title: Measurement of protein phosphorylation stoichiometry by selected reaction monitoring mass spectrometry.
abstract: The stoichiometry of protein phosphorylation at specific amino acid sites may be used to infer on the significance of the modification, and its biological function in the cell. However, detection and quantification of phosphorylation stoichiometry in tissue remain a significant challenge. Here we describe a strategy for highly sensitive, label-free quantification of protein phosphorylation stoichiometry. Method development included the analysis of synthetic peptides in order to determine constants to relate the mass spectrometry signals of cognate peptide/phosphopeptide pairs, and the detection of the cognate peptides by using high resolution Fourier Transform mass spectrometry (FTMS) and selected reaction monitoring mass spectrometry (SRM). By analyzing extracted ion currents by FTMS, the phosphorylation stoichiometries of two tyrosine residues (tyrosine-194 and tyrosine-397) in the protein tyrosine kinase Lyn were determined in transfected human HEK293T cells and two cultured human multiple myeloma strains. To achieve high sensitivity to measure phosphorylation stoichiometry in tissue, SRM methods were developed and applied for the analysis of phosphorylation stoichiometries of Lyn phospho-sites in multiple myeloma xenograft tumors. Western immuno-blotting was used to verify mass spectrometry findings. The SRM method has potential applications in analyzing clinical samples wherein protein phosphorylation stoichiometries may represent important pharmacodynamic biomarkers.
authors.affiliation: McLaughlin Centre for Molecular Medicine, MaRS East Tower, 101 College Street, Toronto, M5G 1L7, Canada.
authors.forename: Lily L
authors.initials: LL
authors.lastname: Jin
authors.forename: Jiefei
authors.initials: J
authors.lastname: Tong
authors.forename: Amol
authors.initials: A
authors.lastname: Prakash
authors.forename: Scott M
authors.initials: SM
authors.lastname: Peterman
authors.forename: Jonathan R
authors.initials: JR
authors.lastname: St-Germain
authors.forename: Paul
authors.initials: P
authors.lastname: Taylor
authors.forename: Suzanne
authors.initials: S
authors.lastname: Trudel
authors.forename: Michael F
authors.initials: MF
authors.lastname: Moran
mesh_terms: D000818:Animals; D002460:Cell Line; D005583:Fourier Analysis; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D009101:Multiple Myeloma / Q000378:metabolism; D009368:Neoplasm Transplantation; D010750:Phosphoproteins / Q000032:analysis / Q000378:metabolism*; D010766:Phosphorylation; D011506:Proteins / Q000032:analysis / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D014443:Tyrosine / Q000378:metabolism; D019061:src-Family Kinases / Q000032:analysis / Q000378:metabolism
chemical_list: D010750:Phosphoproteins; D011506:Proteins; D014443:Tyrosine; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases