1. Proteomics: Technology and Cell Signaling Presenter: Ido Tal Advisor: Prof. Michal Linial 31.5.15 י " ג סיון תשע " ה

2. Outline Proteomics motivation and background Identification and quantification technologies –  Antibodies based (e.g. Western Blot)  2D Gel  Mass Spectrometry (MS)  SILAC MS based study  Identified phosphopeptides in signaling cascade  Understanding the function of proteins.  Identified phospho-sites.  difference in phospho-sites Conclusions and thoughts

3. What are the functions of proteins? Replicating DNA Sensing the environment Control and Regulation Enzymes Transporting molecules Protecting against pathogens etc

5. DNA tells what is possible RNA tells what is probable Genomics Transcriptomics Proteins tell what is actually happen Proteomics

6. Proteome “The total protein complement able to be encoded by a given genome” “Entire protein complement expressed by a genome, or by a cell or tissue type” Prof. Marc Wilkins, 1995

7. Proteome “The proteome of a cell, tissue or body fluid is the collection of all proteins and their modification present in a given state.”

8. What we want to investigate? The sequence (in amino acids) of a protein Post translational modification (PTM) Spatial localization Identification Quantity Protein-protein interactions 3D-structure of all proteins Clinical related (Biomarkers)

9. Identification and quantification technologies Antibodies e.g. Western blot http://www.proteinatlas.org/

10. Identification and quantification technologies 2D gel

11. Identification and quantification technologies Comparative analysis

12. Identification and quantification technologies Mass Spectrometry (MS) SILAC

13. Mass Spectrometry (MS) 1) Proteins extracted and fractioned or enriched for specific proteins or PTMs. 2) Peptides are separated and ionized. 3) Mass spectra of the peptides and their fragments are obtained. 4) The acquired data are analysed.

14. Mass Spectrometry (MS) preparation

15. Ionizer Sample + _ Mass Analyzer Detector MALDI Electro-Spray Ionization (ESI) Time-Of-Flight (TOF) Quadrapole Ion-Trap Electron Multiplier (EM) 2002, Nobel Prize JB Fenn - electrospray ionization (ESI) K. Tanaka - soft laser desorption Mass Spectrometry (MS)

16. AA mass Amino-AcidResidual MWAmino-AcidResidual MW AAlanine71.03712MMethionine131.04049 CCysteine103.00919NAsparagine114.04293 DAspartic acid115.02695PProline97.05277 EGlutamic acid129.04260QGlutamine128.05858 FPhenylalanine147.06842RArginine156.10112 GGlycine57.02147SSerine87.03203 HHistidine137.05891TThreonine101.04768 IIsoleucine113.08407VValine99.06842 KLysine128.09497WTryptophan186.07932 LLeucine113.08407YTyrosine163.06333

17. Mass Spectrometry (MS) mass analyzer

18. Mass Spectrometry (MS) analysis

19. MS/MS

20. MS/MS

21. SILAC - Stable isotope labeling by amino acids in cell culture Technique based on MS that detects differences in protein abundance among samples using non- radioactive isotopic labeling.

22. SILAC

23. SILAC –careful quantifications

24. Education: Physics, Mathematics Chemical Engineering Research Max-Planck-Institute of Biochemistry (Director) Martinsried Germany Matthias Mann

25. PTM Importance Many critical events involved in cellular responses are mediated by changes in post-translational modification (PTM) rather than transcriptional changes. Influence and control Protein modification Enzymatic activity Protein conformation PPI Cellular localization

26. EGF signaling begins with activation of the EGF receptor and extends through a cascade of downstream kinases to mediate the increased phosphorylation of a large number of substrate proteins. EGF signal

27. Previous studies Focus on “early events” Using anti-phosphotyrosine antibodies, which exclude detection of downstream serine/threonine kinase signaling large numbers of phosphorylation sites, but without a functional context. No dynamic view (the sequence of events) Very few proteins identified in the signaling

28. The research question Study the global in vivo phosphoproteome and its temporal dynamics upon growth-factor stimulation.

31.  More than 10,000 phosphopeptides were detected.  Greater than 99% certainty of phosphopeptide identification  identified 6,600 phosphorylation sites on a total of 2,244 proteins.

32. Challenges and dealing Some fragmentations do not contain sufficient information to determine the phospho-site with single a.a. accuracy. Using PTM score for localization, which assigns probabilities to each of the possible sites based on their distinguishing fragment ions. The algorithm make use of the four most intense fragment ions per 100 m/z units.

33. Example of PTM score for localization

34. Challenges and dealing In addition take 22 consensus motifs from 15 of the most common kinases and matched all candidate phospho-site against these motifs. Given the peptide sequence and number of phospho-sites, group all peptide into 4 categories depending on the PTM score and motifs.

35. Localization prob. >0.75 Localization prob. 0.25-0.75 & match one of the 22 motifs Localization prob. 0.25-0.75 & no match one of the 22 motifs

36. Hunter's experiment comparison Hunter (1980)MannPhospho-site 0.05%1.8%pY – phosphotyrosine 10%11.8%pT - phosphothronine 90%86.4%pS - phosphoserine tyrosine phosphorylation tends to occur on less abundant proteins compared to serine and threonine phosphorylation. pY is less stable in phosphoamino acid analysis than pS/pT.

37. Database comparison More than 90% of Mann’s experiment sites were novel with respect to SwissPort database. Close to 90% of Mann’s experiment sites were novel compared with his previous studies.

38. functional context Clustering with Fuzzy c-means – Each profile is assigned a grade of membership for a set of clusters. Found 6 clusters: 4 with upregulated phosphopeptides 2 with downregulated phosphopeptides

41. Functional of regulated phosphopeptides

42. difference in phospho-sites Whether different phospho-sites within the same protein were regulated in the same manner? Most proteins (77%) that had a regulated phosphopeptide also had at least one additional phosphopeptide that behaved differently. i.e., that was either unchanging or else in a different cluster. Most proteins (77%) that had a regulated phosphopeptide also had at least one additional phosphopeptide that behaved differently. i.e., that was either unchanging or else in a different cluster.

43. difference in phospho-sites

45. Difference in phospho-sites For obtain accurate and functionally relevant understanding of activation kinetics we need to measure site specifically. Protein phosphorylation typically serves different functions on different sites of the protein.

46. Open source databases for the collection of Phosphoproteome Phosida allows the retrieval of phosphorylation acetylation N-glycosylation of any protein of interest

47. Open source databases for the collection of Phosphoproteome

48. Open source databases for the collection of PTM PhosphoSitePlus ® (PSP) is an online systems biology resource providing comprehensive information and tools for the study of protein post- translational modifications (PTMs)

49. Conclusions Importance of proteomics. Identification and quantification technologies - MS and SILAC and using them for Identified phosphopeptides in signaling cascade Understanding the function of proteins in signaling using Fuzzy c-means Identified phospho-sites and the function that can hidden in their difference.