1. Session III How we analyzed proteomic data? 台大生技教改暑期課程

2. Topics for session III 1. Image analysis (for 2-DE gel) 2. Mass data analysis 3. Protein structure analysis

3. 1. Image analysis

4. Examples of 2-DE results D Healthy controlPatient Digest to peptide fragment MS analysis

5. Unexpected variation between gels Interior variation between 2-DE experiments –Same loading amount? –Same gel condition? –Same staining condition? Exterior variation after gel developed –Unwanted spots (dye or reagent deposit) –Dirty spots (hair, dust)

6. What image analysis software do? – spot detection – unwanted spot filtering – background subtraction – normalization – image matching – expression comparison – pI/MW calibration – data organization

7. Currently available 2-DE image analysis software – Melanie 4 (Swiss Institute of Bioinformatics, SIB) – Phoretix 2D (Nonlinear dynamics) – Progenesis (Nonlinear dynamics) – Z3 and Z4000 (Compugen) – Delta2D (Sunergia group) – A-GelFox 2D (Alpha innotech) – Flicker (NCI, through internet)

8. Spot detection One of the first and most important steps in 2-DE analysis. Locating the spots in the gel image Locating the spots in the gel image defining their shape defining their shape calculating measurement information (volume and area) calculating measurement information (volume and area)

9. Spot detection

10. Filtering Removal of unwanted spots: What’s unwanted spots: dust on gel dust on gel stain deposit stain deposit bulky spots bulky spots

11. Background subtraction General background subtraction method: No background No background Mode of non-spot Mode of non-spot Manual background Manual background Lowest boundary Lowest boundary Average boundary Average boundary

12. Normalization General normalization method: Total spot volume Total spot volume Single spot Single spot Total volume ratio Total volume ratio

13. Image matching

14. Expression comparison Two fold up or down expression are thought to be significant.

15. pI/MW calibration PI calibrationMW calibration observed or experimental pI/MW

16. Data organization

17. Spot annotation

18. 2. Mass data analysis

19. Useful proteomic resource http://tw.expasy.org/

20. Useful proteomic resource

21. Matrix Science - Mascot http://www.matrixscience.com/

22. Three major functions in Mascot Peptide Mass Fingerprint (PMF): The experimental data are a list of peptide mass values from an enzymatic digest of a protein. (MALDI-TOF) Sequence Query: One or more peptide mass values associated with information such as partial or ambiguous sequence strings, amino acid composition information, MS/MS fragment ion masses, etc. A super-set of a sequence tag query. MS/MS Ion Search: Identification based on raw MS/MS data from one or more peptides. (LC/MS/MS)

23. Difference between MALDI-TOF and LC/MS/MS MALDI-TOF LC/MS/MS

24. 2-1 PMF analysis

25. Raw data for PMF 899.20762980.8123 905.21261471.3723 909.19172312.2317 915.21811533.8486 925.46371881.7635 938.39721528.9462 1044.30072111.9482 1050.31412396.1550 1060.27974689.0698 1066.28897302.0029 1072.29915688.8511 1078.31698919.1113 1084.26571474.5900 1088.27933180.5122 1094.29474573.5195 1104.26381546.4652 1110.28371470.9734 1271.31631498.0187 m/z Relative intensity

26. Mascot PMF query form

27. Mascot PMF parameters Your name; Email Search title Database Taxonomy Enzyme Monoisotopic or Average Modifications Protein Mass Peptide tol. ± Mass values Missed cleavages Data file Query

28. Database Comment EST EST divisions of Genbank, (currently EST_human, EST_mouse, EST_others) MSDBComprehensive, non-identical protein database NCBInrComprehensive, non-identical protein database OWLNon-identical protein database (obsolete) RandomRandom sequences for verifying scoring statistics SwissProtHigh quality, curated protein database

29. Taxonomy Ensure the hit list will only contain entries from the selected species speed up a search bring a weak match

30. Enzyme NameCleaveDon't cleaveN or C term TrypsinKRPCTERM Arg-CRPCTERM Asp-NBD NTERM Asp-N_ambicDE NTERM ChymotrypsinFYWLPCTERM CNBrM CTERM Formic_acidD CTERM Lys-CKPCTERM Lys-C/PK CTERM PepsinAFL CTERM Tryp-CNBrKRMPCTERM TrypChymoFYWLKRPCTERM Trypsin/PKR CTERM V8-DEBDEZPCTERM V8-EEZPCTERM CNBr+Trypsin M CTERM KRPCTERM Nonesee notes semiTrypsinsee notes

31. Enzyme "None" is not an allowed choice for a Peptide Mass Fingerprint, where the specificity of an enzyme is essential. If the search fails to produce a positive match, then try again with semiTrypsin (below) before resorting to "None". "semiTrypsin" means that Mascot will search for peptides that show tryptic specificity (KR not P) at one terminus, but where the other terminus may be a non-tryptic cleavage. This is a half-way house between choosing "Trypsin" and "None".

32. Monoisotopic or Average nominal mass values: nominal mass values: calculated from integer atomic weights. (H=1, C=12, N=14, O=16), not practical in proteomics. Average mass values: Average mass values: equivalent to taking the centroid of the complete isotopic envelope Monoisotopic mass value: Monoisotopic mass value: the mass of the first peak of the isotope distribution.

33. Monoisotopic or Average For peptides and proteins, the difference between an average and a monoisotopic weight is approximately 0.06%. Insulin (5.8 kD)Albumin (66.4 kD)

34. Monoisotopic Tol: 1 Da -1.01 Monoisotopic MW

35. Average Tol: 1 Da Average MW

36. Monoisotopic Tol: 2 Da

37. Average Tol: 2 Da

38. Modifications Most protein samples exhibit some degree of modification. Natural post-translational modifications: Natural post-translational modifications: phosphorylation and glycosylation. Deliberate modifications: Deliberate modifications: deliberately introduced during sample work-up, such as cysteine derivatisation.

39. Modifications Fixed modifications Fixed modifications are applied universally, to every instance of the specified residue(s) or terminus. Example: Carboxymethyl (Cys) means that all calculations will use 161 Da as the mass of cysteine. Variable modifications Variable modifications are those which may or may not be present. Example: if Oxidation (Met) is selected, and a peptide contains 3 methionines, Mascot will test for a match with the experimental data for that peptide containing 0, 1, 2, or 3 oxidised methionine residues.

40. Modifications Fixed modifications Fixed modifications are applied universally, to every instance of the specified residue(s) or terminus. Example: Carboxymethyl (Cys) means that all calculations will use 161 Da as the mass of cysteine. Variable modifications Variable modifications are those which may or may not be present. Example: if Oxidation (Met) is selected, and a peptide contains 3 methionines, Mascot will test for a match with the experimental data for that peptide containing 0, 1, 2, or 3 oxidised methionine residues.

41. Peptide tol. ± %fraction expressed as a percentage mmuabsolute milli-mass units, i.e. units of.001 Da ppmfraction expressed as parts per million Daabsolute units of Da The error window on experimental peptide mass values

42. Missed cleavages Missed cleavage = 0, complete digestion Missed cleavage >=1, incomplete digestion

43. Submit and processing

44. Concise protein summary

45. protein summary

46. PMF protein view (I) Score and Expect Protein name MW and pI coverage

47. PMF protein view (II) Match peptides No match peptides RMS error Protein information

48. 2-2 MS/MS analysis

49. Raw data for MS/MS Parent ion Daughter ion

50. Mascot MS/MS query form

51. Protein summary Most possible candidate

52. MS/MS Protein view (I) The sum of all highest scores within each peptide group

53. MS/MS Protein view (II) Protein score: The sum of all highest scores within each peptide group

54. Peptide view

55. 3. Protein structure analysis

56. Research Collaboratory for Structural Bioinformatics (RCSB)

57. Protein data bank (PDB) Proteosome

58. Example: 3D structure for proteosome

64. Stereo view

65. Rotation

66. Secondary Structure