Focus on right spots using. Ettan DIGE

Transcription

1 Focus on right spots using Ettan DIGE

2 GEHC is part of a family of businesses aligned with our customers needs and acting as one company to drive growth. Commercial Finance Infrastructure Industrial Healthcare NBC Universal Consumer Finance

3 Protein Sciences Products Sample preparation Protein chracterisation >Ettan DIGE >Biacore (SPR) >Calorimetry, Microcal Protein Purification - ÄKTA >ÄKTA >Sephadex, HiTrap, SOURCE >µg-kg 100 % blocked target hours

4 What s different?

5 Proteomics workflows 2DE-MALDI PPF 2DE-ESI (schematic Venn diagram) LC-MALDI Different methods develop different subsets of the proteome with surprisingly little overlaps. Even one and the same sample analyzed on different brands of ESI or MALDI instruments identify predominantly non-redundant proteins. Eventually, analyzing a statistically sufficient number of replicates might improve, but not eliminate this dilemma. The perfect workflow is not in sight, yet.

6 Differential analysis: considerations

7 2-D Electrophoresis - Strengths Physico-chemical parameters of proteins measured Isoforms and post-translational modifications displayed, High resolution, particularly after prefractionation High throughput, parallel runs Crude samples tolerance Multiple detection, blotting, applicable Efficient fraction collector Non-shotgun method Multiplexing, DIGE Reproducibility, internal standard (DIGE)

8 DIGE internal standard Differential analysis using 2D electrophoresis Differences due to disease state, drug treatment, life cycle stage Induced biological change - what we want to measure Experimental factors Differences in IEF/SDS-PAGE conditions Gel distortions user-to-user variation System variation (gel-to-gel variation) Data analysis user-specific editing and interpretation Intrinsic differences that occur within populations eg animal-to-animal, plant-to-plant or culture-to-culture, subjected to identical conditions Inherent biological variation

9 Facts about the origins of experimental variances in bioanalytics Technical variances Sample preparation (SOP)> 5-20% MS-measurement variances< 5-10% Errors in data evaluation (FPR)> 5%???? Biological variances Cell culture samples > 5-20% Samples from inbred mice> % Human tissue samples> 4-5-fold Human cancer tissue samples > 2-20-fold from Helmut E.Meyer, Bochum, scientific talk, Hamburg 2008

10 Tracking of (reproducible?) protein losses

11 Tracking of (reproducible?) protein losses

12 Differential analysis using 2D electrophoresis What results can you expect? Protein is not detected as differentially expressed Protein is detected as differentially expressed Protein is not differentially expressed T F (false positive) Protein is really differentially expressed F (false negative) T T = true result F = false result

13 Differential analysis using 2D electrophoresis H 0 H 1 induced variance Statistics

14 Differential analysis using 2D electrophoresis H 0 Ω² H 1 Detected as differently expressed, excluding false negatives Statistics Differently expressed, but not detected as differently expressed = false negative Not differently expressed, but detected as differently expressed = false positive

15 Ettan DIGE Ettan DIGE is an integrated solution for the detection and quantification of real biological differences in protein expression.

16 DIGE components DIGE Dyes Imaging hardware DIGE analysis software

17 CyDye DIGE Fluor dyes Minimal labelling dyes Label 50 µg of protein 3 colors: Cy 2, Cy3, Cy5 MW matched (~450Da) Charge matched (positive) Label ε-amino group of lysine Sensitivity ng Linear dynamic range over 4 orders of magnitude

18 Minimal Dyes Labelling Chemistry Dye+ N + N + H3 N- protein Dye+ N + N O O O N O ph 8.5 O N H protein NHS reactive group A positively charged dye molecule replaces the positive charged lysine on the protein - no net change in pi

19 Sensitivity Taken from: Proteomics in Drug Research Edited by K. Marcus, K. Stühler, A. van Hall, M. Hamacher, B. Warscheid, H. E. Meyer Copyright 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN:

20 DIGE workflow Multiplexing 2-DE with an internal standard

21 Overlay of normal and transgenic mouse images Increased abundance Equal abundance Reduced abundance Normal mouse = Cy TM 3 labelled - Blue Transgenic = Cy5 Labelled - Red

22 Co-detection and matching Image 1: Pooled internal standard Boundaries transferred to image 2 and 3 Image 2: Sample 1 Image 3: Sample 2 Boundaries are used for quantitation relative to pooled internal standard Matching between gels via pooled internal standard Gel 1 Gel 2

23 Ettan DIGE internal standard Sample 1, gel1 Sample 2, gel 2 Is this system variation or induced biological change? Sample 3, gel 3 Sample 4, gel 4 Conclusion expression increase in samples 3 and 4

24 Ettan DIGE internal standard Standard (Cy2) Sample 1 (Cy3) Sample 2 (Cy5) Gel 1 Standard (Cy2) Sample 3 (Cy3) Sample 4 (Cy5) Gel 2 Conclusion expression decrease in sample 3

25 Standard lysis buffer inc. PI 6 50 µg 6 SYPRO Ruby gels Analyse by PDQuest Caco-2 cells Remark: Influence of difference in lysis conditions is not taken into account in this set-up 300 µg Cy3 Typhoon, DIGE lysis buffer incl. PI 6 50 µg Cy5 Cy5 Cy5 Cy5 Cy5 Cy5 Cy5 Cy5 Cy5 Cy5 Cy5 Cy5 Cy3 Cy3 Cy3 Cy3 Cy3 Cy3 6 DIGE gels (2 50 µg /gel) Analyse by DeCyder

26 Another way to compare overall system performance Distribution of ratios Mean values gel 1, 2, 3 vs. gel 4, 5, DIGE, DeCyder SYPRO Ruby, PDQuest Number of spots in bin Range

27 DIGE internal standard Measuring induced biological change Differences due to disease state, drug treatment, life cycle stage Induced biological change - what we want to measure Experimental factors Differences in IEF/SDS-PAGE conditions Gel distortions user-to-user variation System variation (gel-to-gel variation) Data analysis user-specific editing and interpretation Intrinsic differences that occur within populations eg animal-to-animal, plant-to-plant or culture-to-culture, subjected to identical conditions Inherent biological variation

28 Differential analysis using 2D electrophoresis H 0 Ω² H 1 Detected as differently expressed, excluding false negatives Statistics Differently expressed, but not detected as differently expressed = false negative Not differently expressed, but detected as differently expressed = false positive

29 How many replicates do I need to detect a selected expression change? Stain or label Silver Sypro Ruby DIGE Variation system + biological (%) no. of replicates needed to detect an expression change of small effect 25% 416* 68 8 medium effect 50% 133* 23 4 Strong effect 100% * Expression changes smaller than 2-fold not easy to publish

30 Proteomics: No Mistakes Allowed!

31 Ettan DIGE Applications

32 Ettan DIGE publications: to June Review Research Review Research

33 Ettan DIGE for analysis of human colon cancer Healthy and tumor samples from 6 patients = 6 gels 52 unique proteins identified with significant changes 42 of these were only evident when including the internal standard in the analysis excl. Int Std; 10 DIGE; 42 Friedman et al. Proteomics 2004; 4(3):

34 Silver stained Cy 3 Cell surface Non- fractionated Membrane fraction Cell surface labelling specificity Cytosolic fraction

35 Ettan DIGE & Blue Native Electrophoresis. Protein complexes of Arabidopsis mitochondria (Cy3, green) respiratory system and chloroplasts (Cy5, red) photosynthesis system. Blue Native PAGE course, October 2006, Institute for Plant Genetics, University of Hannover

36 CyDye DIGE Fluors Scarce sample labelling dyes Label 5 µg of protein 2 colors: Cy 3, Cy5 Size matched (~680Da) Charge matched (neutral) Label thiol group of cysteine

37 CyDye DIGE Fluor saturation dyes Labelling chemistry - 2. Coupling Dye HS- protein Dye O O N N H O Maleimide reactive group 37 C, 30 min ph 8.0 O O N N H S O protein Saturation labelling - all available cysteines are labelled Enables more sensitive detection for scarce samples

38 Relative Sensitivity cells + Cy3 Hs766 Courtesy of Kai Stühler, RU, Bochum, DE Silver stain of cells cells + Cy5 Cy5 stain of cells

39 2-D DIGE results Chromatogram Flow through Harvest Mab Select SuRe Affinity chromatography Eluate

40 Current techniques for HCP analysis 1-D SDS PAGE Separation according to size Proteins stained with Coomassie or Silver ELISA Based on antigen-antibody recognition Overall protein detection without separation Drawbacks: Low sensitivity (Coomassie) Narrow dynamic range Low reproducibility Limited separation resolution Drawbacks: Not all HCP are detected - accuracy based on quality of polyclonal antibody against HCP Batch to batch variations of antibody Specificity, sensitivity and cross reactivity must be verified FDA recently expressed their concerns regarding the quality of ELISA HCP antibodies

41 Improved analysis of HCP with 2-D DIGE Increased resolution (2-D separation) A) 2-D DIGE (50 µg) 1-D SDS PAGE (5µg) Coomassie staining B) IgG hc IgG lc Harvest Flow through Eluate

42 Redox-DIGE using cysteine labeling (N-ethylmaleimide) 2D DIGE Red spots: proteins have undergone redox-sensitive thiol mod. Yellow spots: proteins containing occluded thiols from: Hurd TR, Prime TA, Harbour ME, Lilley KS, Murphy MP. Detection of reactive oxygen species-sensitive thiol proteins by redox difference gel electrophoresis. J Biol Chem 282 (2007)

43 Ettan DIGE & Multiplex Western Blotting Nonphosphorylated and phosphorylated forms of GSK3β could be detected in PC-3U cells without TGF-β activation.

44 Value of Ettan DIGE without the benefit of the internal standard 42 proteins would have been overlooked due to large degree of variation Friedman et al. Proteomics 2004, 5, Reduces and simplifies hands-on work Cost efficient