1. Isobaric tags for quantitative analysis Joshua J. Coon U. Wisconsin-Madison

2. Isobaric Tagging Ross,PL. et al MCP 2004

3. iTRAQ Structure

4. Available Reagents

5. Phosphorylation site on LIN28

6. Contemporary proteomics

7. Introduction of internal standard

8. Popular quantitative methods

9. MS1 vs MS2 quantitation

10. Number of quantitative data points

11. Dual cell linear ion trap orbitrap MS

12. Questions

13. Possible sources of error

14. 1. Tag impurities

15. 1. Tag impurities - Correction factors

16. 1. Tag impurities - TagQuant

17. 2. Low-intensity reporter ions

18. 2. Low-intensity reporter ions – Summing

19. 3. Interfering fragment ions

20. 3. Sample Processing

21. 4. Interference

24. Solutions to sources of error

25. Fragmentation method compatibility

26. ETD does not generate intense reporters

27. Gardner, Coon, Brodbelt, et al., Anal Chem, 2009 IRMPD on an dual-cell linear ion trap

28. IRMPD of isobaric tag-labeled peptide

29. Fragmentation method compatibility

30. Dual cell linear ion trap orbitrap MS

31. Performance improvements McAlister, GC. et al Analytical Chemistry 2010

32. Metabolic labeling vs isobaric tags

33. Peptide Identification

34. Peptide and Protein Quantitation

35. Distribution of error

36. Triplicate Analyses

38. Quantitation validation

39. Samples

40. MS workflow

41. Results

42. Cell-type specific genes

43. Cell type specific genes

44. ES / iPS differences

45. Protein-protein interactions

46. Reprogramming networks

47. Triplicate 6-plex TMT experiment and correlation of protein to RNA

49. Ongoing 8-plex experiment

50. Software

53. Cyclin-dependent kinase 2

54. RNA up Protein down

55. 1. Tag impurities

56. SRM and western blot validation