Absolute quantification of two antimicrobial peptides αS2-casein182-207 and αS2-casein151-181 in bovine milk by UHPLC-ESI-MS/MS in sMRM mode

Abstract

To evaluate if the recently identified antimicrobial milk peptides α_S2-casein_182-207 and α_S2-casein_151-181 can be of physiological or technological relevance, their presence in commercial milk was investigated. Thus, a UHPLC-ESI-MS/MS scheduled multiple reaction monitoring method was developed for quantification using stable isotope-labeled peptides. The developed protocol gave very good linear response (R² > 0.99) for both peptides, recovery >95% and coefficients of variations of 5.51% and 4.79%. The limits of detection (0.3 nM α_S2-casein_182-207 and 0.5 nM α_S2-casein_151-181) were low enough to detect both peptides without further enrichment. The highest peptide concentration was recorded in pasteurized milk (1.36 μM α_S2-casein_182-207 and 0.20 μM α_S2-casein_151-181). The concentration of α_S2-casein_182-207 was in a range that was previously reported to act bacteriostatic against Bacillus subtilis. Industrial milk processing led to a significant loss of both peptides resulting in a mean concentration of 0.47 µM α_S2-casein_182-207 and 0.098 µM α_S2-casein_151-181 in ultrahigh-temperature milk.

Keywords: Absolute quantification; Antimicrobial peptides; Milk; UHPLC–ESI–MS/MS-sMRM; α(S2)-casein(182–207).