Tandem mass spectrometric method for definitive localization of phosphorylation sites using bromine signature

Determination of the phosphorylation site in peptides by conventional tandem mass spectrometry is subject to ambiguity due to the neutral loss of the phosphate groups, especially in multiphosphorylated peptides. To prevent the neutral loss, the phosphate groups in phosphoserine or phosphothreonine peptides were replaced by p-bromobenzyl mercaptan via β-elimination and Michael addition. The unique isotopic signature of the Br introduced facilitated definitive localization of phosphorylation sites in multiphosphorylated peptides with highly adjacent serine or threonine residues. This method could be used to confirm phosphorylation sites determined by conventional tandem mass spectrometry after phosphopeptide enrichment.