No abstract

Nonenzymatic post-translational protein modifications (nePTMs) affect the nutritional, physiological, and technological properties of proteins in food and in vivo. In contrast to the usual targeted analyses, the present study determined nePTMs in processed milk in a truly untargeted proteomic approach. Thus, it was possible to determine to which extent known nePTM structures explain protein modifications in processed milk and to detect and identify novel products. The method combined ultrahigh-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry with bioinformatic data analysis by the software XCMS. The nePTMs detected by untargeted profiling of a β-lactoglobulin−lactose model were incorporated in a sensitive scheduled multiple reaction monitoring method to analyze these modifications in milk samples and to monitor their reaction kinetics during thermal treatment. Additionally, we identified the structures of unknown modifications. Lactosylation, carboxymethylation, formylation of lysine and N-terminus, glycation of arginine, oxidation of methionine, tryptophan, and cysteine, oxidative deamination of N-terminus, and deamidation of asparagine and glutamine were the most important reactions of β-lactoglobulin during milk processing. The isomerization of aspartic acid was observed for the first time in milk products, and N-terminal 4-imidazolidinone was identified as a novel nePTM.

Casein phosphopeptides are multiphosphorylated milk peptides, which can have anticariogenic activity and improve mineral absorption by binding bivalent metal ions. The present study investigated phosphopeptides in kefir because fermentation may lead to their enhanced release from milk proteins. After selective enrichment by hydroxyapatite extraction, phosphopeptides and their phosphorylation degree were identified by matrix-assisted desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) before and after enzymatic dephosphorylation. Peptide structures were determined by ultrahigh-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (UHPLC–ESI–MS/MS) revealing 27 phosphopeptides in kefir, including nine peptides containing the motif pSpSpSEE, which binds minerals most efficiently. The majority (18) of phosphopeptides were derived from β-casein, but only three were derived from the most abundant milk protein α s1 -casein. After simulated gastrointestinal digestion, MALDI-TOF-MS analysis detected eight putative phosphopeptides in kefir, four of which were assigned by UHPLC–ESI–MS/MS to α s2 -casein 124–133 , α s2 -casein 137–146 , β-casein 30–40 , and κ-casein 147–161 . These results indicate that kefir is a good dietary source of multiphosphorylated peptides.