title: 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.
authors.affiliation: Instituto de Química Orgánica General, Consejo Superior de Investigaciones Científicas, C/Juan de la Cierva 3, 28006 Madrid, Spain.
authors.forename: Oswaldo
authors.initials: O
authors.lastname: Hernández-Hernández
authors.forename: Rosa
authors.initials: R
authors.lastname: Lebrón-Aguilar
authors.forename: Jesús E
authors.initials: JE
authors.lastname: Quintanilla-López
authors.forename: M Luz
authors.initials: ML
authors.lastname: Sanz
authors.forename: F Javier
authors.initials: FJ
authors.lastname: Moreno
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002364:Caseins / Q000737:chemistry*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry*; D010766:Phosphorylation; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D002364:Caseins; D010446:Peptide Fragments; C065989:caseinomacropeptide