Measurement of Protein Phosphorylation Stoichiometry by SRM‐MS

Protein phosphorylation is a major post‐translational modification involved in most biological processes and numerous diseases. Much biological relevance lies in the knowledge of the extent of phosphorylation at any given protein phosphorylation site, i.e., protein phosphorylation stoichiometry. Major progress in quantitative mass spectrometry has allowed the establishment of protocols that permit accurate determination of protein phosphorylation stoichiometry. The Basic Protocol presented herein describes a label‐free mass spectrometry approach for determining phosphorylation stoichiometry using electrospray nano‐liquid chromatography coupled to selected reaction monitoring (SRM). This entails the calculation of the respective response rates of the phosphorylated and unphosphorylated cognate peptide species using a peptide dephosphorylation reaction. The ratio of these response rates is then applied as a correction factor to the LC‐SRM‐MS signal intensity ratio of the corresponding endogenous phosphopeptide/nonphosphopeptide pair in a biological sample of interest. An Alternate Protocol is also presented that describes how heavy isotope‐labeled synthetic peptides can be used as internal standards to determine the stoichiometry of phosphorylation. Curr. Protoc. Chem. Biol. 4:65‐81 © 2012 by John Wiley & Sons, Inc.