Detection of Two Minor Phosphorylation Sites for Bovine κ-Casein Macropeptide by Reversed-Phase Liquid Chromatography–Tandem Mass Spectrometry

Abstract

This work addresses the characterization of phosphopeptides in bovine κ-casein macropeptide by reversed-phase liquid chromatography-electrospray ionization-tandem mass spectrometry (RPLC-ESI-MS(2)). Two different mass spectrometers, equipped with an ion trap (IT) or a quadrupole time-of-flight (Q-TOF) analyzer, were used to perform an accurate phosphorylation site assignment. A total of 8 phosphopeptides from 26 identified peptides were characterized. MS(2) spectra of phosphopeptides were dominated by the neutral loss of a phosphoric acid molecule (H(3)PO(4)) and sufficient informative fragment ions resulting from peptide backbone cleavages enabling the elucidation of the phosphopeptide sequence. A higher number of sequence informative b and y ions were detected using a Q-TOF instead of an IT analyzer. In addition to the well-established phosphorylation sites at Ser(149) and Ser(127), this study also revealed the presence of two minor phosphorylation sites at Thr(145) and Ser(166). These findings indicate that RPLC-ESI-MS(2) on a Q-TOF analyzer is a useful technique for identifying low-abundance phosphorylation sites in caseins.