A Novel Method of Identifying Phosphorylation Sites Using a Thiophosphorylated Peptide and ESI-MS

Abstract

This chapter discusses the identification of phosphorylation sites using a thiophosphorylated peptide and ESI-MS. Many sites of phosphorylation can be predicted by consensus motifs or homology to other phosphorylated proteins. By thiophosphorylating a synthetic peptide that corresponds to the predicted site, a carrier peptide is produced that protects the putative native phosphopeptide from nonspecific adsorption during isolation and that can replace 32P as a marker. This carrier thiophosphopeptide can be distinguished from the native phosphopeptide by its additional 16 mass units per attached (thio)phosphate. In a study described in the chapter, a thiophosphorylated, synthetic peptide coeluted with, and served as a carrier of, the corresponding endogenously phosphorylated peptide from a C-18 reverse-phase HPLC column. Mass spectrometry distinguished the endogenously phosphorylated peptides from the thiophosphorylated carrier, allowing determination of stoichiometry and identification of sites of phosphorylation on the native peptide. The isolation and analyses involving MS, MS/MS, and subdigestion used only 250 pmoles of the Mr 200,000 protein, CD45. Using this technique, four sites of in vivo phosphorylation were identified in this protein. The corresponding four serines were phosphorylated in vitro by casein kinase II (CKII).