title: Stable isotope-free relative and absolute quantitation of protein phosphorylation stoichiometry by MS.
abstract: Qualitative and quantitative information are crucial to a detailed understanding of the function of protein phosphorylation. MS is now becoming a quantitative approach to analyze protein phosphorylation. All methods that have been described either require the elaborate/expensive use of stable isotopes to compare a limited number of samples or do not provide phosphorylation stoichiometries. Here, we present stable isotope-free MS strategies that allow relative and absolute quantitation of phosphorylation stoichiometries. By using the developed methods, we can normalize to robustly account for run-to-run variations and variations in amounts of starting material. This procedure monitors the unmodified proteolytic peptides derived from the protein of interest and identifies peptides that are suitable for normalization purposes. Also, we can determine changes in phosphorylation stoichiometry by monitoring the changes in the normalized ion currents of the phosphopeptide(s) of interest. Absolute phosphorylation stoichiometry are measured by monitoring the ion currents of a phosphopeptide and its unmodified cognate as the signal intensity changes of both peptide species are correlated. The method is applicable to multiply phosphorylated species (for which one more sample with varying phosphorylation stoichiometry than number of phosphorylation sites is required to correct for the differences in the ionization/detection efficiencies of the phosphopeptide, its partially phosphorylated and unphosphorylated cognates). Last, we can quantitate species with ragged ends resulting from incomplete proteolysis and measure phosphorylation stoichiometries of single samples by controlled dephosphorylation. These approaches were validated and subsequently applied to the phosphorylation of the yeast transcription factor Pho4.
authors.affiliation: Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Hanno
authors.initials: H
authors.lastname: Steen
authors.forename: Judith A
authors.initials: JA
authors.lastname: Jebanathirajah
authors.forename: Michael
authors.initials: M
authors.lastname: Springer
authors.forename: Marc W
authors.initials: MW
authors.lastname: Kirschner
mesh_terms: D002623:Chemistry Techniques, Analytical; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; C060292:PHO4 protein, S cerevisiae; D010446:Peptide Fragments; D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors
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