Identification of multiphosphorylated peptides in milk.

Multiphosphorylated peptides endogenously present in milk exert anticariogenic activity due to their calcium binding capacity. This study performed comprehensive analysis of multiphosphorylated peptides in raw milk using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Since phosphopeptides are often negatively discriminated during ionization, putative phosphopeptides were identified by three different methods: (i) selective detection in 4-chloro-α-cyanocinnamic acid MALDI matrix compared to α-cyano-4-hydroxycinnamic acid; (ii) higher relative signal intensity in negative compared to positive ionization mode; and (iii) detection of signal pairs with mass differences of -80 Da or multiples thereof before and after enzymatic dephosphorylation. Thus, 18 putative phosphopeptides from raw milk were annotated. Peptide structures were then determined by product ion spectra from targeted liquid chromatography electrospray ionization tandem-MS analysis. Thus, β-casein33-48, β-casein29-48, β-casein1-21, β-casein2-25, β-casein1-25, β-casein1-27, β-casein1-28, β-casein1-29, β-casein1-32, αS2-casein1-21, and αS2-casein1-24 were revealed as major peptides with one or four phosphorylation sites in raw milk.