Accurate phosphorylation site localization using phospho-brackets

Highlights: • P-bracket was developed for phosphosite localization using paired phospho-containing site-determining product ions. • In phosphosite localization of 101,520 synthetic phosphopeptides, a false localization rate (FLR) of 0.9% was obtained. • 1,601 and 1,393 HeLa phosphopeptides (identified by Mascot and Sequest, respectively) were accurately localized. • P-bracket, as a stand-alone software withuser-friendly GUIs, is available through http://proteingoggle.tongji.edu.cn. - Abstract: Phosphorylation is one of the most important and widely studied protein post-translational modifications. Tandem mass spectrometry using higher-energy collisional dissociation has evolved into a state-of-the-art analytical platform for both phosphorylation identification and site localization. Tens of thousands of phosphopeptides can now be routinely identified from a single shotgun proteomics study; site localization, however, is much more complicated and many challenges still exist. Here, we report our development of P-bracket using direct experimental evidence of phospho-containing site-determining product ions for accurate site localization without the need for additional FLR control. A P-bracket is defined as a complementary product ion pair that forms a bracket to confine a phosphorylation event to a unique site. P-bracket has been successfully benchmarked with a set of six synthetic phosphopeptides with a single phosphorylation event, a set of 96 synthetic peptides and phosphopeptide reference libraries, and two HeLa phosphopeptide LC-MS/MS (HCD) datasets; Accurate phosphosite localization by P-bracket will greatly enhance identification confidence of phosphopeptides and contribute to structural and functional studies of phosphoproteins.