Phosphopeptide sequencing by in-source decay spectrum in delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Protein phosphorylation underlies numerous cellular signaling processes. Since a reliable prediction of phosphorylation site(s) based on a consensus amino acid sequence is rather difficult to date, determination of phosphorylation site(s) in phosphoproteins is a crucial step toward the understanding of their function at the molecular level. A conventional protocol for the determination of phosphorylation sites utilizes radioactive labeling of a phosphoprotein by P-32 and purification of digested peptides carrying radioactivity, followed by Edman degradation. This method is not only tedious, but also indirect because the evidence will be based on disappearance of a phenylthiohydantoin signal from the degradation cycle where the P-32 radioactivity is eluted, Several methodologies have been developed to determine the phosphorylation sites directly by using mass spectrometry. These include collision-induced dissociation (CLD) and post-source decay (PSD), both of which tend to produce fragment ions less efficiently as the number of residues exceeds 20, Moreover, in both decay processes, there is a tendency for the phosphate group to be removed during the breakdown of the main peptide chain. We report a method that allows direct observation of phosphorylated peptide fragments of phosphopeptides exceeding 20 residues by using an in-source decay fragmentation by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, yielding results which are difficult or impossible to obtain by existing methods using CID or PSD. (C) Academic Press.