As one of the most studied post-translational modifications (PTM),
protein phosphorylation plays an essential role in almost all cellular processes. Current methods are able to predict and determine thousands of phosphorylation sites, whereas stoichiometric quantification of these sites is still challenging. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS)-based targeted proteomics is emerging as a promising technique for site-specific quantification of
protein phosphorylation using proteolytic
peptides as surrogates of
proteins. However, several issues may limit its application, one of which relates to the
phosphopeptides with different phosphorylation sites and the same mass (i.e., isobaric
phosphopeptides). While employment of site-specific product
ions allows for these isobaric
phosphopeptides to be distinguished and quantified, site-specific product
ions are often absent or weak in tandem mass spectra. In this study, linear algebra algorithms were employed as an add-on to targeted proteomics to retrieve information on individual
phosphopeptides from their common spectra. To achieve this simultaneous quantification, a LC-MS/MS-based targeted proteomics assay was first developed and validated for each
phosphopeptide. Given the slope and intercept of calibration curves of
phosphopeptides in each transition, linear algebraic equations were developed. Using a series of mock mixtures prepared with varying concentrations of each
phosphopeptide, the reliability of the approach to quantify isobaric
phosphopeptides containing multiple phosphorylation sites (≥ 2) was discussed. Finally, we applied this approach to determine the phosphorylation stoichiometry of
heat shock protein 27 (HSP27) at Ser78 and Ser82 in
breast cancer cells and tissue samples.