One-source peptide/phosphopeptide ratio standards for accurate and site-specific determination of the degree of...

Reversible protein phosphorylation is a key mediator for intracellular signal transduction. Here we describe an innovative method for the production of pairs of peptide standards designed for quantitative mass spectrometry. These standard pairs can be used for site-specific analysis of the degree of phosphorylation of proteins in a bottom-up approach. The method starts from an isotopically labeled phosphopeptide analogue of the analyte phosphopeptide and ends up with a labeled peptide/phosphopeptide ratio standard in which the molar ratio between the phosphorylated and the unphosphorylated form is exactly defined. The signals of the ratio standard are used to standardize the corresponding analyte signals. This compensates for differences in LC recovery or ionization efficiency between the phosphorylated and unphosphorylated forms. The method can also be extended to quantitative analysis of multisite phosphorylation in a single peptide, which is exemplified for the presence of two phosphorylation sites. Peptide/phosphopeptide ratio standards exhibit high ratio accuracy, since ratio adjustment is performed by volumetric operations only.