Phosphoproteins of the chicken eggshell calcified layer

The chicken eggshell matrix is a complex mixture of proteins and proteoglycans. It also contains phosphoproteins that are thought to affect mineralization of the matrix. Several of the matrix phosphoproteins, such as the major component osteopontin, have already been identified as phosphoproteins in other tissues, but the phosphorylation status of the eggshell matrix forms was unknown. The phosphopeptides, obtained after cleavage of the matrix proteins with several different cleavage methods, were enriched by anion-exchange chromatography and reversible binding to titanium oxide and identified by LC-MSn or pseudo-MSn analysis following neutral loss scanning. Altogether we identified 39 phosphorylated matrix proteins, 22 of which were not known to be phosphorylated before. Eight of the proteins were identified as eggshell matrix components for the first time. Together these proteins contained more than 150 different phosphorylation sites, 103 of which were determined with high confidence. Among the major phosphorylated proteins of the chicken eggshell matrix were osteopontin and the eggshell-specific proteins ovocleidin-17, ovocleidin-116, and ovocalyxin-32.