One‐source peptide/phosphopeptide standards for accurate phosphorylation degree determination

Reversible protein phosphorylation is a key mediator for intracellular signal transduction. Here we report an innovative method for accurate, site‐specific protein phosphorylation degree determination by nanoLC‐ESI‐MS/MS. A stable isotope‐labeled pair of peptide/phosphopeptide standards with volumetrically defined molar ratio is used as reference, providing an internal standard for both the analyte peptide and the phosphopeptide. For the preparation of one‐source peptide/phosphopeptide standards, an aliquot of the labeled phosphopeptide standard is quantitatively dephosphorylated, yielding an equimolar solution of the peptide standard. Subsequently, the two solutions are mixed at a 1:1 or other volumetric ratio, which equals the molar ratio. This procedure assures a defined concentration ratio of both components that is independent from their absolute concentration. We demonstrate the applicability of the one‐source peptide/phosphopeptide standard method by determining the phosphorylation degree of the signalling proteins STAT5A/B and STAT6.