Identification of phosphorylation sites in peptides using a gas-phase sequencer

A simple procedure is described for determining the location of phosphorylation sites in phosphopeptides. The method employs measurement of 32P-labeled inorganic phosphate release during Edman degradation cycles using a gas-phase sequencer. The procedure is based on extracting peptides and inorganic phosphate from portions of the sample filter at strategic cycles in the sequence analysis followed by determination of the relative amounts of phosphate and phosphopeptide. One advantage of this technique is the very high recovery of the phosphate associated with the peptide, 80-97% in this study. In the course of this work, it was also found that phosphoserine residues themselves caused reduced efficiency of the Edman degradation as compared with unesterified serine residues. The present procedure has the merit of being simple and easy to apply.