1. Proteomics and mass spectrometry Manimalha Balasubramani

2. Outline Mass spectrometers Protein identification Quantitative proteomics Protein-protein interactions

3. A mass spectrum 799.01179.21559.41939.62319.82700.0 Mass (m/z) 6.3E+4 0 10 20 30 40 50 60 70 80 90 100 % Intensity 1258.5603 1303.7007 878.4913 983.4860 1031.5374 1254.5614 1114.5428 1657.7953 842.4926 1586.8064 1232.5907 1964.8882 924.5113 1153.5334 965.4456 1035.5696 833.0566 1315.5780 1280.5370 1074.5405 2120.9883 1360.7209 1191.6130 1689.7865 870.5201 2518.1062 1800.9324 1395.7062 1848.9419 1475.7374 1630.7738 2211.0520 1593.7693 2393.0823 2169.9207 2439.0872 2021.9116

4. Basically measures mass Adapted from google

5. Components… Adapted from an Analytical chemistry textbook

6. Ionization process ESI Nobel prize in Chemistry, 2002 Matrix Assisted Laser Desorption Ionization ElectroSpray Ionization MALDI

7. MALDI – Matrix Assisted Laser Desorption Ionization

8. ESI – Electro Spray Ionization

9. Mass analyzers – several designs Adapted from Aebersold, R.; Mann, M. Nature 2003, 422, 198-207

10. GPCL inventory  ABI Voyager DE PRO, walk-up use  ABI 4700 Proteomics Analyzer  Thermoelectron LCQ Deca with Surveyor HPLC  ABI Qstar Elite with Ultimate 3000 HPLC  Bruker micrOTOF with Ultimate 3000 HPLC  Bruker 12 Tesla FTMS with Ultimate 3000 HPLC

11. Time-of-flight (TOF) analyzers MALDI TOF Voyager DE PRO ESI TOF Ultimate 3000 with micrOTOF

12. MALDI TOF - principle KE = zeV = 1mv 2 2

13. MS of serum albumin MALDI TOF ESI TOF

14. Tandem mass spectrometer MALDI TOF/TOF MS and MS/MS

15. Ion Trap MS, MS 2, MS 3, ….MS n

16. Quadrupole-q-TOF ESI QqTOF

17. …installation phase…. FT MS

18. …bottom line…..Resolution and mass accuracy…

19. FWHM Full width at half maxima of a peak

20. Resolution and mass accuracy R = M Δm R = resolution M = mass of the peak of interest Δ m = width in daltons of the peak Δm measured at 50% peak height is the Full Width at Half Maxima (FWHM)

21. Mass accuracy is measured as parts per million value ppm = 10 6 Δm = 10 6 M R

22. outline Mass spectrometers Protein identification Quantitative proteomics Protein-protein interactions

23. Peptide Mass Fingerprinting - PMF Database entry NCBI From: http://gobi.ym.edu.tw/course/mass/2004-0325.pdf

24. Informatics  Search engines Mascot, Matrix Science Sequest, Thermoelectron  Free-ware Protein prospector (http://prospector.ucsf.edu/) TPP tools (http://tools.proteomecenter.org/TPP.php)

25. Database searching using MASCOT Overview of the experiment Submission of data to MASCOT webserver

26. 1D SDS PAGE of proteins Adapted from Aebersold, R.; Mann, M. Nature 2003, 422, 198-207

27. Mass spectrum Mass to charge ratio (m/z) Intensity

28. Peak list  Compiled from the mass spectra Mass list Mass list and intensity  Submitted to the search engine

29. http://www.matrixscience.com/

30. Mascot scoring A frequency factor matrix, F, is created, in which each row represents an interval of 100 Da in peptide mass, and each column an interval of 10 kDa in intact protein mass. As each sequence entry is processed, the appropriate matrix elements fi,j are incremented so as to accumulate statistics on the size distribution of peptide masses as a function of protein mass. The elements of F are then normalised by dividing the elements of each 10 kDa column by the largest value in that column to give the Mowse factor matrix M: After searching the experimental mass values against a calculated peptide mass database, the score for each entry is calculated according to: Where MProt is the molecular weight of the entry and the product term is calculated from the Mowse factor elements for each match between the experimental data and peptide masses calculated from the entry.

31. List of common contaminants  Trypsin autolysis peptides  Matrix peaks  Keratin from skin, hair  Other contaminants

32. Protein Identification Adapted from Aebersold, R.; Mann, M. Nature 2003, 422, 198-207

33. Tandem mass spectrum http://qbab.aber.ac.uk

34. Tandem mass spectrum

35. Database Searching Peptide Mass Fingerprinting Sequence tag approach De novo sequencing inspect raw data http://qbab.aber.ac.uk Tandem mass spectra (MS/MS) can be used for peptide sequencing

36. Mascot Search Results Search title : SampleSetID: 362, AnalysisID: 567, MaldiWellID: 15790, SpectrumID: 17225, Path=\Mani\102004\New Analysis 1 Database : NCBInr 20040606 (1846720 sequences; 611532004 residues) Timestamp : 20 Oct 2004 at 14:52:50 GMT Top Score : 681 forgi|180570, creatine kinase [Homo sapiens] Probability Based Mowse Score Score is-10*Log(P), where P is the probability that the observed match is a random event. Protein scores greater than 75 are significant (p<0.05).

37. Top hits from Mascot Search – there are multiple accession numbers for the same protein

38. Search returns a cluster of proteins with the same matching peptides

39. Nominal mass (M r ): 42591; Calculated pI value: 5.34 Observed Mass & pI: 43kd, 6.2-6.27 Creatine kinase - B [Homo sapiens] Match to: gi|21536286 ; Score: 681 Sequence Coverage: 46% 1 MPFSNSHNAL KLRFPAEDEF PDLSAHNNHM AKVLTPELYA ELRAKSTPSG 51 FTLDDVIQTG VDNPGHPYIM TVGCVAGDEE SYEVFKDLFD PIIEDRHGGY 101 KPSDEHKTDL NPDNLQGGDD LDPNYVLSSR VRTGRSIRGF CLPPHCSRGE 151 RRAIEKLAVE ALSSLDGDLA GRYYALKSMT EAEQQQLIDD HFLFDKPVSP 201 LLSASGMARD WPDARGIWHN DNKTFLVWVN EEDHLRVISM QKGGNMKEVF 251 TRFCTGLTQI ETLFKSKDYE FMWNPHLGYI LTCPSNLGTG LRAGVHIKLP 301 NLGKHEKFSE VLKRLRLQKR GTGGVDTAAV GGVFDVSNAD RLGFSEVELV 351 QMVVDGVKLL IEMEQRLEQG QAIDDLMPAQ K Creatine kinase B is the highest scoring protein

40. outline Mass spectrometers Protein identification Quantitative proteomics Protein-protein interactions

41. Sample preparation Quantitative Proteomics

42. From 2D gels ….to MALDI or ESI MS Control Test Cy3 Cy5 Pool Image analysis with Delta2D, Decodon Quantitate Export spot list to robotic picker

43. 2 nd Dimension – SDS PAGE 1 st Dimension - Isoelectric focussing Spot picking Trypsin gel digest..its high-throughput…

44. Colorectal cancer markers Mass spectral analysis MS MS/MS m/z Database Search Protein Identified Yes Immunohistochemistry 2D 1D1D Immunoblotting Validation Isolate Nuclear Matrix In-gel Tryptic digest No Tumor specific markers CC3, CC4, CC5, CC6a, CC6b de novo sequencing Balasubramani et al., Cancer Res., 2006

45. Shotgun proteomics Adapted from Aebersold, R.; Mann, M. Nature 2003, 422, 198-207

46. …typical workflow to identify biomarkers that distinguish indolent versus aggressive forms of cancer.. Group A, Indolent Group B, Aggressive Fractionate Eg. Immunodeplete, subcellular Fractionate Eg. Immunodeplete, subcellular Tryptic peptides Label with iTRAQ reagent 115 Label with iTRAQ reagent 116 Combine labeled digests LC fractionate MS and MS/MS Protein ID and Quantitate

47. Sample handling HPLC 1D or 2D LC MALDI In-solution Isoelectric focussing

48. Protein-protein interaction studies  Immunoaffinity pull-downs  Tandem affinity purification

49. GPCL Billy W Day Paul Wood Mirunalni Thangavelu Tamanna Sultana Emanuel M Schreiber Chris Bolcato Chris Myers Patrick Miller Robert Wolfe

50. definitions  The amu is defined as 1/12 th the mass of one neutral 6 C 12 atom  Amu is also called the dalton  1 amu =1/12 ( 12g 12 C/mol 12 C 6.0221 x 10 23 atoms 12 C/mol 12 C 1.6605 x 10 -24 g/atom 12 C

51. Isotopic species of M  (M + H) + (M + 1H)/1H +  (M + 2H) 2+ (M + 2H)/2H +  (M + 3H) 3+ (M + 3H)/3H +