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Proteomic Analysis:  Proteomic Analysis:Basics and ApplicationsBasics and Applications           Ignasi Forné        Febru...
IntroductionGenomics
IntroductionTranscriptomics
IntroductionProteomics
Introduction    Genomics             Transcriptomics                      Proteomics    Location, Time, Interaction partne...
Proteomics: Basics                      Fractionation              Fractionation                      Enrichment          ...
Proteomics: Basics                            Fractionation              Fractionation                            Enrichme...
Proteomics: Basics                            Fractionation              Fractionation                            Enrichme...
Proteomics: Basics                            Fractionation              Fractionation                            Enrichme...
Proteomics: Basics                                                                                                     nES...
Proteomics: Basics                                                                                                  nESI  ...
Proteomics: Basics                                                                                                     nES...
Proteomics: Basics                     nESI                                 DDA     C18‐HPLC                              ...
Proteomics: Basics   Peptide charge and mass     p         g                             (M+2H)2+ [                       ...
Proteomics: Basics  _ _ _ F: ITMS + c NSI d w Full ms2 472.77@cid35.00                                      575.30     100...
Proteomics: Basics                                                      y5                dance                           ...
Proteomics: Basics                                   Ser                   Ile               Val               Lys        ...
Proteomics: Basics                                    Ser                                   Ile                      Val  ...
Proteomics: Basics                                               Ser                                    Ile               ...
Proteomics: Basics                                     Ser                         Ile                       Val          ...
Proteomics: Basics                     nESI                                 DDA     C18‐HPLC                              ...
Proteomics: Basics                                              nESI                                                   DDA...
Proteomics: Basics                                                                                                        ...
Proteomics: BasicsPeptide QuantificationPeptide Quantification      Intensity                         Peptide 1 L Sample A...
Proteomics: BasicsPeptide QuantificationPeptide Quantification                              Label Free                    ...
Proteomics: Basics                     Ong et al, Nat Chem Biol 2005
Proteomics: Basics                     de Godoy et al, Nature 2008
Proteomics: BasicsIn depth analysisIn depth analysis             Cox et al, Ann Rev Biochemistry 2011   Michalski et al, J...
Proteomics: ApplicationsExpression Proteomics WT                                                                          ...
Proteomics: Applications  Interactomics                                                                                   ...
Proteomics: ApplicationsPTM Analysis   Ub Ligase                                              GG                         G...
Proteomics: ApplicationsPTM AnalysisControl                                                                       P       ...
Proteomics: Missing valuesProteome           run 1                     run 2                    run 3             run 4   ...
Proteomics             Nature Methods: Method of the Year 2012
Proteomics: Targeted AnalysisProteome           run 1                     run 2                     run 3            run 4...
Proteomics: Targeted Analysis                                   Ser                   Ile               Val               ...
Proteomics: Targeted Analysis                                    Ser                                    Ile               ...
Proteomics: Targeted Analysis                           p            [                 ]                                  ...
Proteomics: Targeted Analysis                                Gallien et al, J Mass Spectrometry 2011
Proteomics: Targeted Analysis                                Picotti et al, Cell 2009
Proteomics: Targeted Analysis                                Picotti et al, Cell 2009
Structural Proteomics                        Hein et al, Handbook of System biology 2013
Structural Proteomics                        Examples                                   Herzog et al Science 2012
Summary  • MS‐based Proteomics:          • Identification          •Q           Quantification                ifi i       ...
Thanks!!                           Prof. Axel Imhof           And the Colleagues at the Imhof‘s and the ZfP              (...
Questions??
Proteomic Analysis:  Proteomic Analysis:Basics and ApplicationsBasics and Applications           Ignasi Forné        Febru...
Proteomics analysis: Basics and Applications
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Proteomics analysis: Basics and Applications

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Proteomics analysis: Basics and Applications

  1. 1. Proteomic Analysis:  Proteomic Analysis:Basics and ApplicationsBasics and Applications Ignasi Forné February 14, 2013
  2. 2. IntroductionGenomics
  3. 3. IntroductionTranscriptomics
  4. 4. IntroductionProteomics
  5. 5. Introduction Genomics Transcriptomics Proteomics Location, Time, Interaction partners, PTMs, Dynamics, Turnover Adapted from Hein et al, Handbook of System biology 2013
  6. 6. Proteomics: Basics Fractionation Fractionation Enrichment Enrichment Cells/Tissue Proteins Peptides
  7. 7. Proteomics: Basics Fractionation Fractionation Enrichment Enrichment Cells/Tissue Proteins Peptides nESI C18‐HPLC Mass Spectrometer (m/z) Time
  8. 8. Proteomics: Basics Fractionation Fractionation Enrichment Enrichment Cells/Tissue Proteins Peptides nESI C18‐HPLC Mass Spectrometer (m/z) Time
  9. 9. Proteomics: Basics Fractionation Fractionation Enrichment Enrichment Cells/Tissue Proteins Peptides nESI C18‐HPLC Mass Spectrometer (m/z) Time
  10. 10. Proteomics: Basics nESI DDA C18‐HPLC Full MS top 1 MS2 top 2 MS2 top 3 p MS2 .... MS2 top n-1 MS2 Mass Spectrometer (m/z) top n MS2 Time Time Full MS p [ ] MS2 MS2 MS2 MS2T: FTMS + p NSI Full ms [300.00-2000.00] 472.7700 472.7700 100 _ _ _ top 1 top 2 top 3 top n 100 F: ITMS + c NSI d w Full ms2 472.77@cid35.00 90 575.30 90 100 80 428.25 90 ce 80 70 Relative Abundanc 60 80 70 473.2710 Relative Abundance 50 70Relativ Abundance 60 40 60 50 944.5325 513.2991 30 50 ve 769.4453 769 4453 40 691.3953 20 40 473.7723 10 472.3233 30 703.36 30 471.8120 474.2738 517.17 0 20 242.05 343.35 20 472 473 474 1046.2464 232.18 / 10 0 686 31 713.30 686.31 10 1106.5581 1381.7826 1844.3618 873.53 1306.7396 1537.8872 0 1890.9115 0 200 400 600 800 400 600 800 1000 1200 1400 1600 1800 2000 m/z m/z Time
  11. 11. Proteomics: Basics nESI DDA C18‐HPLC Full MS top 1 MS2 top 2 MS2 top 3 p MS2 .... MS2 top n-1 MS2 Mass Spectrometer (m/z) top n MS2 Time Time Full MS MS2 MS2 MS2 MS2T: FTMS + p NSI Full ms [300.00-2000.00] 472.7700 944.5324 top 1 top 2 top 3 top n 100 100 F: ITMS + c NSI d w Full ms2 944.53@cid35.00 90 1108.53 90 100 80 80 90 Relative Abundanc e 70 945.0824 810.08 70 60 80 945.5353 Relative Abundance 50 70Relativ Abundance 60 761.14 40 60 50 944.5325 944.2667 513.2991 30 50 ve A 769.4453 769 4453 945.7967 40 691.3953 20 621.24 946.5384 40 10 30 9 30 1266.51 943.5265 0 20 944 945 946 947 20 353.26 1046.2464 m/z 493.13 493 13 915.90 1381.7826 10 1469.38 1469 38 10 1106.5581 1844.3618 1714.74 1306.7396 1537.8872 1890.9115 0 0 500 1000 1500 400 600 800 1000 1200 1400 1600 1800 2000 m/z m/z Time
  12. 12. Proteomics: Basics nESI DDA C18‐HPLC Full MS top 1 MS2 top 2 MS2 top 3 p MS2 .... MS2 top n-1 MS2 Mass Spectrometer (m/z) top n MS2 Time Time Full MS MS2 MS2 MS2 MS2 513.2990T: FTMS + p NSI Full ms [300.00-2000.00] 100 472.7700 top 1 top 2 top 3 top n 100 90 F: ITMS + c NSI d w Full ms2 513.30@cid35.00 513.6329 80 534.53 90 100 598.58 dance 70 80 90 60 Relative Abund 80 70 50 Relative Abundance 70Relativ Abundance 40 513.9668 60 30 60 50 944.5325 513.2991 20 50 655.13 655 13 ve 769.4453 769 4453 514.3010 40 691.3953 10 513.1269 512.7917 514.6352 40 0 30 513.0 513.5 514.0 514.5 30 449.42 m/z 20 20 230.02 717.35 1046.2464 10 343.04 343 04 10 1106.5581 1381.7826 1844.3618 825.52 926.25 1050.54 1306.7396 1537.8872 0 1890.9115 0 200 400 600 800 1000 400 600 800 1000 1200 1400 1600 1800 2000 m/z m/z Time
  13. 13. Proteomics: Basics nESI DDA C18‐HPLC Full MS top 1 MS2 top 2 MS2 top 3 p MS2 .... MS2 top n-1 MS2 Mass Spectrometer (m/z) top n MS2 Time Time DDA Video
  14. 14. Proteomics: Basics Peptide charge and mass p g (M+2H)2+ [ The charge p ] 100 472.7700 (m2-m1)/z= 0.5  z=2 90 [m2-m1 = 1] 80 z=2  m= M+2H+ ance 70Relativ Abunda 60 473.2710 50 The mass ve 40 0 m/z= (M+2H+)/2 30 m/z=472.7700 20 473.7723 H+ =1 0073 =1.0073 10 472.3233 471.8120 474.2738 0 M 943.5254 M= 943 5254 472 473 474 m/z /
  15. 15. Proteomics: Basics _ _ _ F: ITMS + c NSI d w Full ms2 472.77@cid35.00 575.30 100 428.25 90 80 undance 70 60 lative Abu 50 40 Rel 30 703.36 517.17 20 242.05 343.35 232.18 10 686.31 713.30 873.53 0 200 400 600 800 m/z
  16. 16. Proteomics: Basics y5 dance y4 Rellative Abund y3 y7 b3 y2 b4 y6 b2 b5 b7 b6 m/z
  17. 17. Proteomics: Basics Ser Ile Val Lys Gly Val Met Glu y5 dance y4 Rellative Abund y3 y7 b3 y2 b4 y6 b2 b5 b7 b6 m/z
  18. 18. Proteomics: Basics Ser Ile Val Lys Gly Val Met Glu Ser Val Ile Gly Ser Val Ile Met Val Glu Gly dance Ser Val Ile Met Val Rellative Abund Gly Gly Ser Val b3 Gly Ser Val Ile Met Ser Gly b4 b2 b5 b7 b6 m/z
  19. 19. Proteomics: Basics Ser Ile Val Lys Gly Val Met Glu Ile Met Val Glu Lys y5 Met Val Glu Lys Ser Val Ile Met Val Glu Lys dance y4 Rellative Abund Val Glu Gl Lys L Val Ile Met Val Glu Lys Glu Lys y3 y7 y2 y6 m/z
  20. 20. Proteomics: Basics Ser Ile Val Lys Gly Val Met Glu y5 Ile dance y4 Rellative Abund Met y3 Val y7 b3 Ser y2 Val b4 y6 Ile b2 Met b5 Val Ser Glu b7 b6 m/z
  21. 21. Proteomics: Basics nESI DDA C18‐HPLC Full MS top 1 MS2 top 2 MS2 top 3 p MS2 .... MS2 top n-1 MS2 Mass Spectrometer (m/z) top n MS2 Time Time The Charge The Mass of the Peptide  The Masses of the Fragments
  22. 22. Proteomics: Basics nESI DDA C18‐HPLC Full MS top 1 MS2 top 2 MS2 top 3 p MS2 .... MS2 top n-1 MS2 Mass Spectrometer (m/z) top n MS2 Time Time Peptide identification Protein identification Relative Abundance Sequence Database Search R m/z Score Calculation Target-Decoy-based Peptide to protein mapping FDR thresholding th h ldi FDR thresholding Adapted from Hein et al, Handbook of System biology 2013
  23. 23. Proteomics: Basics nESIPeptide QuantificationPeptide Quantification C18‐HPLC p [ ] 472.7700 100 90 80 ance 70 Relative Abunda 60 473.2710 50 40 30 20 473.7723 10 472.3233 Intensity 0 471.8120 472 473 474 474.2738 Mass Spectrometer (m/z) Mass Spectrometer (m/z) / Peptide 1 L Sample A Time m/z
  24. 24. Proteomics: BasicsPeptide QuantificationPeptide Quantification Intensity Peptide 1 L Sample A Time m/z
  25. 25. Proteomics: BasicsPeptide QuantificationPeptide Quantification Label Free (across runs) ( ) Peptide 1 L Sample B Intensity Peptide 1 L Sample A Time Peptide 1 H Sample B run 2 run 2 MS2 Reporter Ions run 1 Isotopic Labelling (SILAC, Dimethyl,  (SILAC Dimethyl Heavy peptides) m/z
  26. 26. Proteomics: Basics Ong et al, Nat Chem Biol 2005
  27. 27. Proteomics: Basics de Godoy et al, Nature 2008
  28. 28. Proteomics: BasicsIn depth analysisIn depth analysis Cox et al, Ann Rev Biochemistry 2011 Michalski et al, J Proteome Res 2011
  29. 29. Proteomics: ApplicationsExpression Proteomics WT WT+KO Lys Protein  Identification  PINK1 KO KO +  Lys +8 In‐gel  digestion Quantification  LC‐MSMS SILAC 2 BSA 1.5 WT > KO Keratin 1 se log 10 Ratio H/L Revers 0.5 Serie1 0 -2 -1.5 -1 -0.5 0 0.5 1 1.5 g -0.5 -1 KO> WT -1.5 log 10 Ratio H/L Forw ard Schreiner et al, Mol Biol Cell 2012
  30. 30. Proteomics: Applications Interactomics ‐/+P Arg, Lys Protein  Protein  Extract 1 Identification Arg +10, Lys +8 P +  Protein  Extract 2 In‐gel  Quantification  digestion LC‐MSMS SILAC 2 1.5 INTERACTORS Proteins confirmed in erse (SILAC 6) both SILAC 1 1 and 6 lo 10 Ratio H/L Reve 0.5 05 0 Serie1 -1.5 -1.25 -1 -0.75 -0.5 -0.25 0 0.25 0.5 0.75 1 1.25 1.5 -0.5 og -1 -1.5 -2 log 10 Ratio H/L Forward (SILAC 1)
  31. 31. Proteomics: ApplicationsPTM Analysis Ub Ligase  GG GG‐K  Identification  In‐gel  GG +  digestion LC‐MSMS DCC complex Quantification GG Villa et al, Mol Cell 2012
  32. 32. Proteomics: ApplicationsPTM AnalysisControl P Protein  P‐site  Extract  P Identification  In‐solution P Phosphopeptide  digestion LC‐MSMS enrichment  +  P Protein  Extract  P Quantification  P Labelfree PStimulus l Ac Ac Ac‐K Protein  Extract  Extract Ac Identification  In‐solution Ac digestion Acetylpeptide enrichment  LC‐MSMS +  Protein  Extract  Extract Quantification  Ac Labelfree Ac
  33. 33. Proteomics: Missing valuesProteome run 1 run 2 run 3 run 4 Time Course 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 5 Protein 10 ...
  34. 34. Proteomics Nature Methods: Method of the Year 2012
  35. 35. Proteomics: Targeted AnalysisProteome run 1 run 2 run 3 run 4 Time Course Ti C 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 5 Protein 10 ...
  36. 36. Proteomics: Targeted Analysis Ser Ile Val Lys Gly Val Met Glu y5 dance y4 Rellative Abund y3 y7 b3 y2 b4 y6 b2 b5 b7 b6 m/z
  37. 37. Proteomics: Targeted Analysis Ser Ile Val Lys Gly Val Met Glu Ile Met Val Glu Lys y5 Met Val Glu Lys dance y4 Rellative Abund Val Glu Gl Lys L y3 m/z
  38. 38. Proteomics: Targeted Analysis p [ ] 472.7700 472 7700 100 Ile Met Val Glu Lys 90 y5 Met Val Glu Lys 80 ce Relativ AbundancRelativ Abundance 70 60 Val Glu Lys y4 473.2710 50 y3 ve ve 40 30 20 473.7723 10 472.3233 472 3233 471.8120 474.2738 0 472 473 474 / m/z SRM Video Picotti et al, Nat Methods 2012
  39. 39. Proteomics: Targeted Analysis Gallien et al, J Mass Spectrometry 2011
  40. 40. Proteomics: Targeted Analysis Picotti et al, Cell 2009
  41. 41. Proteomics: Targeted Analysis Picotti et al, Cell 2009
  42. 42. Structural Proteomics Hein et al, Handbook of System biology 2013
  43. 43. Structural Proteomics Examples Herzog et al Science 2012
  44. 44. Summary • MS‐based Proteomics: • Identification •Q Quantification ifi i • Shotgun/Discovery • Targeted • Structural Proteomics and Interactomics
  45. 45. Thanks!! Prof. Axel Imhof And the Colleagues at the Imhof‘s and the ZfP  (Molecular Biology Dept. Biomedical Center‐LMU)
  46. 46. Questions??
  47. 47. Proteomic Analysis:  Proteomic Analysis:Basics and ApplicationsBasics and Applications Ignasi Forné February 14, 2013

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