Estimate phosphopeptide identification rate by tandem mass spectrometry

Mass spectrometry has played a significant role in the identification of unknown phosphoproteins and sites of phosphorylation in biological samples. Analysis of protein phosphorylation, in particular large scale phosphorylation analysis – phosphoproteomics, has recently been enhanced by efficient enrichment, fast and accurate instrument, and better software, but challenges remain because of low stoichiometry of phosphorylation and poor efficiency in ionization and fragmentation due to neutral loss. To evaluate current mass spectrometric performance, we present here a method to estimate phosphopeptide identification efficiency by tandem mass spectrometry. Phosphopeptides were directly isolated from whole plant cell extracts, dephosphorylated, and then incubated with three purified kinases, CK2, MAPK6, and SnRK2.6, along with 16O4- and 18O4-ATP separately, for in vitro kinase reactions. Phosphopeptides were further enriched and analyzed by LC-MS. Phosphopeptide identification rate was estimated by comparing phosphopeptides identified by tandem mass spectrometry with phosphopeptide pairs generated by stable isotope labeled kinase reactions. Overall we found that the current high speed and high accurate mass spectrometry can only identify 20-40% of total phosphopeptides mainly due to relatively poor fragmentation and low abundance.