1. Proteomics What is it? How is it done? Are there different kinds? Why would you want to do it (what can it tell you)?

2. DNA RNA Protein The Central Dogma of Molecular Biology transcription translation Key Concept: Proteins do the work everything else is mostly information Key Concept: Proteomics is the “high throughput” analysis of proteins.

3. DNAGenomics RNAGenomics/Transcriptomics ProteinProteomics MetabolitesMetabolomics The Central Dogma-omics

4. DNAGenomics ~6-45,000 parts RNAGenomics/Transcriptomics ~100,000 parts ProteinProteomics ~1,000,000 parts MetabolitesMetabolomics >500,000,000 parts The Central Dogma-omics Key Concept: Complexity increases the farther away from information you get Key Concept: Chemical complexity also increases the farther away you get

5. Protein Machines Key Concept: Biochemical functions are carried out by multi-protein machines The polyAdenylation Machinery The Proteosome Key Concept: A Protein Function can be inferred by it’s binding partners Key Concept: Knowledge of a Machine’s components is required to understand how it works and how it is regulated

6. Protein Networks 2 Steps Key Concept: Protein Machines are organized into larger Networks

7. Proteins are Organized in Super Networks Key Concept: The proteome is HIGHLY Networked

8. Key Concept: Proteins vary widely in concentration

9. Major Types of Proteomics Interactomics: Mapping Protein:Protein Interactions -Yeast 2-hybrid techniques -high throughput protein identification by Mass Spectrometry Survey Proteomics: Qualitative or Quantitative Analysis of the protein component -whole organism, tissue, cell type, or subcellular compartment -2D gel electrophoresis ->MS -typically a few 100 proteins -Multidimensional LC->MS/MS -typically a few 1000 proteins Identification of Biomarkers

10. Information Trade Offs Vida infra G. MacBeath

11. Proteins are Organized in Super Networks Key Concept: The proteome is HIGHLY Networked

12. Time of Flight Mass Spectrometer Key Concept: All Mass Spectrometers have at least 2 parts: an ion source and a mass analyzer Detectors Flight tube Timed ion selector Laser Sample plate Reflector Accelerating field + + + + + + + Key Concept: Mass Spectrometers can only measure charged species.

13. A Quadrupole Mass Filter

14. Ion Trap Mass Spectrometer Key Concept: Mass Spectrometers can only measure ions and more accurately, the behavior of ions in an electric field

15. A Generic Mass Spectrum Key Concept: A mass spectrum is a graphical representation of the the data, with intensity on the y axis and m/z on the x axis

16. Important Properties of a Mass Spectrum Mass is expressed as m/z (mass/charge). m=1000 z=1 m/z =1001 m=1000 z=2 m/z =501 m=1000 z=3 m/z =334.3 Intensity is usually normalized to 100 Intensity can be expressed as ion current or ion counts The intensity is related to abundance, but only when you are referring to the same compound. Mass accuracy is an important parameter that affects downstream analysis. Mass accuracy is usually expressed as ppm. EM=1000.1 TM=1000 ppm= (0.1/1000) x 10 6 100 ppm

17. How to Identify Proteins Key Concept: Each unique protein will give rise to a unique set of peptides Proteolysis

18. Peptide Mass Fingerprinting Key Concept: A single protein yields many proteolitic fragments Proteolysis

19. Peptide Mass Fingerprinting Key Concept: A mass spectrometer is a very accurate scale!

20. Peptide Mass Fingerprinting

21. ENSP00000031135 ENSP00000084795 ENSP00000198939 ENSP00000201886 ENSP00000202773 ENSP00000216019 ENSP00000216038 ENSP00000216520 ENSP00000216832 ENSP00000223129 ENSP00000225430 ENSP00000225792 ENSP00000233078

22. Peptide Mass Fingerprinting ENSP00000031135 ENSP00000084795 ENSP00000198939 ENSP00000201886 ENSP00000202773 ENSP00000216019 ENSP00000216038 ENSP00000216520 ENSP00000216832 ENSP00000223129 ENSP00000225430 ENSP00000225792 ENSP00000233078 ENSP00000254108 ENSP00000254636 ENSP00000254719 ENSP00000254942 ENSP00000259848 ENSP00000259925 ENSP00000261366 ENSP00000261700 ENSP00000216019 ENSP00000262570 ENSP00000262584 ENSP00000262637 ENSP00000262709

23. Peptide Mass Fingerprinting ENSP00000031135 ENSP00000084795 ENSP00000198939 ENSP00000201886 ENSP00000202773 ENSP00000216019 ENSP00000216038 ENSP00000216520 ENSP00000216832 ENSP00000223129 ENSP00000225430 ENSP00000225792 ENSP00000233078 ENSP00000263698 ENSP00000263746 ENSP00000264258 ENSP00000264293 ENSP00000269349 ENSP00000269576 ENSP00000270799 ENSP00000281154 ENSP00000283179 ENSP00000216019 ENSP00000290341 ENSP00000294823 ENSP00000296490 ENSP00000254108 ENSP00000254636 ENSP00000254719 ENSP00000254942 ENSP00000259848 ENSP00000259925 ENSP00000261366 ENSP00000261700 ENSP00000216019 ENSP00000262570 ENSP00000262584 ENSP00000262637 ENSP00000262709

24. Peptide Mass Fingerprinting ENSP00000031135 ENSP00000084795 ENSP00000198939 ENSP00000201886 ENSP00000202773 ENSP00000216019 ENSP00000216038 ENSP00000216520 ENSP00000216832 ENSP00000223129 ENSP00000225430 ENSP00000225792 ENSP00000233078 ENSP00000263698 ENSP00000263746 ENSP00000264258 ENSP00000264293 ENSP00000269349 ENSP00000269576 ENSP00000270799 ENSP00000281154 ENSP00000283179 ENSP00000216019 ENSP00000290341 ENSP00000294823 ENSP00000296490 ENSP00000254108 ENSP00000254636 ENSP00000254719 ENSP00000254942 ENSP00000259848 ENSP00000259925 ENSP00000261366 ENSP00000261700 ENSP00000216019 ENSP00000262570 ENSP00000262584 ENSP00000262637 ENSP00000262709 ENSP00000202773 ENSP00000216019 ENSP00000216038 ENSP00000216520 ENSP00000216832 ENSP00000223129 ENSP00000225430 ENSP00000225792 ENSP00000233078 ENSP00000233084 ENSP00000233468 ENSP00000240851 Key Concept: Each mass pulls out ~11,000 candidate proteins

25. Peptide Mass Fingerprinting ENSP00000031135 ENSP00000084795 ENSP00000198939 ENSP00000201886 ENSP00000202773 ENSP00000216019 ENSP00000216038 ENSP00000216520 ENSP00000216832 ENSP00000223129 ENSP00000225430 ENSP00000225792 ENSP00000263698 ENSP00000263746 ENSP00000264258 ENSP00000264293 ENSP00000269349 ENSP00000269576 ENSP00000270799 ENSP00000281154 ENSP00000283179 ENSP00000216019 ENSP00000290341 ENSP00000294823 ENSP00000254108 ENSP00000254636 ENSP00000254719 ENSP00000254942 ENSP00000259848 ENSP00000259925 ENSP00000261366 ENSP00000261700 ENSP00000216019 ENSP00000262570 ENSP00000262584 ENSP00000262637 ENSP00000202773 ENSP00000216055 ENSP00000216038 ENSP00000216019 ENSP00000216832 ENSP00000223129 ENSP00000225430 ENSP00000225792 ENSP00000233078 ENSP00000233084 ENSP00000233468 ENSP00000240851 ENSP00000244357 Key Concept: Ideally only one protein should be uniquely identified

26. Key Concept: Fragments can be matched to a database of proteins. The number of indentified proteins is related to the mass accuracy.

27. Peptide Mass Fingerprints often FAIL to give Significant Results -Protein Mixtures “confuse” the statistical algorithms -Not enough high quality peaks -Too many “popular masses” -Possible to “play” with the search parameters to give a statistically meaningful result. Can be overcome by performing peptide fragmentation (AKA MS/MS)

28. Thermo Demo Movie

29. Proteolysis What is MS/MS -MS/MS is a method in which a peptide is fragmented and the masses of the fragment ions measured.

30. What is MS/MS -MS/MS is a method in which a peptide is fragmented and the masses of the fragment ions measured.

31. How do you interpret an MS/MS spectra?? S A M P L E R S A M P L E + R S A M P L E R S A M P S A M L E R P L E R + + +

32. S A M P L E R +

33. Steps for Manual Interpretation 1.Label the precursor masses 2.Label any obvious water losses (m/z with  18) 3.Look for any peaks with  28 (a – b ion pairs) 4.Look up the mass of the first b ion in the dipeptide chart. 5.Begin building the b ion series and use the y ion series for confirmation using the tables. 6.Extend till the you reach the presurser mass. (the last amino acid in the b ion series should be a Lys or Arg and will be +18 relative to the mass in the table.)

34. 213.1 99.1

35. 213.1 99.1

36. AA CodeMW AA CodeMW AA CodeMW AA CodeMW G57.02 T101.04D115.02H137.05 A71.037 C103.01Q128.05F147.06 S87.03 L113.08K128.09R156.10 P97.05 I113.08E129.04Y163.06 V99.06N114.04M131.04W186.08 99.1 213.1

37. AA CodeMW AA CodeMW AA CodeMW AA CodeMW G57.02 T101.04D115.02H137.05 A71.037 C103.01Q128.05F147.06 S87.03 L113.08K128.09R156.10 P97.05 I113.08E129.04Y163.06 V99.06N114.04M131.04W186.08 V 99.1 213.1

38. AA CodeMW AA CodeMW AA CodeMW AA CodeMW G57.02 T101.04D115.02H137.05 A71.037 C103.01Q128.05F147.06 S87.03 L113.08K128.09R156.10 P97.05 I113.08E129.04Y163.06 V99.06N114.04M131.04W186.08 N V 114

39. AA CodeMW AA CodeMW AA CodeMW AA CodeMW G57.02 T101.04D115.02H137.05 A71.037 C103.01Q128.05F147.06 S87.03 L113.08K128.09R156.10 P97.05 I113.08E129.04Y163.06 V99.06N114.04M131.04W186.08 V N Q 114 128

40. AA CodeMW AA CodeMW AA CodeMW AA CodeMW G57.02 T101.04D115.02H137.05 A71.037 C103.01Q128.05F147.06 S87.03 L113.08K128.09R156.10 P97.05 I113.08E129.04Y163.06 V99.06N114.04M131.04W186.08 I/L V N Q 114 128 113

41. V N Q I/L G T SE I K AA CodeMW AA CodeMW AA CodeMW AA CodeMW G57.02 T101.04D115.02H137.05 A71.037 C103.01Q128.05F147.06 S87.03 L113.08K128.09R156.10 P97.05 I113.08E129.04Y163.06 V99.06N114.04M131.04W186.08

42. V N Q I/L G T SE I K AA CodeMW AA CodeMW AA CodeMW AA CodeMW G57.02 T101.04D115.02H137.05 A71.037 C103.01Q128.05F147.06 S87.03 L113.08K128.09R156.10 P97.05 I113.08E129.04Y163.06 V99.06N114.04M131.04W186.08 V N Q I/L

43. 175.00 417 584.10 826.25

44. 157.04

45. Proteolysis Key Concept: A sample analyzed by MALDI can generate between 10-20 MS/MS spectra before it is consumed.

46. How to Identify Proteins Key Concept: A few MS/MS spectra or even MS/MS spectra from a single protein can be manually interpreted CE Protein Bands are cut from gel, trypsinized and analyzed by mass spectrometry -direct sequencing -search a database (probability based ID)

47. MS/MS allows for Shot Gun Proteomics Key Concept: Shot Gun Proteomics lets you look at many things at once Proteolysis

49. Protein ID by Mass Spectrometry

50. ~10,000 MS/MS per hour Key Concept: LC-MS/MS data is time restricted by the elution profile of the peptides. Maldi is restricted by sample consumption.

51. V N Q I/L G T SE I K AA CodeMW AA CodeMW AA CodeMW AA CodeMW G57.02 T101.04D115.02H137.05 A71.037 C103.01Q128.05F147.06 S87.03 L113.08K128.09R156.10 P97.05 I113.08E129.04Y163.06 V99.06N114.04M131.04W186.08 Key Concept: de Novo interpretation is computationally intensive. Most software programs do it by Spectral Matching

52. acquired spectrum Theoretical spectrum (y/b ions) 100% 0% 1 0 Spectral Matching

53. acquired spectrum theoretical spectrum (y/b ions) 100% 0% 1 0 x Spectra matched

54. Protein ID by Mass Spectrometry