Ser71 of αS1-Casein is Phosphorylated in Breast Milk-Evidence from Targeted Mass Analysis

Abstract

Scope: The casein phosphoproteins in mother's milk supply calcium and phosphate ions and make them biologically available to the newborn. Human αS1-casein is of particular interest being also an autoantigen and proinflammatory cytokine. Phosphorylation of αS1-casein by casein kinase 2 completely abolishes binding to toll-like receptor 4 and proinflammatory effects. It is, however, not known, which amino acids are affected. Therefore, breast milk samples were analyzed in an effort to detect the phosphorylation sites of αS1-casein.

Methods and results: Breast milk samples were tryptically digested. Target tandem MS analysis confirmed the known phosphorylation sites S33 and S41; evidence for pS89 was found in some samples. Experimental support for the presence of pS31 and pS34 was weak. Phosphorylation of a new site in αS1-casein, S71, was reproducibly measured in all samples, albeit at much lower intensity than pS33 and pS41.

Conclusion: Phospho-occupancy rates varied greatly and could not be confidently correlated to other parameters within the cohort of 20 donors. The new phosphosite S71 is located in the neighborhood of the serine-rich region and may contribute to the cluster of high charge density at normal milk pH, likely exerting an influence on protein tertiary structure and thus function.

Keywords: breast milk; mass spectrometry; phosphorylation; phosphosite S71; αS1-casein.