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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.

Objectives The study was designed to evaluate the dose-effect of PEP2DIA(r)(PEP), a milk protein hydrolysate on glycemic control of type 2 diabetic Goto-Kakizaki (GK) rats treated for 6 weeks. Methods Rats (n = 10) were treated with PEP solubilized in spring water by oral gavage once a day for 6 weeks. The effects of a 6-week-administration of PEP (63 mg/kg, 88.6 mg/kg and 126 mg/kg per os) were evaluated on sucrose tolerance and insulin response to sucrose in type 2 diabetic GK rats from weaning (3 weeks) to adult age (9 weeks). Circulating GLP-1 concentration and DDP-4 activity, α-glucosidase activity in duodenum and jejunum, Fatty Acid Synthase (FAS) and Sterol regulatory element-binding protein 1 (SREBP-1c) gene expressions in liver and retroperitoneal adipose tissue were also measured. PEP is a patented milk protein hydrolysate containing AP dipeptides which inhibit alpha glucosidase. The alpha-glucosidase inhibitor Acarbose (40 mg/kg) was used as reference. Results The 6 week-treatment with PEP at 3 doses improved sucrose tolerance with the best effect at the dose of 63 mg/kg. Insulin response to sucrose was lower than control after PEP treatment at all the doses tested with the strongest decrease with 63 mg/kg of PEP. This decrease in insulin response seems to be at least in part the consequence of an improvement of the insulin resistance of the GK rats. At the lowest dose tested (63 mg/kg), FAS and SREBP-1c gene expressions were significantly decreased in retroperitoneal adipose tissue of GK rats, suggesting that PEP inhibited lipogenesis. PEP treatment induced strong increases in GLP-1 plasma level at all the doses tested but the difference reached significance only with 63 and 126 mg/kg of PEP. An inhibition of alpha-glucosidase in duodenum but not in jejunum was observed after the 6-week-treatment with PEP. Moreover, in retroperitoneal adipose tissue, PEP at the lowest dose tested (63 mg/kg), significantly decreased gene expression of both SREBP-1c and FAS, suggesting a beneficial effect on triglyceride accumulation in adipose tissue Conclusions These results showed that PEP2DIA(r) could have a beneficial effect on glycemic control of type 2 diabetic GK rats and suggest that this milk protein hydrolysate should be beneficial Impaired Fasting Glucose or Impaired Glucose Tolerance subjects for delaying the progression to overt type 2 diabetes. Funding Sources INGREDIA S.A.

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