1. Proteomics

2. Composition – Splice variants – Post translational modifications Interactions Distribution Activity Dynamics High content information

3. TOP DOWN VS BOTTOM UP

4. Mass Spectrometry

5. Ionization methods for peptides Electrospray ionization (ESI) Matrix assisted laser desorption ionization (MALDI)

6. Tandem Mass Spectrometry (MSMS) Molecular Systems Biology 2008 4:222

7. QqTOF Layout

8. Ion Trap Methods in Enz. 402:109

9. Schematic of the LTQ Orbitrap Velos MS instrument with three new hardware implementations. Olsen J V et al. Mol Cell Proteomics 2009;8:2759-2769 ©2009 by American Society for Biochemistry and Molecular Biology

10. Peptide Fragmentation Adv Protein Chemistry & Structural Biology, Vol. 80, 1-44, 2010

11. Archives of Physiology and Biochemistry, 2009; 115(5): 311–319

12. Typical work flow Mass spectrometry Proteins Peptides

13. From proteome lists to biological impact– tools and strategies for the analysis of large MS data sets PROTEOMICS Volume 10, Issue 6, pages 1270-1283, 13 JAN 2010 DOI: 10.1002/pmic.200900365 http://onlinelibrary.wiley.com/doi/10.1002/pmic.200900365/full#fig1 Volume 10, Issue 6, http://onlinelibrary.wiley.com/doi/10.1002/pmic.200900365/full#fig1

15. Proteins: Considerations Virtually any source e.g. tissues, fluids, cells, cellular components, supernatants Free from degradation. Rapid harvest and processing or storage. A few micrograms required for analysis but must be reasonably concentrated Free from inhibitory materials Dynamic range of materials

16. Compositional Questions What proteins are present? Are their protein differences between two samples? – Diseased vs normal – Differentiated vs undifferentiated – Resting vs activated – Wild type vs knockout

17. Separation Approaches Separation methods – 1-2 D PAGE – Multidimensional LC – Capillary electrophoresis – Affinity Antibody Lectin Substrate cofactor PTM

18. 1and 2D LC Orthogonal separations – Protein level in principle not generally in practice – MUDPIT on line – 1 st dimension offline Many types of separation

19. 2D SDS PAGE Strengths – fair resolution – Meta data size and isoelectric point Molecular heterogeneity (PTMs) Limitations – Load – Solubility – Isoelectric point extremes – molecular size – variability

20. 2D Difference Gel electrophoresis (DIGE) AppliedBiomics.com

21. Biosynthetic Labeling Based on incorporation of stable isotope labels Benefits – Uniform labeling – Minimally manipulative – Samples processed as single batch Considerations – Incorporation efficiency Division Turnover rate – Catabolism – PTMs – Cost – Cell growth in SF or reduced media

22. Nat. Inst. Aging

23. Triple SILAC Proteome Res., Article ASAP DOI: 10.1021/pr200740a October 20, 2011

24. Stable Isotope Labeling Dual tags – e,.g. ICAT SH specific Reduces complexity but may limit number of useful peptides for ID Multiplex (up to 10) – ABI iTRAQ – PE ExacTag

25. iTRAQ—Isobaric Tags for Relative and Absolute Quantification Adv Protein Chemistry & Structural Biology, Vol. 80, 1-44, 2010

26. Comparison of SILAC and iTRAQ SILACiTRAQ Basis of labellingBiosyntheticChemical Sample labellingSimpleLabour intensive RequirementsActive metabolismProteins Defined mediaProteins MultiplexingYes SignalDilutedSingle IDInference some timesConfirmed Potential for Biaslowhigher ReutilisationNot applicable Cellular capacityNot applicable

27. Phosphoproteomics

28. Proteomics 2007, 7, 2751 Isolation methods

29. Proteomics 2007, 7, 2751 Derivatization methods

30. Comparison of phosphorylation enrichment methods Proteomics 2007, 7, 2751

31. TCR Activation specific phospho sites Sci. Signal. 2009 Vol. 2, p. ra46

32. Selected Reaction Monitoring Targeted for selected analytes. Not a discovery approach for protein identification Increases sensitivity 30-50 fold Qualitative or quantitative Offers multiplexing capacity 20-30 analytes per run

33. Selected reaction monitoring applied to proteomics Journal of Mass Spectrometry Volume 46, Issue 3, pages 298-312, 10 MAR 2011 DOI: 10.1002/jms.1895 http://onlinelibrary.wiley.com/doi/10.1002/jms.1895/full#fig1 Volume 46, Issue 3, http://onlinelibrary.wiley.com/doi/10.1002/jms.1895/full#fig1

34. Selected Reaction Monitoring

35. Selected reaction monitoring applied to proteomics Journal of Mass Spectrometry Volume 46, Issue 3, pages 298-312, 10 MAR 2011 DOI: 10.1002/jms.1895 http://onlinelibrary.wiley.com/doi/10.1002/jms.1895/full#fig5 Volume 46, Issue 3, http://onlinelibrary.wiley.com/doi/10.1002/jms.1895/full#fig5

37. Bioorthogonal Chemistry

39. Bioorthogonal CLICK Chemistry

41. Activity Based Protein Profiling

42. Mechanism of probe action Inactive enzyme Active enzyme Labelled Unlabelled

43. Nomura et. al Nature Rev. Cancer 10:630 (2010)

44. Marker 2E4A-SH 2E4B-SH LG8A-SH LG8B-SH Normal Lethal 12k 17k 24k 31k 38k 52k 76k 102k 150k 225k Marker 2E4 Cell lysateLG8 Cell lysate 2E4-Serine Hydrolase LG8-Serine Hydrolase 12k 17k 24k 31k 38k 52k 76k 102k 150k 225k Marker 2E4 Cell lysateLG8 Cell lysate 2E4-Serine Hydrolase LG8-Serine Hydrolase Commassie

45. Urine Profiling in Renal Transplant

46. Summary :Activity based protein profiling Adjunct to other high content approaches Feasible to apply directly to clinical samples for rapid monitoring and quantitation of activity Full coverage of activities not attainable at this time Potential for identification of new processes Feasibility of point of care analysis in near real time Potential for arrayed format for multiplexing Utility in infectious diseases?

47. Examples

48. Cell Migration Requires Dynamic and Regional Processes

49. MW + Lys

50. What does enrichment Mean?

51. Candidate Selection 119 selected based on “enrichment” Screened ~90 clones targeting 22 proteins using shRNAmir mediated silencing 15 targets with 2 or more clones inhibiting migration by >50% and outside 3 SD of assay variation.

52. NK Synapse (NKIS)

54. IQGAP1 involvement in MTOC and granule polarization in NK‐cell cytotoxicity European Journal of Immunology Volume 41, Issue 9, pages 2763-2773, 3 AUG 2011 DOI: 10.1002/eji.201040444 http://onlinelibrary.wiley.com/doi/10.1002/eji.201040444/full#fig9 Volume 41, Issue 9, http://onlinelibrary.wiley.com/doi/10.1002/eji.201040444/full#fig9

55. NK Granule Release LC MSMS Contents ~400 proteins Membrane and associated proteins

56. Acknowledgements MCPSB – Namita Kanwar – Dmitry Shanshun – Paymen Ezzati – Peter Nickerson – John Cortens – Xiaobo Meng – Ravi Dwivedi – William Summers – Patty Sauder – Dustin Lippert – Mario Navarrete Oleg Krokhin Chris Bleackley ThermoFisher Pierce – Monica O’Hara – Ryan Bomgarden

57. Morphological and phenotypic changes associated with EMT Acloque H et al. J Clin Invest. 2009; 119:1438 i-ii Interactions ii-iii ECM iii Polarity iv Mobility Control TGF Control TGF Beta

58. SILAC Labelleing + TGFβ1 - TGFβ1 72h K and R C 12, N 14 K and R C 13, N 15 Combine SDS PAGE LC MS/MS

59. Data analysis 3500 proteins identified with confidence scores >95% Only proteins observed in biological replicates included in current analysis Those outside 95% interval of the population (z score >1.96) selected 45 up regulated 36 down regulated Log 2 ratio Frequency