For the comparative analysis of phosphoproteins, two samples are digested with trypsin or Lys-C and the resulting peptides from each are then converted to d0- and d3-methyl esters, respectively. The two sample are then mixed together and subjected to nano-flow immobilized metal affinity chromatography (IMAC) to selectively capture phosphopeptides in the sample (1). These are then eluted to a nanoflow HPLC column that is interfaced to electrospray ionization on a tandem linear ion trap-Fourier transform mass spectrometer (LTQ-FTMS). This instrument operates at a resolution of 100,000, measures masses to three decimal places, and records the molecular masses of phosphotryptic tryptic peptides in each sample at the high attomole level (2). Phosphorylation levels in the two samples are deduced from the ratios of ion abundances observed for d0- and d3- labeled peptides in each sample. Sequence information on these peptides is obtained by performing ether collision activated dissociation (CAD) or electron transfer dissociation (ETD) (3) experiments automatically under computer control at the rate of 4/sec on the LTQ. Parent proteins are identified by searching the resulting spectra against protein databases with SEQUEST software. The above approach has been employed: (a) to sequence phosphopeptides presented to the immune system on cancer cells, (b) to characterize phosphorylation sites on histones as a function of the cell cycle, gene expression and gene silencing (4), (c) to identify proteins involved in maturation of human sperm (5), and (d) to characterize phosphorylation sites on proteins involved in cell migration.
1) Phosphoproteome Analysis by Mass Spectrometry and Its Application to Saccharomyces cerevisiae S.B. Ficarro, M.L. McCleland, P.T. Stukenberg, D.J. Burke, M.M. Ross, J. Shabanowitz, D. F. Hunt, and F.M. White, Nature Biotechnology, 2002, 20, 301-305.
2) Syka J.E.P., Marto J.A., Bai D.L., Hornung S., Senko M.W., Schwartz J.C., Ueberheide B., Garcia B., Busby S., Muratore T., Shabanowitz J., Hunt D.F., A Novel Linear Quadrupole Ion Trap/FT Mass Spectrometer: Performance Characterization and Use in the Comparative Analysis of Histone H3 Post-Translational Modifications, J Proteome Res, 2004: In Press.
3) Peptide Sequence Analysis by Electron Transfer Dissociation: A Non-Ergodic Process for the Masses. J.E.P. Syka, J.J. Coon, M. Schroeder, D.L. Bai, J. Shabanowitz and D.F. Hunt. Proc. Natl. Acad. Sci. USA, 2004, In Press.
4) Site-Selective Histone Methylation in Chromatin, S.D. Briggs, T. Xiao, Z.-W. Sun, J.A. Caldwell, J. Shabanowitz, D.F. Hunt, C.D. Allis, B.D. Strahl, Nature, 2002, 418, 498.
5) Phosphoproteome Analysis of Capacitated Human Sperm. Evidence of Tyrosine Phosphorylation of a Kinase-Anchoring Protein 3 and Valosin-Containing Protein/p97 During Capacitation, Ficarro S, Chertihin O, Westbrook A, White F, Jayes F, Kalab P, Marto JA, Shabanowitz J, Herr J, Hunt DF, Visconte PE, J. Biol. Chem., 2003, 278, 11579-11589.