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. 2. 3.

Osmotic lysis (cultured cells)
Suspend cells in hypoosmotic solution.

Repeated freezing and thawing (bacteria)
Freeze using liquid nitrogen

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. 2. 3. 4. 5.

Sonication probe (cell suspension)
Avoid overheat, cool on ice between burst.

French pressure (microorganism with cell wall)
Cells are lysed by shear force.

Mortar and pestle (solid tissue, microorganism)
Grind solid tissue to fine powder with liquid nitrogen.

Sample grinding kit (for small amount of sample)
For precious sample.

Glass bead (cell suspension, microorganism)
Using abrasive vortexed bead to break cell walls.

4. The method of cell lysis The control of pH The control of temperature Avoidance of proteolytic degradation . 3.Cell disruption (continued) Key variable for successful extraction from crude material 1. 2.

5. 2.Removal of contaminants Major type of contaminants: DNA/RNA Lipids polysaccharides Solid material Salt 1. . 4. 3.

They precipitate with the proteins when sample applying at basic end of IEF gel How to remove: 1.DNA/RNA contaminant DNA/RNA can be stained by silver staining. precipitation of proteins 2. They cause horizontal streaking at the acidic part of the gel. sonication (mechanical breakage) 4. DNase/RNase treatment 3. DNA/RNA extraction method (phenol/chroloform) .

Removal of other contaminants Removal of lipids: >2% detergent Precipitation Removal of solid material Centrifugation Removal of polysaccharides: Enzymatic precedure Precipitation Removal of salts Microdialysis Precipitation .

DNase . coli extract on 7 cm pH 3-10 NL + DNase .Effect of DNase Treatment E.

but many proteins not precipitated More effective than either alone.Protein precipitation Ammonium sulfate (salting out) TCA precipitation Acetone and/or ethanol TCA plus acetone Not efficient. good for basic proteins . de-salting necessary Can be hard to resolubilize Leaves SDS behind.

coli lysate .Effect of sample precipitation E. coli lysate precipitated with TCA/acetone and resuspended Crude E.

2-mercaptoethanol) Carrier ampholytes (0.8 % IPG buffer) Sonication can help solubilization Sample can be heated only prior to addition of urea . or 7 M urea / 2 M thiourea Detergent (CHAPS.…) Reductant (DTT.8 M) .Protein solubilization Urea (8-9.

Extraction:Comparison Urea vs Urea/Thiourea 8 M urea Rat liver 7 M urea / 2 M thiourea .

. but efficacy as reductant is in doubt. but negative charge makes it unsuitable for 1st dimension. very hazardous Good reductant.Reductants DTT (dithiothreitol) most commonly used DTE (dithioerythreitol) 2-mercaptoethanol tributylphosphine triscarboxyethylpho sphine triscyanoethylphosp hine interchangeable with DTT required at high concentration. Uncharged. contains impurities. but may have solubilization benefits (?). soluble. Poorly soluble.

but avoid Tris base .) High pH Most commonly used Inactivates serine and cysteine proteases Is inactivated by DTT and 2mercaptoethanol More soluble. Inhibits metalloproteases May show up in 2-D pattern Inhibits most proteases. less toxic than PMSF. but can cause charge modifications(?).Protease inhibitors PMSF (phenylmethyl sulfonyl fluoride) AEBSF (Pefabloc) EDTA Peptide protease inhibitors (leupeptin aprotinin etc.

coli extract pH 4-7 no salt 30 mM NaCl .Effect of salt E.

can cause losses .De-salting techniques Dialysis Spin dialysis Gel filtration Precipitation/ resuspension Slow Detergents can concentrate with protein Protein losses Complicated.

5 10 Dialyzed sample pH 5 6 7.Effect of dialysis Pre-dialysis sample 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) Homogenized in 8 M urea. 4% CHAPS Homogenized in 2% SDS Heated at 95 ºC 3 min Homogenate precipitated with 80% acetone. 4% CHAPS First dimension is pH 3-10 L run on IPGphor in 8 M urea. 0. 65 mM DTT . 10% TCA.5% IPG buffer. Resuspended in 8 M urea. 2% CHAPS.

First Dimension .

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 .First Dimension: Denaturing IEF High molar (8 mol/L) urea.

IEF with Carrier Ampholytes gel Pharmalytes Ampholines electric field long IEF time where R = H or .COOH.(CH ) . x = 2 or 3 decreasing pI .

Plot of the net charge of a protein versus the pH of its environment .

11 .10 NL (non-linear gradient) pH 6 . 13 cm and 18 cm strips: pH 4 .7 pH 3 .Immobiline DryStrips: 1st Generation 11 cm strips: pH 4 .7 pH 3 .11 7 cm.10 L (linear gradient) pH 3 .10 pH 6 .

Comparison: E. Coli Protein Extract in IPG pH 6-11 7 cm 11 cm 13 cm 18 cm .

Wide and Narrow pH Gradients pH 3 7 8 9 9 pH 3 44 5 56 6 7 8 10 10 Wide gradients are applied for: entire protein spectrum Narrow gradients are applied for: increased resolution increased loading capacity to detect and analyse more proteins 44 9 55 66 7 7 8 8 9 .

Mouse liver proteins From A.6 1491 IPG 5. (1999) IPG 4 .7 1429 .5 .6. Görg et al.5 218 IPG 5 .7 Number of spots 1564 IPG 4 .

Görg et al.Ups of Spots Mouse liver proteins IPG 4-7 IPG 4-7 IPG 5-6 From A.5-6.7 .Increased Resolution: Blow . (1999) IPG 5.

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 .

18 and 24 cm strip holders Cup-loading stripholders for all lengths Built-in power supply delivering 8000 V. 11 .IPGphor features Platform accommodates up to 12 strip holders 7. 13 . 10 phases each (ramp or step) Safety lid .25 °C Programmable “delayed start” rehydration period 10 possible programs.5 mA Built-in Peltier cooling. 1. 18 .

Görg et al. 30 V applied during rehydration from A.Life Science News 1 (1998) 4-6 .Effect of Rehydration under Low Voltage Mouse liver proteins 18 cm IPG strips A. No voltage applied B.

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 .

First dimension: denaturing isoelectric focusing separation according to the pI 2. .Principle of 2-D Electrophoresis 1. Second dimension: SDS electrophoresis separation according to the MW The 2-D electrophoresis gel resolves thousands of protein spots.

[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 .Common reagents of PAGE Monomer: Acrylamide Basic unit in PAGE gel Neurotoxic Bridge: Bis.

Chemical structures Acrylamide Bis.N'-methylene-bis(acrylamide)] Ammonium persulfate TEMED (tetramethylethylenediamine) . [N.

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 pipetting the agarose overlay applying the IPG strip low 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. 4. 2. 7. lipoproteins and Ca2+ binding proteins and other difficult-to-stain proteins Do not stain DNA/RNA MS compatible Expensive . 6. No overstainng. 1-4000 dynamic range. 5. 3. Simple protocol. Less protein to protein variation Stains glycoproteins.

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 .

Need special image acquiring instrument. MS compatible 2. MS compatible 2. MS compatible 2.Comparison of protein staining methods for proteomics Methods SYPRO Ruby Sensitivity 1 ng Charcteri 1. Low cost Silver stain by Merril 1 ng Silver stain by Gottlieb Coomassie Blue G-250 Coomassie Blue R-250 1 ng 10 ng 50-100 ng . MS compatible 2. Glycoprotein and other low abundance proteins can be detected 1. High sensitivity 3. High sensitivity 2. 1. High sensitivity 1. Easy to handle 1.

Image Analysis .

Imaging analysis Imaging Desktop scanner Fluorescence imager Phosphorimager Bio-Rad FXPro Plus .

“Typhoon” .Variable Mode Imager Multi-color fluorescence Phosphor-imaging Chemiluminescence .

g.Technology Image analysis e. 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 .

0 9.0 1.0 8. hydrophilic) 66 3C 3 9 45 45 37 30 30 30 46 36 45 47 42 15 20. hydrophilic) 97 66 97 B (LPS-treated.5 6.4 3.2 4.2 4.4 14.650 individual protein spots .0 10.0 5.1 20.1 48 19 41 14.0 7.0 5.0 5.0 9.0 3.Imaging analysis of difference 2-DE A (Control.0 8.5 6.0 7.0 5.0 10.

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3D Viewer Control 900 LPS-treated .Spot 900.

Peptide fragment fingerprint . Cut protein spot 2. Spot onto MALDI chip 3. Protein digestion Protease 4.Protein Identification by MALDI-TOF 1. MALDI-TOF analysis 6. Peptide purification 5.

Referances More help: from the Amersham Pharmacia Biotech Handbook: On the Internet: http://www.de/blm/ deg 2004 .com Angelika Görg’s manual on her Website: http://www.weihenstephan.apbiotech.