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.

Promoting the Calcium-Uptake Bioactivity of Casein Phosphopeptides in vitro and in vivo

Casein phosphopeptides have been studied widely for their ability to chelate calcium. However, systematic studies on the effects of casein phosphopeptides (CPP) on calcium absorption in vitro and in vivo are scarce. The purities of two commercially available products, CPP1 and CPP2, are 18.37 and 25.12%, respectively. Here, the in vitro calcium binding capacity of CPP2 was 142.56 ± 7.39 mg/g, which was higher than that of CPP1 (107.15 ± 6.27 mg/g). The calcium transport results in a Caco-2 monolayer model indicated that, relative to controls, CPP1 and CPP2 increased calcium transport by 21.78 and 53.68%, respectively. Subsequent animal experiments showed that the CPP2-Ca-H group (1% Ca, 0.4% CPP2) had significant increases in the femur index, serum Ca2+ and serum osteocalcin levels, and femoral Ca content. The CPP2-Ca-H animal also had decreased serum alkaline phosphatase levels, parathyroid hormone content, and urinary pyridinoline content. Overall, our results demonstrated that CPP2 had stronger effects on promoting calcium uptake than CPP1.

Fabrication of ferritin-casein phosphopeptides-calcium shell-core composite as a novel calcium delivery strategy

Plant ferritin endows a natural cage-like nanospace for carrying bioactive ingredients. By taking advantage of the calcium binding ability of casein phosphopeptide (CPP) and the cage like conformation of plant...

Antioxidant Properties of Casein Phosphopeptides (CPP) and Maillard-Type Conjugated Products

A casein phosphopeptide (CPP) fraction derived from tryptic hydrolysis of bovine casein was evaluated for antioxidant activity. Conjugations or mixtures of CPP with polysaccharide, galactomannan (Gal), or xyloglucan (Xyl) were prepared to evaluate potential enhancement of CPP antioxidant activity. The effect of calcium was also investigated. The CPP preparation alone was effective at scavenging hydroxyl radicals and sequestering Fe2+ to protect against Fenton reaction-induced deoxyribose oxidation in non-site-specific (up 63.3% inhibition) and site-specific (up 32.1% inhibition) binding assays, respectively. CPP also effectively quenched 2,2’-azinobis-3-ethylbenzothiazoline-6-sulfonic acid radicals (ABTS•+) to an extent of 67.6% scavenging in an aqueous system. In a soybean lecithin liposome system, CPP exhibited effective protection against peroxyl radical-induced liposomal peroxidation (38.3% of control in terms of rate of propagation). Conjugating CPP with Gal or Xyl polysaccharides using Maillard reaction conditions significantly reduced activity in the Fenton reaction-deoxyribose assays, while exhibiting no effect on the antioxidant activity of native CPP in both the ABTS and liposome assays, respectively. These results represent comparative antioxidant capacity of the native CPP and associated conjugates in phases that varied in relative hydrophilic and hydrophobic character. We conclude that CPP has the potential to act as both a primary and secondary antioxidant by displaying transition metal ion sequestering activity and free radical quenching activity. Improvements in antioxidant activity of CPP by Maillard-type conjugation with Xyl or Gal were relatively small and model-specific.