Two dimensional gel electrophoresis

What is 2D gel electrophoresis? Separation and identification of proteins in a sample by displacement in 2 dimensions oriented at right-angle to one another. First dimension: Isoelectric focussing Second dimension: SDS PAGE

Overview of 2D gel electrophoresis

Basis of 2D gel electrophoresis Highly unlikely for proteins to be similar in two distinct properties Isoelectric point Protein mass in SDS PAGE Protein complex mass in native PAGE

Advantages of 2D gel electrophoresis Resolution : 104 - 105 proteins from a sample. Preset conditions ( pH ranges, size of the gel, staining methods etc) can be changed to increase resolution. Delivers a map of intact protein that can be stored and analyzed at will. One of the core technology of proteomics.

Limitations Hard to resolve very acidic or very basic proteins very low or very high molecular weight. Poor resolution hydrophobic or membrane bound nuclear proteins Sample size proteins cannot be amplified like DNA. Difficulty in process automation

2D gel electrophoresis steps Sample preparation First dimension separation Isoelectric focussing Second dimension separation SDS PAGE Detection of spots Analysis of protein spots

Sample preparation

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

Sample preparation A. Contaminants and their removal procedure Particle Effect Removal Charged molecules E.g.: Salts, buffer components & nucleotides Poor focussing Dialysis or gel filtration SDS Proteins move towards positive electrode Acetone precipitation DNA Clog the gel pores Interfere with staining DNase or sonication Particulate matter Centrifugation

Sample preparation B. Solubilise proteins: Lysis Solution Composition Component Function Final Concn Urea (fresh & deionised) Solubilises proteins 8M-9M Detergents: Non-ionic/ zwitter ionic (CHAPS, CHAPSO, NP-40) Maintains proteins in solution during rehydration 0.5-4% Reducing Agent: DTT/ DTE Cleaves sulfide bonds 20-100mM Ampholytes (3-10) Maintaining the pH gradient 0.2-2% CHAPS: 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate DTT: Dithrioerythritol; DTE: epimer of DTT

Sample preparation: Protocol

Isoelectric focussing

Isoelectric focussing Separation on basis of pI, not Mw Requires very high voltages (5000V) Requires a long period of time (10h) Presence of a pH gradient is critical Uses ampholytes to establish pH gradient

Isoelectric focussing Isoelectric point is the pH of a solution at which the net charge of protein is zero. In electrophoresis there is no motion of the particles in an electric field at the isoelectric point. NH3+ COOH pH < pI Net positive charge COO- pH = pI NH2 pH > pI Net negative charge

Isoelectric focussing pH gradients Soluble Ampholytes heterogeneous polymers of charged compounds in a regular (tube) polyacrylamide gel Variability: batch to batch pH range about 4-8 at equilibrium Immobilised pH gradient Acrylamide derivatives with charged side chains immobilised on a strip Stable and reproducible gradient Higher mechanical strength Higher protein loading capacity

Isoelectric focussing IPG strips (3 mm x 18 cm x 0.5 mm) Narrow range Medium range Broad range 4 7 3.5 4.5 5.5 6.7 4.0 5.0 6.0 3 10 6 11

Isoelectric focussing Load sample per groove Peel off protective film from strip Place IPG strip gel facing down in groove Rehydrate overnight (~22 hrs) at room temperature

Isoelectric focussing IEF run Slowly increase the voltage and apply a high voltage at end to obtain sharp narrow zones of protein

Isoelectric focussing Equilibration Reduction : 50mM DTT in equilibration buffer for 15 minutes Maintains proteins in a reduced state Alkylation (optional): 125mM iodoacetamide in equilibration buffer for 15 minutes Alkylating sulfhydryl group reduces vertical streaking Equilibration buffer: 6M urea, 30% glycerol; 1.6% SDS; 0.002% bromophenol blue; 45 mM Tris base (pH 7.0)

SDS PAGE

SDS-PAGE Second dimension SDS-PAGE steps Equilibrating IPG strips after IEF. Applying IPG strips to the second dimension SDS gel. Performing SDS-PAGE

SDS-PAGE: Step1 After IEF run Remove the IPG strip from the tray Place IPG strip facing up in the equilibration buffer + -

SDS-PAGE: Step 2 IPG strip in Equilibration buffer + - IPG strip in Equilibration buffer SDS-PAGE 0.5% agarose in running buffer Marker in paper IPG strip is placed on top of pre-cast SDS-PAGE gel and electric current applied

SDS-PAGE: Step 3 Separation on 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 Degree of resolution determined by %acrylamide & electric field strength

Detection

Sensitivity (ng/spot) Detection Stain Sensitivity (ng/spot) Advantages Coomassie R-250 50-100 Simple, fast, consistent Silver stain 1-4 Very sensitive, awkward Copper stain 5-15 Reversible, simple, fast Zinc stain SYPRO ruby 1-10 Very sensitive, fluoroscent Western blotting Directly used for sequencing

Coommassie stain Silver stain Detection Coommassie stain Silver stain

Copper stain SYPRO flourescent Detection Copper stain SYPRO flourescent

Insufficient equilibration, insufficient SDS Trouble shooting 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

Analysis

Analysis of spots 2D gel software

Analysis of spots 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

Analysis of spots Common Software Features Image contrast and coloring Gel annotation (spot selection & marking) Automated peak picking Spot area determination (Integration) Matching 2 gels Stacking/Aligning/Comparing gels

Analysis Excision the spots of interest Pick up the protein gel spot from gel Manual Automatic In-gel digestion: Washing process Dehydratation and drying Trypsin digestion (50 ng trypsin, 37C 16h) Extraction Desalt and concentrate the peptide

Analysis Identification of eluted protein spots MALDI-TOF (Matrix Assisted Laser Desorption/Ionization-Time Of Flight) MS (Mass Spectrometry), Peptide mass fingerprint

Applications Analyzing proteome profiles Detecting post- or co-translational modifications Discovering new drug targets Studying protein expression in normal, disease, or developmental states Identifying novel proteins

(?????) Normal cells Tumor SDS-PAGE isoelectrofocusing Laser-captured microdissector (LCM) (?????) Clinical specimens Cryostat 2D gel electrophoresis Immage system

Recap

Schedule of a 2D Experiment Day 1: Sample preparation and IEF 1. Load protein sample onto IPG strip (IEF) 2. Run the IEF (about 24 hours) 3. Polyacrylamide gel casting Day 2: Equilibrium IPG strip and running SDS-PAGE 1. Remove IPG strip from IEF machine 2. Equilibrium IPG strip 3. Put IPG strip onto SDS-PAGE 4. Run the SDS-PAGE (overnight)

Schedule of a 2D Experiment Day 3: Staining, image scanning and image analysis 1. Remove the gel from the cassette 2. Stain the gel by SYPRO Ruby or silver 3. Scan the gel image 4. Image analysis Day 4: In-gel digestion, MALDI-TOF and database search 1. Pick the protein gel spot from gel 2. In-gel digestion 3. Spot the sample onto MALDI chip 4. MALDI-TOF analysis 5. Database search

Database of 2D maps

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