Casein phosphoproteome: Identification of phosphoproteins by combined mass spectrometry and two-dimensional gel electrophoresis

We report a fast and easy‐to‐use procedure that combines polyacrylamide gel electrophoresis with matrix assisted laser desorption/ionization‐time of flight‐mass spectrometry (MALDI‐TOF) and nanoelectrospray‐tandem mass spectrometry (nES‐MS/MS) analysis for the identification of casein components and defined phosphorylated sites. This methodology ensured identification of more than 30 phosphorylated proteins, five β‐, fifteen αs1‐, ten αs2‐, and four κ‐casein (CN) components, including nonallelic, differently phosphorylated, and glycosylated forms. The sugar motif covalently bound to κ‐CN was identified as chains, trisaccharide GalNAc, Gal, NeuGc, and tetrasaccharide 1GalNAc, 1Gal, 2NeuGc. Also identified was a biantennary chain made up of both chains of trisaccharide 1GalNAc, 1Gal, 1NeuGc, and tetrasaccharide 1GalNAc, 1Gal, 2NeuGc moiety on a single κ‐CN component. The phosphate group on site Ser12 of tryptic peptide 8–22 of most phosphorylated αs1‐CN (11 phosphate groups) was localized and the oligosaccharide sequence of the main tryptic glycopeptides of two κ‐CN components was determined by means of MS/MS analysis.