

pI = 8.6

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Isoelectric Focusing
Separation of basis of pI, not Mw Requires very high voltages (5000V) Requires a long period of time (10h) Presence of a pH gradient is critical Degree of resolution determined by slope of pH gradient and electric field strength Uses ampholytes to establish pH gradient

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Ampholytes vs. IPG

Ampholytes are small, soluble, organic molecules with high buffering capacity near their pI (not characterized) Used to create pH gradients via user Gradients not stable Batch-to-batch variation is problematic
Lecture 1.3

An immobilized pH gradient (IPG) is made by covalently integrating acrylamido buffer molecules into acrylamide matrix at time of gel casting Stable gradients Pre-made (at factory) Simplified handling
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IPG Strips
Strip Length Gel Length Strip Width Gel Thickness pH Gradients Standard Overlapping 7.9 cm 7.3 cm 3.3 mm 0.5 mm 11.8 cm 11.0 cm 3.3 mm 0.5 mm 17.8 cm 17.1 cm 3.3 mm 0.5 mm

3-10,4-7 3-10,4-7 3-10,4-7 3-6,5-8 3-6,5-8 3-6,5-8

CH2=CH-C-NH-R || O

Acrylamido buffer

R = weakly acidic or basic buffering group

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Narrow-Range IPG Strips

pH 4 pH 5

pH 4

pH 9

pH 5

pH 6

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IEF Phase of 2D GE

Rehydrate IPG strip & apply protein sample

Lecture 1.3

Place IPG strip in IEF apparatus and apply current

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Steps in 2D GE
Sample preparation
Isoelectric focusing (first dimension) SDS-PAGE (second dimension) Visualization of proteins spots Identification of protein spots Spot pattern evaluation/annotation
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SDS PAGE

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SDS PAGE Tools

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SDS PAGE Principles

SO Na 4
+

Sodium Dodecyl Sulfate

_ _ _ _ _ _ _

C A T H O D E

_ _ _ _ _ _

_ _ _ _ _ _ _ _ _ _

_ _

_ _

_ _ _ _ _

+ + + + + + +

A N O D E

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SDS-PAGE Principles
Loading Gel

Running Gel

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Mobility & Acrylamide%

200 200 200 45 200 45 6.5 45 45 45

200

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Electrophoretic Mobility
45kD 10kD 25kD

m = n/E = q/f
m = electrophoretic mobility n = velocity of molecule E = electric field q = charge of molecule f = frictional coefficient

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SDS-PAGE
Separation of basis of MW, not pI Requires modest voltages (200V) Requires a shorter period of time (2h) Presence of SDS is critical to disrupting structure and making mobility ~ 1/MW Degree of resolution determined by %acrylamide & electric field strength
Lecture 1.3 32

SDS-PAGE for 2D GE
After IEF, the IPG strip is soaked in an equilibration buffer (50 mM Tris, pH 8.8, 2% SDS, 6M Urea, 30% glycerol, DTT, tracking dye) IPG strip is then placed on top of pre-cast SDS-PAGE gel and electric current applied This is equivalent to pipetting samples into SDS-PAGE wells (an infinite #)
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SDS-PAGE for 2D GE

equilibration

SDS-PAGE

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2D Gel Reproducibility

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Trouble Shooting 2D GE
Horizontal streaks Vertical streaks

Sample not completely solubilized prior to application on IPG, sample poorly soluble in rehydration solution, ionic impurities, ionic detergents

Insufficient equilibration, insufficient SDS

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Advantages and Disadvantages of 2D GE

Provides a hard-copy record of separation Allows facile quantitation Separation of up to 9000 different proteins Highly reproducible Gives info on Mw, pI and post-trans modifications Inexpensive
Lecture 1.3

Limited pI range (4-8) Proteins >150 kD not seen in 2D gels Difficult to see membrane proteins (>30% of all proteins) Only detects high abundance proteins (top 30% typically) Time consuming
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2D Gel Protocols & Courses

Online Protocols
http://ca.expasy.org/ch2d/protocols/ http://www.abdn.ac.uk/~mmb023/protocol.htm http://www.aber.ac.uk/~mpgwww/Proteome/Tut_2D.html http://www.noble.org/PlantBio/MS/protocols.html

1 Day and 1 Week Courses

http://us.expasy.org/bprg/training/(Geneva) http://www.pence.ca (Toronto)
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Steps in 2D GE
Sample preparation
Isoelectric focusing (first dimension) SDS-PAGE (second dimension) Visualization of proteins spots Identification of protein spots Spot pattern evaluation/annotation
Lecture 1.3 39

Protein Detection
Coomassie Stain (100 ng to 10 mg protein) Silver Stain (1 ng to 1 mg protein) Fluorescent (Sypro Ruby) Stain (1 ng & up)
C2H 5 CH2 N O3S C CH3 C2H5 N CH2 SO3

Coomassie R-250
N H 5 C2 Lecture 1.3 C2 H5 40

Gel Stains - Summary

Stain
Coomassie R-250 Colloidal Coomassie Silver stain Copper stain Zinc stain SYPRO ruby

Sensitivity (ng/spot)
50-100 5-10 1-4 5-15 5-15 1-10

Advantages
Simple, fast, consistent Simple, fast Very sensitive, awkward Reversible, 1 reagent negative stain Reversible, simple, fast high contrast neg. stain Very sensitive, fluorescent

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Stain Examples

Coomassie

Silver Stain

Copper Stain

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Stain Examples

Normal liver Tumor Both

SYPRO fluorescent stain

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Detection via Western Blot

Glioma Silver Stain

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Glioma Western Blot (anti-p53 antibody)

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Imaging/Scanning Tools

Phosphoimager for 32P and 35S labelled 1D or 2D gels

Fluoroimager for SYPRO labelled 1D or 2D gels

Densitometer or Photo Scanner

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Steps in 2D GE
Sample preparation
Isoelectric focusing (first dimension) SDS-PAGE (second dimension) Visualization of proteins spots Identification of protein spots Spot pattern evaluation/annotation
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2D-GE + MALDI (PMF)

Trypsin + Gel punch

p53

Trx

G6PDH
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2D-GE + MS-MS

Trypsin + Gel punch

p53

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Typical Results
401 spots identified 279 gene products Confirmed by SAGE, Northern or Southern Confirmed by amino acid composition Confirmed by amino acid sequencing Confirmed by Mw & pI
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Steps in 2D GE
Sample preparation
Isoelectric focusing (first dimension) SDS-PAGE (second dimension) Visualization of proteins spots Identification of protein spots Spot pattern evaluation/annotation
Lecture 1.3 50

2D Gel Software

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Commercial Software
Melanie 4 (GeneBio - Windows only)
http://ca.expasy.org/melanie

ImageMaster 2D Elite (Amersham)

http://www.imsupport.com/

Phoretix 2D Advanced
http://www.phoretix.com/

PDQuest 6.1 (BioRad - Windows only)

http://www.proteomeworks.bio-rad.com/html/pdquest.html

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Common Software Features

Image contrast and coloring Gel annotation (spot selection & marking) Automated peak picking Spot area determination (Integration) Matching/Morphing/Landmarking 2 gels Stacking/Aligning/Comparing gels Annotation copying between 2 gels
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2D Gel Analysis Freeware

CAROL http://gelmatching.inf.fu-berlin.de/Carol.html
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2D Gel Analysis Freeware

FLICKER http://www-lecb.ncifcrf.gov/flicker
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Flicker
Permits comparison of 2 images from internet sources on web browser Comparison via adjustable flicker rate of overlaid gel images Images may be enhanced by spatial warping, 3D projections or relief map, image sharpening, contrast enhancement, zooming, complement grayscale transform
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2D Gel Analysis Freeware

Melanie Viewer http://ca.expasy.org/melanie/Viewer.htm

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2D Gel Analysis Freeware

http://www.gelscape.ualberta.ca:8080/index.html
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Federated 2D Gel Databases

Remotely queryable via the web Attainable through SWISS-PROT search Linked to other 2D databases via web Image mapped 2D gel spots to support graphical image query Directly reachable from within 2D gel analysis software
Appel, R.D. et al. Electrophoresis 17: 540-546 (1996)
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2D Gel Databases

http://ca.expasy.org/ch2d/2d-index.html
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2D Gel Databases

http://ca.expasy.org/ch2d/2d-index.html
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Swiss 2D-PAGE

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Swiss 2D-PAGE

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Swiss 2D-PAGE

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Competing Technologies

Capillary Electrophoresis (single & tandem)

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ICAT vs 2D Gels

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ICAT

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MudPIT
IEX-HPLC RP-HPLC

Trypsin + proteins

p53

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2D Gels vs Protein Arrays

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A Triumph For Gels (Actually Western Blotting)

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Yeast Proteome Analysis

Ghaemmaghami S, et al., Nature 425:737-741 (2003).

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Tap Tagged Western

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Tap-Tagged Western Sensitivity

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Yeast Proteome Results

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The Yeast Proteome

80% of the proteome is expressed during normal growth conditions Abundance of proteins ranges from fewer than 50 to more than 106 molecules per cell Many proteins, including essential proteins and most transcription factors, are present at levels that are not readily detectable by other proteomic techniques
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Conclusions
2D gel electrophoresis is still the most popular and powerful method for protein display, separation, visualization and quantitation Offers good to excellent sensitivity and is now very reproducible 2D GE is still essential for proteomics Running and analyzing 2D gels requires skill, patience and good software
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Conclusions
Web tools are now available that permit partial analysis and comparison of 2D gels Commercial software still is required in most cases to complete full-scale analysis Web-enabled gel databases are now democratizing & popularizing 2D gel analysis Competing technologies are now emerging that may offer advantages over 2DE
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