Identification, production and bioactivity of casein phosphopeptides - A review.

Milk and dairy products are significant sources of proteins and peptides impacting human health. In this way, the interest in CPPs, bioactive phosphorylated peptides resulting from the hydrolysis of caseins, has grown in the past years. CPPs were mainly studied for their capacity to chelate and increase the bioavailability of essential minerals involved in multiple physiological processes. Moreover, CPPs harbour interesting antioxidant and anti-inflammatory properties. Recent in vivo and in vitro studies demonstrated that these different roles are strongly linked to the intrinsic properties of CPPs and CPP concentrate preparations. This review first comments on the different methods of CPP analytical characterization, focusing on recent techniques. Then, the CPP release occurring during the gastrointestinal digestion was reviewed, followed by the different CPP obtention processes and their impact on their physicochemical characteristics. Finally, the different bioactive roles attributed to CPPs, including mineral chelating properties, are discussed. We show that CPPs have a promising role in treating various pathologies, notably to compensate for deficiencies in certain nutrients and an anti-oxidant and anti-inflammatory role. Nevertheless, the mechanisms by which CPPs exert their role remain to be elucidated, and this requires precise characterization of CPPs. This work highlights the key parameters to be considered to study and produce CPPs and the different ways to be investigated in the future to elucidate their roles in vivo and characterize their potential for human health.

Partial-, Double-Enzymatic Dephosphorylation and EndoGluC Hydrolysis as an Original Approach to Enhancing Identification of Casein Phosphopeptides (CPPs) by Mass Spectrometry.

The identification of phosphopeptides is currently a challenge when they are part of a complex matrix of peptides, such as a milk protein enzymatic hydrolysate. This challenge increases with both the number of phosphorylation sites on the phosphopeptides and their amino acid length. Here, this paper reports a four-phase strategy from an enzymatic casein hydrolysate before a mass spectrometry analysis in order to enhance the identification of phosphopeptides and phosphosites: (i) the control protein hydrolysate, (ii) a two-step enzymatic dephosphorylation of the latter, allowing for the almost total dephosphorylation of peptides, (iii) a one-step enzymatic dephosphorylation, allowing for the partial dephosphorylation of the peptides and (iv) an additional endoGluC enzymatic hydrolysis, allowing for the cleavage of long-size peptides into shorter ones. The reverse-phase high-pressure liquid chromatography-tandem mass spectrometry (RP-HPLC-MS/MS) analyses of hydrolysates that underwent this four-phase strategy allowed for the identification of 28 phosphorylation sites (90%) out of the 31 referenced in UniprotKB/Swiss-Prot (1 June 2021), compared to 17 sites (54%) without the latter. The alpha-S2 casein phosphosites, referenced by their similarity in the UniProt database, were experimentally identified, whereas pSer148, pThr166 and pSer187 from a multiphosphorylated long-size kappa-casein were not. Data are available via ProteomeXchange with identifier PXD027132.