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

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

doi:10.3390/foods10092134

Abstract

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.

Highlights

Introduction published maps and institutional affilMilk is, above all, the main food of the newborn in mammals and mandatory for its good growth, containing all of the nutrients necessary, such as lipids, carbohydrates, vitamins and proteins
Due to the data-dependent analysis mode used to manage the mass spectrometry (MS) and MS/MS data acquisition, the number of MS and MS/MS scans provides a rapid overview of the peptide heterogeneity of the different samples analyzed
A low number of MS scans, which denotes a high number of ions with an intensity above the threshold defined for fragmentation, is associated with a high number of MS/MS scans, since numerous ions have been selected in MS mode and fragmented

Summary

Introduction

Introduction published maps and institutional affilMilk is, above all, the main food of the newborn in mammals and mandatory for its good growth, containing all of the nutrients necessary, such as lipids, carbohydrates, vitamins and proteins. Milk proteins made up of 20% whey proteins and 80% caseins are used by the dairy industry for their nutritional and techno-functional properties. These proteins are a source of multiple molecules with biological activity that can be generated during their hydrolysis. The caseins are phosphorylated proteins with a specific cluster of phosphorylation (pSer-pSer-pSer-Glu-Glu) that can bind several minerals, such as calcium, iron, zinc and magnesium [2,3,4].

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