Genomics, Transcriptomics and Proteomics

Transcription

1 Genomics, Transcriptomics and Proteomics GENES ARNm PROTEINS Genomics Transcriptomics Proteomics (10 6 protein forms?) Maturation PTMs Partners Localisation

2 EDyP Lab : «Exploring the Dynamics of Proteomes»

3 EDyP Lab : Exploring the Dynamics of Proteomes

4 EDyP Lab : Exploring the Dynamics of Proteomes Biomarkers Proteome Dynamics of Intracellular Compartments Genome Annotation EDyP-AMT EDyP-PTM EDyP-Info EDyP-Platform

5 Our «Toolbox» for Proteomic Studies Lys Arg Lys Trypsin Arg Lys Lys

6 Our «Toolbox» for Proteomic Studies nanolc Sensitivity + Sampling nanoesi-ms/ms

7 Our Toolbox for Proteomic Studies MS spectra (quantification) MS/MS spectra (identification)

8 Our Toolbox for Proteomic Studies Proteins Proteome (MS/MS spectra)

9 Our Toolbox for Proteomic Studies 1996 : Edman Sequencing «High throughput»: 1 protein/day (µg/protein)

10 Our Toolbox for Proteomic Studies 2008 : nanolc-ms/ms «High throughput» : 400 protéines/h (100 ng) (100s attomoles/protein) 1996 : Edman Sequencing «High throughput»: 1 protein/day (µg/protein)

11 EDyP Lab : Exploring the Dynamics of Proteomes Biomarkers Proteome Dynamics of Intracellular Compartments Genome Annotation EDyP-AMT EDyP-PTM EDyP-Info EDyP-Platform

12 Proteomics data for genome annotation Proteins Proteome MS/MS spectra

13 Proteomics data for genome annotation Proteins Genome Proteome MS/MS spectra Proteome MS/MS spectra Ferro, Tardif et al. J Proteome Res (in press)

14 Proteomics data for genome annotation TGAACAATATCTCTGGGTTATTTAAAAAAAAGTTCAGGAACAATATCTCTGGGTTATTTAAAAAAAATCCGTTA C H N C E E R P Q G P M W E C N A K T W P M E V C P P V T C I L N D Q K R H F Y W E D T V S A P T P P V T C R Q K R H F Y W E D T V S A P T W E P «Peptide-hit» PQG QGP PQG GPM PMW PMW MWE WEC Ferro, Tardif et al. J Proteome Res (in press)

15 Proteomics data for Arabidopsis thaliana genome annotation PepLine Map

16 Proteomics data for Arabidopsis thaliana genome annotation PepLine Map GenoAnnot Map [GenBank import] AT1G subunit of Tic complex (TIC110)

17 Proteomics data for Arabidopsis thaliana genome annotation PepLine Map GenoAnnot Map [GenBank import] AT1G Hydrogen antiporter (Q9LQ76 in UniProt)

18 EDyP Lab : Exploring the Dynamics of Proteomes Biomarkers Proteome Dynamics of Intracellular Compartments Genome Annotation EDyP-AMT EDyP-PTM EDyP-Info EDyP-Platform

19 Someone who could have been

20 Someone who could have been

21 Someone who, finally, decided to be a Cell Biologist Gagnon et al., 2002, Cell 110,

22 Desjardins et al

23 EDyP Lab : Exploring the Dynamics of Proteomes Biomarkers Proteome Dynamics of Intracellular Compartments Genome Annotation EDyP-AMT EDyP-PTM EDyP-Info EDyP-Platform

24 Translational Research Protein Biomarkers for Pathologies Early detection of the pathology Follow up of the medicamentous treatment (personnalized medicine) Introduction of new clinical Biomarkers is slowing!!! A major part of the problem is the lack of a biomarker Pipeline

25 Pipeline Set-up for Bladder Cancer Biomarker Discovery DECanBio European project - 8 partners Clinicians, Biochemists, Analysts, Statisticians, Bioinformaticians 4 years ( ) ; 3 M Novel MS-based strategies to Discover and Evaluate Cancer Biomarkers in urine Application to Diagnosis of Bladder Cancer

26 Pipeline Set-up for Bladder Cancer Biomarker Discovery Human biofluid / tissue Validated Biomarkers

27 Pipeline Set-up for Bladder Cancer Biomarker Discovery Samples 10s Human biofluid / tissue Analytes 100s Biomarker discovery Biomarker candidates 10s Semi quantitative mass spectrometry method No a priori on the identity of the proteins Adapted from Rifai, Gilette and Carr et al., Nature Biotech, 2006, 24,

28 Pipeline Set-up for Bladder Cancer Biomarker Discovery Samples 10s Human biofluid / tissue Analytes 100s 100s Biomarker discovery Biomarker candidates 10s Biomarker evaluation Verified Biomarkers 2-5 Can we replace antibody based methods by MS???

29 Pipeline Set-up for Bladder Cancer Biomarker Discovery Samples 10s Human biofluid / tissue Analytes 100s 100s Biomarker discovery Biomarker candidates 10s Biomarker evaluation Verified Biomarkers 2-5 Can we replace antibody based methods by MS??? MS Targeted Method : Quantification of the Biomarker candidates Accurate and multiplex Method

30 Pipeline Set-up for Bladder Cancer Biomarker Discovery Samples 10s Human biofluid / tissue Analytes 100s 100s Biomarker discovery Biomarker candidates 10s Biomarker evaluation Verified Biomarkers s Biomarker validation? Validated Biomarkers

31 Challenges for Quantitative Proteomics in Clinical Studies Increase dramatically the throughput of quantitative proteomics

32 Challenges for Quantitative Proteomics in Clinical Studies Quantify low abundant proteins with good accuracy Proteins measured in Plasma span > 10 orders of magnitude in abundance!!!

33 DECanBio Biomarker Discovery phase Urine proteome (hundreds of Proteins) Semi-quantitative Proteomics, without any a priori Dozens of samples from healthy persons and bladder cancer patients

34 DECanBio Biomarker Discovery : AMT Strategy

35 DECanBio Biomarker Discovery : AMT Strategy AMT phase 1 Urine (pools) Biochemical Fractionation + SDS-PAGE Tryptic Peptides LC-FT-MS/MS m/z ; z ; Tr Urine AMT Data base

36 DECanBio Biomarker Discovery : AMT Strategy AMT phase 2 Urine (one patient) SDS-PAGE (stacking gel) Tryptic Peptides LC-FT-MS m/z ; z ; Tr Urine AMT Data base

37 DECanBio Biomarker Discovery : AMT Strategy AMT phase 2 Urine (one patient) SDS-PAGE (stacking gel) Tryptic Peptides LC-FT-MS m/z ; z ; Tr Identif. Abundance Urine AMT Data base

38 DECanBio Biomarker Discovery : AMT Strategy Monoisotopic Molecular Weight (Mw) 344,633 isotopic distributions 2,140 AMT matches Normalized Elution Time (NET)

39 DECanBio Biomarker Discovery : AMT Strategy Peptide Index 1511 Mass = Scan = 2445 NET = Abund =

40 DECanBio Biomarker Discovery : AMT Strategy Scatterplot 3D 500ng injected Triplicates

41 DECanBio Biomarker Discovery : AMT Strategy Dual column nanolc AMT phase 2 Urine (one patient) 1 run every 60 min SDS-PAGE (stacking gel) Quality Control: 1 CytC digest every 5 runs runs / day Triplicates 3-6 patients / day Tryptic Peptides About 100 patients / month LC-FT-MS m/z ; z ; Tr Identif. Abundance Urine AMT Data base

42 About Protein lists «The computer says I need to upgrade my brain to be compatible with proteomic data analysis»

43 DECanBio Biomarker Discovery : Which Preanalytical conditions? Oneway Analysis of Median_Abundance By Condition Median_Abundance Block Centered Ech # A1 A2 B1 B2 Condition All Pairs Tukey-Kramer 0.05 Ech #2 Ech #3 Block Sample Inhib. Temp

44 AMT Databases can be «translated»!!! Géneration of AMT Database : Tedious!!! Urine AMT Database (Grenoble) Urine AMT Database (Moscow) The only parameters that cannot be modified : mobile phase and «chemistry» of the solid support (the nanolc system, the flow-rate, the diameter of the column, the slope of the gradient can be changed)

45 DECanBio Biomarker Evaluation Samples 10s Human biofluid / tissue Analytes 100s 100s Biomarker discovery Biomarker candidates 10s Biomarker evaluation Verified Biomarkers 2-5 Can we use mass spectrometry instead of antibodies for the Biomarker evaluation step???

46 Evaluation of the Biomarker Candidates Accurate quantification Method Dozens of Biomarker Candidates Multiplex Hundreds of samples (healthy / patients) High throughput Quantification in Biofluids Specific Quantification of low abundant proteins Sensitive

47 Accuracy and Precision of a Quantification Method Y = Measured Target Protein X = Added Target Protein

48 Accuracy and Precision of a Quantification Method Y = Measured Protein Y = 1,0. X R = X = Added Protein

49 Accuracy and Precision of a Quantification Method Y = Measured Protein Y = a. X X = Added Protein

50 Accuracy and Precision of a Quantification Method Y = Measured Protein Y = 1,0. X X = Added Protein

51 Isotope dilution-based Proteomics for «absolute Quantification» Urine Biochemical fractionation AQUA Peptide Digestion (trypsin) Peptide standard ( 13 C) NanoLC-MS Gerber et al. PNAS (2003)

52 Isotope dilution-based Proteomics for «absolute Quantification» LLSDDVDQTR *LLSDDVDQTR NanoLC chromatogram LLSDDVDQTR * LLSDDVDQTR MS scan

53 «Absolute Quantification» of TSST1 in urine using AQUA peptides Strataclean TM Trypsin Artificially Contaminated Urine (Toxin TSST1)

54 «Absolute Quantification» of TSST1 in urine using AQUA peptides AQUA peptide Strataclean TM Trypsin Artificially Contaminated Urine (Toxin TSST1) NanoLC-MS (QToF)

55 Accuracy and Precision of the AQUA Strategy TSST-1 Toxin Quantification Peptide LPTPIELPLK Measured TSST-1 (ng/ml urine) Added TSST-1 (ng/ml urine) AQUA Y = X

56 Isotope dilution-based Proteomics for «absolute Quantification» Urine Biochemical fractionation «Target» Protein recovery assumed to be 100%!!! AQUA Peptide Digestion (trypsin) Digestion yield assumed to be 100%!!! Peptide standards ( 13 C) Gerber et al. PNAS (2003) NanoLC-MS

57 Isotope dilution-based Proteomics for «absolute Quantification» Urine QconCAT Biochemical fractionation Polypeptide standard 13 C Digestion (trypsin) NanoLC-MS Beynon et al. Nature Methods (2005)

58 «Absolute» Quantification of TSST1 using a QconCAT construct QconCAT Strataclean TM Trypsin Artificially Contaminated Urine (Toxin TSST1) NanoLC-MS (QToF)

59 Accuracy and Precision of the QconCAT Strategy TSST-1 Toxin Quantification Peptide LPTPIELPLK Measured TSST-1 (ng/ml urine) QconCAT Y = 0,19.X Added TSST-1 (ng/ml urine) AQUA Y = X

60 In Quest of a Gold Standard for absolute Quantification PSAQ Protein standard 13 C PSAQ : Protein Standard Absolute Quantification In vitro synthesis (Roche) and purification of isotopically labelled ( 13 C Lys and 13 CArg) full lenght proteins Brun et al. MCP (2007)

61 In Quest of a Gold Standard for absolute Quantification PSAQ Urine Protein standard 13 C Biochemical fractionation Digestion (trypsin) NanoLC-MS Brun et al. MCP (2007)

62 «Absolute» Quantification of Toxins in urine using PSAQ PSAQ (TSST1*) Strataclean TM Trypsin Artificially Contaminated Urine (Toxin TSST1) NanoLC-MS (QToF)

63 Accuracy and Precision of the PSAQ Strategy TSST-1 Toxin Quantification Peptide LPTPIELPLK Measured TSST-1 (ng/ml urine) PSAQ Y = 1.08.X QconCAT Y = 0,19.X Added TSST-1 (ng/ml urine) AQUA Y = X

64 In Quest of a Gold Standard for absolute Quantification PSAQ Urine Protein standard 13 C Biochemical fractionation «Target» Protein recovery = PSAQ recovery Digestion (trypsin) «Target»ProteinDigestion yield = PSAQ Digestion yield Brun et al. MCP (2007) NanoLC-MS

65 «Absolute» Quantification of Toxins in Serum using PSAQ

66 Accuracy and Precision of the PSAQ Strategy Estimated SEA Toxin (ng/ml of serum) Added SEA Toxin (ng/ml of serum)

67 Perspectives for PSAQ methodology Technology Combine PSAQ with Multiple Reaction Monitoring (MRM)

68 6.00E E E E E E E / CRKB R 2 = ng injectés Sensitivity, specificity and multiplexing using MRM Signal Intensity Intensity, cps XIC of 616.8/742.4 XIC of 616.8/857.4 Max cps XIC of 616.8/ XIC : 652.4/ XIC:65 XIC 160of 616.8/ /10 XIC 120 XIC:6 XIC 100 of 616.8/ / / Time, min Aire( C p s ) Aire(Cps) / CRKB 6.00E E E+04 R 2 = E E E E ng injectés min Time (min) Target Protein : Human Creatine Kinase / CRKB 6.00E E+04 Aire(Cps) 4.00E E E E E+00 R 2 = ng injectés

69 Perspectives for PSAQ methodology Technology Combine PSAQ with Multiple Reaction Monitoring (MRM) Set up a PSAQ in vitro high throughput production platform Call for an International effort Applications DECanBio : Use PSAQs for the evaluation of the Biomarker candidates InterLab standard material Systems Biology Anti-doping control Limitations Post- translational Modifications Control of the PSAQ folding Accurate Quantification of the PSAQ standard

70 EDyP Lab : Exploring the Dynamics of Proteomes