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

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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.