Identification of Bovine Casein Phosphorylation Using Titanium Dioxide Enrichment in Combination with Nano Electrospray Ionization Tandem Mass Spectrometry

Protein phosphorylation is an important post-translational modification that regulates milk protein structure and function. The objective of this study was to analyze the presence of phosphorylated casein. Bovine milk proteins were first separated by SDS polyacrylamide gel electrophoresis. After in gels digestion and extraction, phosphorylated peptides were enriched by titanium dioxide and identified by ultra performance liquid chromatography coupled with nano electrospray ionization tandem mass spectrometry. This method ensured the identification of 20 phosphorylated peptides, including 7 phosphorylated forms of αs1-casein, 8 αs2-casein, and 5 β-casein. Eight phosphorylated sites derived from 3 αs1-caseins, 3 αs2-caseins, and 2 β-caseins were also identified, and localized on residues Ser61, Ser63 and Ser130 in αs1-casein; Thr145, Ser146 and Ser158 in αs2-casein; and Ser50 and Thr56 in β-casein. These findings provide valuable information for investigating casein phosphorylation of the bovine milk.