2D Gel Analysis David Wishart University of Alberta Edmonton, AB June 2005 2D Gel Analysis David Wishart University of Alberta Edmonton, AB david.wishart@ualberta.ca Lecture 1.3 (c) 2005 CGDN

Separation & Display Tools 1D Slab Gel Electrophoresis 2D Gel Electrophoresis Capillary Electrophoresis HPLC (SEC, IEC, RP, Affinity, etc.) Protein Chips Lecture 1.3

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 Lecture 1.3

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 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 Lecture 1.3

Steps in 2D GE Lecture 1.3

Sample Preparation Sample preparation is key to successful 2D gel experiments Must break all non-covelent protein-protein, protein-DNA, protein-lipid interactions, disrupt S-S bonds Must prevent proteolysis, accidental phosphorylation, oxidation, cleavage, deamidation Lecture 1.3

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 Lecture 1.3

Cell Disruption Methods Vigorous Methods Sonication French press Glass bead disruption Enzymatic lysis Detergent lysis Freeze-thaw Osmotic lysis Gentle Methods Lecture 1.3

Sample Preparation PMSF Pefabloc EDTA EGTA leupeptin Dialysis Proteolysis Protection Contaminant Removal PMSF Pefabloc EDTA EGTA leupeptin Dialysis Filtration Centrifugation Chromatography Solvent Extraction Lecture 1.3

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 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) Lecture 1.3

Detergent Fractionation Cells Extraction with Digitonin/EDTA supernatent pellet Extraction with TritonX100/EDTA Cytoplasmic Fraction supernatent pellet Organelle Membranes Extraction with SDS/EDTA supernatent pellet Nuclear Cytoskeletal (in SDS) Lecture 1.3

Subcellular Fractionation Human mitochondrial proteins Human nuclear proteins Lecture 1.3

Differential Solubilization Protein sample Extraction with 40mM Tris Base supernatent pellet Extraction with 8M Urea, 4% CHAPS Fraction 1 supernatent pellet Fraction 2 Extraction with 5M Urea, 2M Thiourea 2% CHAPS, 2% SB3 supernatent pellet Fraction 3 Extract with SDS Lecture 1.3 Fraction 4

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

2D Gel Principles SDS PAGE Lecture 1.3

Isoelectric Focusing (IEF) Lecture 1.3

IEF Principles A N O D E + _ C T H Increasing pH pI = 8.6 pI = 6.4 Lecture 1.3

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 Lecture 1.3

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 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 Lecture 1.3

IPG Strips Acrylamido buffer Strip Length 7.9 cm 11.8 cm 17.8 cm Gel Length 7.3 cm 11.0 cm 17.1 cm Strip Width 3.3 mm 3.3 mm 3.3 mm Gel Thickness 0.5 mm 0.5 mm 0.5 mm pH Gradients Standard 3-10,4-7 3-10,4-7 3-10,4-7 Overlapping 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 Lecture 1.3

Narrow-Range IPG Strips pH 4 pH 5 pH 4 pH 9 pH 5 pH 6 Lecture 1.3

IEF Phase of 2D GE Rehydrate IPG strip & apply protein sample Place IPG strip in IEF apparatus and apply current 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 Lecture 1.3

SDS PAGE Lecture 1.3

SDS PAGE Tools Lecture 1.3

SDS PAGE Principles Sodium Dodecyl Sulfate SO Na C A T H O D E N _ + 4 Sodium Dodecyl Sulfate C A T H O D E N _ + _ _ _ _ _ _ _ _ Lecture 1.3

SDS-PAGE Principles Loading Gel Running Gel Lecture 1.3

Mobility & Acrylamide% 200 200 200 45 200 45 200 45 45 6.5 45 Lecture 1.3

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 Lecture 1.3

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

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 #) Lecture 1.3

SDS-PAGE for 2D GE equilibration SDS-PAGE Lecture 1.3

2D Gel Reproducibility Lecture 1.3

Insufficient equilibration, insufficient SDS Trouble Shooting 2D GE Horizontal streaks Sample not completely solubilized prior to application on IPG, sample poorly soluble in rehydration solution, ionic impurities, ionic detergents Vertical streaks Insufficient equilibration, insufficient SDS Lecture 1.3

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 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 Lecture 1.3

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) 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 Lecture 1.3

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) CH N C N CH N C H H C CH SO O S C H 2 3 5 Coomassie R-250 Lecture 1.3

Gel Stains - Summary Stain Sensitivity (ng/spot) Advantages Coomassie R-250 50-100 Simple, fast, consistent Colloidal Coomassie 5-10 Simple, fast Silver stain 1-4 Very sensitive, awkward Copper stain 5-15 Reversible, 1 reagent negative stain Zinc stain 5-15 Reversible, simple, fast high contrast neg. stain SYPRO ruby 1-10 Very sensitive, fluorescent Lecture 1.3

Stain Examples Coomassie Silver Stain Copper Stain Lecture 1.3

Stain Examples Normal liver Tumor Both SYPRO fluorescent stain Lecture 1.3

Detection via Western Blot Glioma Silver Stain Glioma Western Blot (anti-p53 antibody) Lecture 1.3

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 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 Lecture 1.3

2D-GE + MALDI (PMF) Trypsin + Gel punch p53 Trx G6PDH Lecture 1.3

2D-GE + MS-MS Trypsin + Gel punch p53 Lecture 1.3

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 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 Lecture 1.3

2D Gel Software Lecture 1.3

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 Lecture 1.3

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 Lecture 1.3

2D Gel Analysis Freeware CAROL http://gelmatching.inf.fu-berlin.de/Carol.html Lecture 1.3

2D Gel Analysis Freeware FLICKER http://www-lecb.ncifcrf.gov/flicker Lecture 1.3

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 Lecture 1.3

2D Gel Analysis Freeware Melanie Viewer http://ca.expasy.org/melanie/Viewer.htm Lecture 1.3

2D Gel Analysis Freeware http://www.gelscape.ualberta.ca:8080/index.html Lecture 1.3

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) Lecture 1.3

2D Gel Databases http://ca.expasy.org/ch2d/2d-index.html Lecture 1.3

2D Gel Databases http://ca.expasy.org/ch2d/2d-index.html Lecture 1.3

Swiss 2D-PAGE Lecture 1.3

Swiss 2D-PAGE Lecture 1.3

Swiss 2D-PAGE Lecture 1.3

Competing Technologies Capillary Electrophoresis (single & tandem) Lecture 1.3

ICAT vs 2D Gels ICAT Lecture 1.3

MudPIT IEX-HPLC RP-HPLC Trypsin + proteins p53 Lecture 1.3

2D Gels vs Protein Arrays Lecture 1.3

A Triumph For Gels (Actually Western Blotting) Lecture 1.3

Yeast Proteome Analysis Ghaemmaghami S, et al., Nature 425:737-741 (2003). Lecture 1.3

Tap Tagged Western Lecture 1.3

Tap-Tagged Western - Sensitivity Lecture 1.3

Yeast Proteome Results Lecture 1.3

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 Lecture 1.3

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 Lecture 1.3

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 Lecture 1.3