: Sample(s) DIGE MDLC MALDI-MS n ESI-MS n DeCyder D MALDI PMF DeCyder MS GE Healthcare milestones in -D electrophoresis 9 Amersham Biosciences launche

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慧纳喇
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1 A A (differential proteomics) (expression proteomics) (functional proteomics) Differential Proteome Analysis Disease (Cell or tissue) Control (Cell or tissue) IEF PAGE Analytical -DE Subtractive Analysis Analytical -DE Variation, Additional spots, Missing spots, Intensity differences Preparative -DE In-gel digestion Peptide mixture MALDI-TOF-MS, ESI-MS-MS PMF, Sequencing Database searching Identity of the query protein Validation of protein, Function of protein

2 : Sample(s) DIGE MDLC MALDI-MS n ESI-MS n DeCyder D MALDI PMF DeCyder MS GE Healthcare milestones in -D electrophoresis 9 Amersham Biosciences launched Ettan DIGE Amersham Pharmacia Biotech launched Typhoon Pharmacia Biotech launched DALT II system 99 Pharmacia Biotech introduced IPGphor 99 Pharmacia Biotech introduced ImageMaster suite 99 Pharmacia Biotech introduced Immobiline DryStrip 9 LKB introduced Immobiline 99 Pharmacia Fine Chemicals introduced Pharmalyte 9 The first multiple separation system, ISO-DALT, was developed 9 The Multiphor flatbed electrophoresis unit is launched. In production for years in! LKB introduced Ampholine, the first commercial carrier ampholyte : : SDS D - ( ) D : : SDS urea, NP- SDS sample ph ph ph front-end ( MDLC) ph Principle according to P.H. O Farrell and J. Klose (9)

IEF ph (IPG) Pharmalyte Ampholine gel (. mm ) electric field long IEF time where R = H or - (CH ) x -COOH, x = or : Immobiline CH =CH-CO-NH-R, R decreasing pi ph Immobiline IPG DeStreak, ph -D!

3 IEF ph (IPG) Pharmalyte Ampholine gel (. mm ) electric field long IEF time where R = H or - (CH ) x -COOH, x = or : Immobiline CH =CH-CO-NH-R, R decreasing pi ph Immobiline IPG DeStreak, ph -D! ph : Immobiline DryStrip ph. NL Immobiline DryStrip ph.. Immobiline DryStrip ph.. Immobiline DryStrip ph NL Immobiline DryStrip ph NL Wide ph gradient: NL ph ph Mouse liver extract IPG cm, ph - run with IPGphor From A. Görg Proteomics Department Technische Universität Munich IPG ph ph ph-nl Ettan IPGphor IPGphor, - V, : V -.ma,. ma W C± C cm IPG

4 Equilibration of IPG strips Prior to SDS electrophoresis the strips are equilibrated with: % SDS, mm Tris-HCl ph., M urea, % glycerol, BPB + ( x min) % DTT ( x min). % iodoacetamide to cover and unfold polypeptides with SDS and DTT glycerol and urea for minimizing EEO effects to remove excess DTT to prevent point streaking Görg A et al.: Manifold Elimination of point streaking on silver stained two-dimensional gels by -cm IPG addition of iodoacetamide to the equilibration buffer. Electrophoresis. (9) - New: Membrane proteins: % SDS and larger volume of solution McDonough J, Marbán E. Optimization of IPG strip equilibration for the basic membrane protein mabc. Proteomics () 9-9. Ettan DALTtwelve IPG strip xcm -cm,, ~L,, Ettan DALTsix ImageScanner III xcm -cm, ~L, - A O.D. bit CCD CCD CCD %,

gel treated gel Silver stain differences Are spot differences due to induced

5 ImageMaster Platinum. ImageMaster Platinum.: n n n n n -- IEF/SDS-PAGE ( ) Ettan TM DIGE Cy Cy Conventional -D control gel treated gel Silver stain differences Are spot differences due to induced biological changes or differences in the way the gels have been cast/run/stained? Cy Cy Cy Cy D Typhoon DeCyder

6 D DIGE Internal standard -D DIGE: without internal standard? Sample (Cy TM ) Sample (Cy) Gel A C C C T T T Sample (Cy) Sample (Cy) Conclusion Pooled sample (internal standard) Gel B Samples and show increased abundance to each other and over the same protein in samples and Internal standard -D DIGE: with the internal standard Standard (Cy TM ) Sample (Cy) Sample (Cy) Without internal standard Gel A With internal standard ( time / dose course) : BSA, Conalbumin, GAPDH, Trypsin inhibitor E. coli. run on gels!. Standard (Cy) Sample (Cy) Sample (Cy) Gel B Conclusion expression decrease in sample Virtual elimination of gel:gel variation reveals induced biological change with statistical accuracy capable of revealing differences in abundance of less than % between samples E. Coli lysate spiked with four different proteins ph ph Box, Spot No. -BSA Box, Spot No. Figure : Location of the four changing spot clusters. Box Box - BSA, Spot (pi.) No. Box - Conalbumin (pi.9) Box - GAPDH (pi.) Box - Trypsin Inhibitor (pi.) Box, Spot No. - Conalbumin - GAPDH - Trypsin inhibitor Proteome analysis of human colon cancer by D DIGE and mass spectrometry Healthy and tumor samples from patients = gels unique proteins identified with significant changes of these were only evident when using the internal standard From Vanderbilt University Medical Center IS Cy excl. Int Std, P Healthy Cy P Tumor Cy DIGE, Friedman et al. Proteomics ; (): 9- Alban et.al, Proteomics,, -

Ettan TM DIGE components CyDye TM DIGE fluors Highly fluorescent dyes designed specifically for this application Sensitive, photostable and spectrally distinct DIGE enabled Typhoon TM Imager Meets

7 Ettan TM DIGE components CyDye TM DIGE fluors Highly fluorescent dyes designed specifically for this application Sensitive, photostable and spectrally distinct DIGE enabled Typhoon TM Imager Meets the exacting needs of this unique technology DeCyder TM software Version. Designed specifically for this multiplexing technology CyDye DIGE Minimal labeling dyes Size and charge matched colors: Cy, Cy, Cy Label ε-amino group of lysine ~ % of protein labeled Start with µg of protein Sub nanogram sensitivity CyDye DIGE Relative Sensitivity Scarce sample labeling dyes Size matched, neutral charge colors: Cy, Cy Label thiol group of cysteine ~ % of protein labeled Start with µg of protein Picogram sensitivity Courtesy of Kai Stühler, RU, Bochum, DE. cells + Cy Hs Silver stain of. cells. cells + Cy Cy stain of. cells

8 DeCyder GEL A Sample Sample DIA In-gel co-detection GEL B Sample Sample GEL C DIA Sample Sample DIA DIGE DIGE The only way to do D is DIGE! Master Internal Std BVA Cross-gel matching Internal Std Protein difference ratios Internal Std D DIGE, DeCyder D,,,,,,,, DIGE / /, Transgenic knockout/ knockin mice /, (LCM)

9 way ANOVA p=.x - way ANOVA p=.x - way ANOVA p=.x - way ANOVA p=.x - Parkinson s disease Occurrence >% of population over yrs increases with age Onset mean age, mean duration yrs Less frequently, onset below yrs Models MPTP -OHDA Symptoms akinesia, rigidity, tremor Treatment L-DOPA therapy and/or dopamine receptor agonists Long-term symptoms on-off phenomenon (rigidity-dyskinesia) Days post MPTP lesion DIGE experimental design for the analysis of MPTP treatment effects Control day days days DIGE experiment Prepare a pooled internal standard Control samples Treated samples Gel A STANDARD Control Treated Gel B STANDARD Control Treated Gel C. Incorporate an internal standard. Randomize STANDARD Treated Control Gel D. Include biological replicates STANDARD Treated Control Pooled internal standard GROUPS a) saline treated controls, b) MPTP day, c) MPTP days, d) MPTP days DIGE experimental design for analysis of the effect of MPTP treatment. Gel Cy Cy Cy Pool CA D Pool CB D Pool CC D Pool A E Pool B E Pool C CD Pool A F Pool B CE 9 Pool C E Pool A CF Pool B F Pool C Workflow for the -D DIGE experiment. DIGE analysis Exp. Parkinson s disease Days post-mptp Total Increased Decreased treatment Identified a number of of differentially expressed proteins involved in in the the proteasome complex dihydropyrimidinase-like protein dihydropyrimidinase-like protein

Zhang, Fuchu He Proteomic analysis of individual variation in normal livers of human beings

10 buffer Murea, M thiourea, % CHAPS, mm of Tris, mm of magnesium acetate, one complete proteinase inhibitor cocktail tablet Department of Genomics and Proteomics, per ml lysis buffer DIGE Xuequn Zhang, Fuchu He Proteomic analysis of individual variation in normal livers of human beings using difference gel electrophoresis Proteomics,, D DIGE Cy(blue) Cy(green) Cy(red) 9 CV DeCyder CV 9

11 E G -D DIGE Ettan TM DIGE E Y. Oda*, T. Owa*, T. Sato*, H.Yamanaka+,Y. Shinohara+, A.Yokoi*, J. Kuromitsu*, T. Nagasu*, and A. Alba+ *Eisai Co., Ltd. Laboratory of Seeds Finding Technology, Tokyo, Japan; +Amersham Biosciences KK, Tokyo, Japan;+Amersham Biosciences, Grove Centre, Amersham, Buckinghamshire, UK E HCT E E* µg Cy Cy cm, ph-.% SDS-PAGE Ettan DIGE E E Ettan DIGE -D DIGE Ettan DIGE DeCyder -D A. E* E B. E C. Inactive= E* Active= E Accessible DIGE : Save Gel, Save Time -D DIGE colour -D groups groups x biological replicates x biological replicates = samples = samples samples per gel = gels sample per gel = gels gel replicates x gel replicates Gel number = gels total Gel number = gels total Time = Days Time = Days RMB/gel, RMB/gel, RMB/CyDye for gels Cost = RMB Cost = RMB DIGE -D DIGE D Ettan TM DIGE -D DIGE DeCyder TM *) Friedman et al. Proteomics ; (): -

12 Pre-fractionation possibilities Protein chromatography e.g. IEX, GF, HIC, RPC; Centrifugation, FFE, or detergent fractionation of subcellular components e.g. mitochondria, nuclei, cytosol ( information on location of proteins!) By affinity Immunoprecipitation or affinity chromatography of complexes or selective removal of abundant proteins Blue native electrophoresis According to charge: IEF in free flow apparatus, between isoelectric membranes, or in Sephadex AKTA) Albumin, transferrin, haptoglobin, IgA, IgG) (Amicon Ultra-) Resource Q column) Nacl,,, nm and M) DIGE Western blot Decyde

13 ,,,nm Western Blot Tatsuhiko Kakisaka, et al; J. Of Chromatography B-,, doi:./j.jchromb...9; Plasma proteomics of pancreatics of pancreatic cancer patients by multi-dimentional LC and D/DIGE: up regulation of LRG in pancreatic cancer (IMAC) (IDA) (NTA) Fe+ Ga+

Blotting Application A B ECL Plex Cy Cy Cy Cy CyDye C D = = ECL Plus Principle of Chemiluminescent Western Blotting Protein bound to blocked membrane Primary Ab Secondary Ab - HRP Peroxide Acridinium

~ pg Primary Antibody Usage Up to /, Up to /, Up to /, /-/ Secondary Antibody Usage Up to /, Up to /, Up to /, /-/ Emission Duration - hrs - hrs - hrs Min months Reprobing Recommended Yes Yes Yes

14 Blotting Application A B ECL Plex Cy Cy Cy Cy CyDye C D = = ECL Plus Principle of Chemiluminescent Western Blotting Protein bound to blocked membrane Primary Ab Secondary Ab - HRP Peroxide Acridinium esters ECL Plus substrate acridinium based Light H O Hyperfilm ECL / CCD ECL Range Specifications HRP Substrate ECL Advance ECL Plus ECL ECL Plex ImageQuant Sensitivity (pg antigen) * ~ pg ~ pg ~ pg ~ pg Primary Antibody Usage Up to /, Up to /, Up to /, /-/ Secondary Antibody Usage Up to /, Up to /, Up to /, /-/ Emission Duration - hrs - hrs - hrs Min months Reprobing Recommended Yes Yes Yes (No) Recommended Membrane Hybond P or Hybond ECL Hybond P for Chemi Hybond ECL for ChemiFluor Hybond ECL Hybond ECL and LFP membrane Recommended Detection Method Hyperfilm ECL / CCD Fluorescent Scanner / Hyperfilm ECL / CCD Hyperfilm ECL / CCD Fluorescent Scanner Recommended Applications Highest sensitivity, primary antibody and/or target is low abundance High sensitivity & chemifluorescent detection on scanners Routine Western blotting Quantitative Western Multiple detections of antigens Highest Sensitivity * In the model system

15 Ettan DIGE TM : Patient Normal Innovate Bio-Drug Discovery Proteomics Several Candidates Genetic Engineering Candidates Preparation Purification Separation Purity Fermentor Fermentation Functional Understanding in vivo, animal level Functional Understanding Functional Understanding Western Blot in vivo, cell level in vitro Production

Protein Labeling and Detection

Protein Labeling and Detection ECL plex Product Specialist 1/ Western Blot 2/ Western Blotting- PI 3/ Western Blotting 1. 2 1 2. 3. 4. Y 3 4 5. 4/ 1. SDS- (SDS-PAGE) SDS ( β- ) --SDS 5/ SDS-PAGE - - - (Tris-HCL ph 6.8) glycine/chloride