Analysis of protein phosphorylation by a combination of elastase digestionand neutral loss tandem mass spectrometry

Loss of phosphoric acid is the most effective fragmentation reaction of pSe r- and pThr-containing phosphopeptides of small size (up to 10-15 residues) in low-energy collision-induced dissociation. Therefore, tandem mass spect rometry with neutral loss scanning was evaluated for its utility to analyze protein phosphorylation using protein kinase A (PKA) catalytic subunit, wh ich is phosphorylated at Thr197 and Ser338, as an example. Analysis of tryp tic digests of phosphoproteins by tandem mass spectrometry with scanning fo r neutral loss of phosphoric acid resulted in spectra with poor signal-to-n oise ratio, mainly because of the large size of the phosphopeptides formed (>2 kDa). This unfavorable size was caused by the distribution of tryptic c leavage sites in PKA and by interference of phosphorylation with tryptic cl eavage. To generate a set of smaller peptide fragments, digestion was perfo rmed using the low-specificity protease elastase. Analysis of the total ela stase digest with neutral loss scanning resulted in observation of a set of partially overlapping phosphopeptides with high abundance, providing a com plete coverage of PKA phosphorylation sites. The peptide size generated by elastase (0.5-1.5 kDa) is ideally suited for this scan mode, which was foun d to provide the highest specificity for detection of singly charged phosph opeptides (neutral loss of 98). Identification of the PKA phosphorylation s ites was performed by mass spectrometric sequencing of the elastase-derived phosphopeptides, which provided highly informative product ion spectra.