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

ABSTRACT: 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

SUBMITTER: Domanski D 

PROVIDER: S-EPMC2909760 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature