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

Barbara Deracinois (1) , Aurélie Matéos (2) , Audrey Romelard , Audrey Boulier , Julie Auger , Alain Baniel , Rozenn Ravallec (1) , Christophe Flahaut (1)

1 BioEcoAgro - Equipe 7 - Health benefit of protein hydrolysates and agro-food co-products: control of production, characterization and valorisation
2 BioEcoAgro - Equipe 9 - Structure-function relationship at the service of the reasoned formulation of food and ingredients

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.

Mots clés: casein phosphopeptides; endoGluC hydrolysis; enzymatic dephosphorylation; mass spectrometry

Domaines: Sciences du Vivant [q-bio]; Sciences de l'environnement

Barbara Deracinois, Aurélie Matéos, Audrey Romelard, Audrey Boulier, Julie Auger, et al.. Partial-, Double-Enzymatic Dephosphorylation and EndoGluC Hydrolysis as an Original Approach to Enhancing Identification of Casein Phosphopeptides (CPPs) by Mass Spectrometry. Foods, 2021, 10 (9), pp.2134. ⟨10.3390/foods10092134⟩. ⟨hal-03653112⟩