1. Integrative Proteomics 01/24/2001 Mass Spectrometry for Protein Identification Brian Cox Integrative Proteomics Translation elongation factor EF-Tu

2. Integrative Proteomics 01/24/2001 Protein Characterization by Mass Spectrometry Protein identification –correlative database matching Quality control –recombinant protein, protein modification reactions Post-translational modifications –phosphorylation, glycosylation, methylation, etc Identification of Domains –Functional structural domains, pro-enzyme to enzyme cleavage

3. Integrative Proteomics 01/24/2001 Sarcoplasmic Reticulum Terminal Cisternae Fraction Myosin PI 3 Kinase TAK-1 TGF beta activated kinase Sarcaluminum precursor Diamine oxidase 6-Phosphofructokinase, aconitase GRP 78 HSP 70, GRP 75 Calsequestrin, SK muscle SR 53 kDa, ATP synthase alpha Glyceraldehyde 3-phosphate dehydrogenase Malate dehydrogensase L-lactate dehydrogenase Calretenin Succinate dehydrogenase GTP:AMP Phosphotransferase SFI 3-hydroxyacyl-CoA dehydrogenase type II SFI Myosin light chain SFI Myosin regulatory l, light chain two SFI Cytochrome C Chaperonin 10 Nitric oxide synthase Calcium transporting ATPase (SERCA1) 6-Phosphofructokinase Glycerol-3-phosphate dehydrogenase ADP, ATP carrier protein ATP synthase Alpha subunit ATP synthase Alpha/Beta subunit ATP synthase Beta subunit Actin SFI SFI ADP, ATP carrier protein SFI Cytochrome C SFI Complex I-MLRQ } Similar spectra } Similar spectra S P HSP60, Rabbit serum albumin ATP synthase beta Creatine kinase, Citrate synthase Fructose bisphosphate aldolase Aspartate aminotransferase Actin Voltage independant pH sensitive K + channel 3-hydroxyacyl-CoA dehydrogenase type II

4. Integrative Proteomics 01/24/2001 0 Salt Elution [NusA] Ligand RNA polymerase subunit  ’ RNA polymerase subunit  Hypothetical ORF RNA polymerase subunit  SUHB Affinity Chromatography - E. coli NusA

5. Integrative Proteomics 01/24/2001 2D Gel Analysis

6. Integrative Proteomics 01/24/2001 Identifying and Mapping Protein Domains by Partial Proteolysis NusA [Trypsin] Proteolytic Products 0

7. Integrative Proteomics 01/24/2001 How Do I Identify Isolated Proteins? Genetics –applicable to simple organisms –time consuming Antibody screening –identifies only known, usually well characterized proteins Edman sequencing –requires large quantities of sample Mass Spectrometry –Correlative mass matching (sequence must be known) –MS/MS fragment data (sequence)

8. Integrative Proteomics 01/24/2001 MS Protein Identification MALDI-ToF Triple quad Ion Trap Q-ToF FT-MS Liquid Solid time consuming, PTMs, protein sequencing high throughput, sensitive Instruments

9. Integrative Proteomics 01/24/2001 MALDI-ToF vs Electrospray Which do I use? Experimental design –What kind of sample will I have Reason for analysis –Unknown protein, quality control or mapping of a known protein Species –Is the genome fully sequenced?

10. Integrative Proteomics 01/24/2001 MALDI-ToF MS 100 well sample plate Bruker Daltonics Reflex III © 384 well sample plate

11. Integrative Proteomics 01/24/2001 MALDI-TOF Typically a resolved (isolated) protein Protein identified by correlative mass mapping of peptides generated by a known chemistry –Enzymatic or chemical Protein sequence must be in a database for identification Identification meausred by a statistical matching score

12. Integrative Proteomics 01/24/2001 Electrospray MS Applied Biosystems | MDS SCIEX API 3000™ LC/MS/MS System Applied Biosystems | MDS SCIEX QSTAR™ Hybrid LC/MS/MS Quadrupole TOF System

13. Integrative Proteomics 01/24/2001 Electrospray MS Usually requires a chromatographic resolving step as only a limited number of peptides can be analyzed at one time Full cell lysate or sub-fraction can be analyzed Generate MS/MS data (fragmentation of peptide for de novo sequence or small sequence tags) Fragmentation can generate enough data that only one or two peptides are required for a positive identification

14. Integrative Proteomics 01/24/2001 MALDI-ToF Schematic Detectors Flight tube Timed ion selector Laser Sample plate Reflector Accelerating field + + + + + + +

15. Integrative Proteomics 01/24/2001 In-Gel Sample Preparation CE Candidate protein bands are excised, reduced, alkylated, and digested overnight with trypsin. Tryptic peptides are recovered, purified and analyzed by MALDI- ToF mass Spectrometry.

16. Integrative Proteomics 01/24/2001 Reflector MALDI Spectrum

17. Integrative Proteomics 01/24/2001 Reflector MALDI Spectrum

18. Integrative Proteomics 01/24/2001 Correlative Database Searching Based on the principal that each protein generates its own unique “fingerprint” of proteins after cleavage by an enzymatic or chemical method into smaller peptides A list of experimental masses can then be compared to lists of calculated masses for each known sequence

19. Integrative Proteomics 01/24/2001 Correlative Database Searching Sequence database used to generate lists of peptide masses based on chemistry rules –Enzymatic (trypsin) or chemical cleavage (CNBr) Parameters of search –Chemistry of peptide generation, database restrictions: mol. wt., taxa, error tolerance for peptide matching

20. Integrative Proteomics 01/24/2001 Correlative Database Searching Mann et al, 1993 used intact mass and peptide masses to identify unknown proteins. Error was from 0.5 to several Daltons for peptides due to poor resolution, average masses only Protein with most peptides matching was ranked first, true mass correlation Problem was large proteins (>100kDa) generate large number of peptide masses, spurious matches Use restriction of maximum protein size to prevent this Total protein sequences available was only 26,000

21. Integrative Proteomics 01/24/2001 Correlative Database Searching Available number of sequences as of Nov. 1999 NCBInr415408 Available number of sequences as of June 2000 NCBInr510935 Available number of sequences as of Jan. 2001 NCBInr606272

22. Integrative Proteomics 01/24/2001 Correlative Database Searching Improvements in instrument mass accuracy and resolution Countered by dramatic increase in number of sequences available to search Develop new searching algorithms using weighted statistical value of a peptide, larger mass peptide contains more “unique information” Knowledge of distribution frequencies of peptide masses allowed for calculation of probability for a match (Perkins et al 1999 and Eriksson et al 2000) –using database size (number of peptides), error tolerance, number of matching peptides (weighted)

23. Integrative Proteomics 01/24/2001 Correlative Database Searching More parameters for calculation of a match –Correlation coefficient (r 2 ) for matching peptides to error tolerance –Amino acid composition from chemical tags or MS/MS spectrum –Calculation of a Z score for true probability certainty of the match ”…a Z score is estimated when the search result is compared against an estimated random match population. Z score is the distance to the population mean in unit of standard deviation.” Proteometrics at the 48 th ASMS annual meeting

24. Integrative Proteomics 01/24/2001 Reflector MALDI Spectrum

25. Integrative Proteomics 01/24/2001 Reflector MALDI Spectrum

26. Integrative Proteomics 01/24/2001 Reflector MALDI Spectrum

27. Integrative Proteomics 01/24/2001 Proteometrics home page Profound Search

28. Integrative Proteomics 01/24/2001 Profound Search

29. Integrative Proteomics 01/24/2001 Profound Search

30. Integrative Proteomics 01/24/2001 Profound Search

31. Integrative Proteomics 01/24/2001 Profound Search

32. Integrative Proteomics 01/24/2001 Profound Search

33. Integrative Proteomics 01/24/2001 Protein Prospector Homepage MS-Fit Search

34. Integrative Proteomics 01/24/2001 MS-Fit Search

35. Integrative Proteomics 01/24/2001 MS-Fit Search

36. Integrative Proteomics 01/24/2001 MS-Fit Search

37. Integrative Proteomics 01/24/2001 MS-Fit Search

38. Integrative Proteomics 01/24/2001 MS-Fit Search

39. Integrative Proteomics 01/24/2001 Homologous Proteins Matching peptide to Xenopus protein Human protein spectrum

40. Integrative Proteomics 01/24/2001 Homologous Proteins Xenopus Human EST Linear sequence of protein Peptide match number

41. Integrative Proteomics 01/24/2001 MS/MS Fragmentation of parent ion (a peptide) to produce sequence dependent data Fragmentation induced in MALDI as post source decay In electrospray by CID (collision induced decay) Searching done by similar principles

42. Integrative Proteomics 01/24/2001 MALDI-ToF Schematic Detectors Flight tube Timed ion selector Laser Sample plate Reflector Accelerating field

43. Integrative Proteomics 01/24/2001 Peptide Fragmentation P. Roepstorff and J. Fohlman, Biomed. Mass Spec. 11 (1984) 601. K. Biemann, Biomed. Env. Mass Spec. 16 (1988) 99.

44. Integrative Proteomics 01/24/2001 Peptide Fragmentation NH3- D R V Y I H P F H L -COOH P F H L -COOH Y I H P F H L -COOH NH3- D R V NH3- D R V Y I H R V Y I H PH y series b series

45. Integrative Proteomics 01/24/2001 Peptide Fragmentation

46. Integrative Proteomics 01/24/2001 Peptide Fragmentation

47. Integrative Proteomics 01/24/2001 Peptide Fragmentation : FYEEVHDLER R E L D H V E FY E E V H D HD VHD EVHD EEVHD B ions Y ions

48. Integrative Proteomics 01/24/2001 Peptide Fragmentation

49. Integrative Proteomics 01/24/2001 Peptide Fragmentation

50. Integrative Proteomics 01/24/2001 Peptide Fragmentation

51. Integrative Proteomics 01/24/2001 Peptide Fragmentation

52. Integrative Proteomics 01/24/2001 Peptide Fragmentation

53. Integrative Proteomics 01/24/2001 Electrospray HPLC UV trace MS scan (deconvoluted) MS/MS of selected ion

54. Integrative Proteomics 01/24/2001 Electrospray Continuous scanning for peptides above a threshold intensity Selected peptides under go MS/MS Return to scan to find next candidate Slow sample introduction for increased peak width off HPLC to allow more time of MS/MS experiments MS/MS process less than a few seconds Data used for search is a peptide mass plus any sequence tags determined from MS/MS

55. Integrative Proteomics 01/24/2001 Unmatched spectrum PSD candidates

56. Integrative Proteomics 01/24/2001 PSDs of candidate peptides

57. Integrative Proteomics 01/24/2001 Coverage of EST ESTPSD Linear sequence of protein

58. Integrative Proteomics 01/24/2001 Conclusion Mass spectrometry will continue to be a powerful tool for the analysis of biopolymers Further improvements in instrumentation and computational sciences will increase the utility and versatility of mass spectrometry Simplification of process will lead to greater use and availability of this technology –e.g. wide variety molecular biology kits currently available

59. Integrative Proteomics 01/24/2001 Contact Us Integrative Proteomics is located in Downtown Toronto For employment inquires please see our web page at www.integrativeproteomics.com