Assay development for the determination of phosphorylation stoichiometry using multiple reaction monitoring methods with and without phosphatase treatment: application to breast cancer signaling pathways.

We have developed a phosphatase-based phosphopeptide quantitation (PPQ) method for determining phosphorylation stoichiometry in complex biological samples. This PPQ method is based on enzymatic dephosphorylation, combined with specific and accurate peptide identification and quantification by multiple reaction monitoring (MRM) with stable-isotope-labeled standard peptides. In contrast with classical MRM methods for the quantitation of phosphorylation stoichiometry, the PPQ-MRM method needs only one nonphosphorylated SIS (stable isotope-coded standard) and two analyses (one for the untreated sample and one for the phosphatase-treated sample), from which the expression and modification levels can accurately be determined. From these analyses, the percent phosphorylation can be determined. In this manuscript, we compare the PPQ-MRM method with an MRM method without phosphatase and demonstrate the application of these methods to the detection and quantitation of phosphorylation of the classic phosphorylated breast cancer biomarkers (ERalpha and HER2), and for phosphorylated RAF and ERK1, which also contain phosphorylation sites of biological importance. Using synthetic peptides spiked into a complex protein digest, we were able to use our PPQ-MRM method to accurately determine the total phosphorylation stoichiometry on specific peptides as well as the absolute amount of the peptide and phosphopeptide present. Analyses of samples containing ERalpha protein revealed that the PPQ-MRM method is capable of determining phosphorylation stoichiometry in proteins from cell lines, and is in good agreement with determinations obtained using the direct MRM approach in terms of phosphorylation and total protein amount.