biostudies-literatureDeracinois BRégion Hauts-de-FranceANRC.P.E.R2134https://www.ebi.ac.uk/biostudies/studies/S-EPMC8471704biostudies-literatureUnknown10(9)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, alloFoods (Basel, Switzerland)Partial-, Double-Enzymatic Dephosphorylation and EndoGluC Hydrolysis as an Original Approach to Enhancing Identification of Casein Phosphopeptides (CPPs) by Mass Spectrometry.PMC8471704AlibiotechAllInPepANR-11- EQPX-0037Deracinois BBoulier AFlahaut CRomelard ARavallec RBaniel AMateos AAuger JfalsePartial-, 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, allo2021-01-01T00:00:00Z2021 Sep2026-05-01T03:10:28.689Z2022-02-11T11:28:51.528ZS-EPMC84717043457424510.3390/foods10092134