Identification of phosphorylation sites in proteins by nanospray quadrupole ion trap mass spectrometry

A method is described for identifying serine phosphorylation sites in proteins, based on conventional (32)P labeling followed by electrophoretic separation, 'in-gel' digestion with a protease, peptide extraction, reversed-phase high-performance liquid chromatographic separation and collection and off-line analysis of the radioactive fractions by nanospray ion trap mass spectrometry. The method was successfully applied to the identification of three phosphorylation sites in two proteins which were subjected to in vitro phosphorylation under physiological conditions. Different combinations of the various scanning modes of the ion trap, including high-resolution, multiple subfragmentation (or MS(n)) and fast scan analysis, were employed to identify the phosphopeptides, determine their sequence and localize the exact site of phosphorylation. 'Blind' fragmentation using fast scans was used to analyze a phosphopeptide which was undetectable in other scanning modes. The sequence, phosphorylation site and double cysteine modification of the potassium adduct of a peptide containing 35 residues were also determined by multiple fragmentation. The results not only support the validity of the proposed method for routine identification of phosphorylation sites, but also demonstrate the exceptional capability of off-line ion trap mass spectrometry in combination with nanospray ionization for performing very detailed studies on the structure of peptides.