Identification of Phosphoserine and Phosphothreonine as Cysteic Acid and β-Methylcysteic Acid Residues in Peptides by Tandem Mass Spectrometric Sequencing

Abstract

Tandem mass spectrometry has long been an intrinsic tool to determine phosphorylation sites in proteins. However, loss of the phosphate moiety from both phosphoserine and phosphothreonine residues in low-energy collision-induced dissociation is a common phenomenon, which makes identification of P-Ser and P-Thr residues complicated. A method for direct sequencing of the Ser and Thr phosphorylation sites by ESI tandem mass spectrometry following β-elimination/sulfite addition to convert −HPO₄ to −SO₃ has been studied. Five model phosphopeptides, including three synthetic P-Ser-, P-Thr-, or P-Ser- and P-Thr-containing peptides; a protein kinases C-phosphorylated peptide; and a phosphopeptide derived from β-casein trypsin digests were modified and then sequenced using an ESI-quadrupole ion trap mass spectrometer. Following incubation of P-Ser- or P-Thr-containing peptides with Na₂SO₃/NaOH, 90% P-Ser and 80% P-Thr was converted to cysteic acid and β-methylcysteic acid, respectively, as revealed by amino acid analysis. The conversion can be carried out at 1 μM concentration of the peptide. Both cysteic acid and β-methylcysteic acid residues in the sequence were shown to be stable and easily identifiable under general conditions for tandem mass spectrometric sequencing applicable to common peptides.