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

{"sci_version": "1.2", "schema": "https://corpus.bntrg.co.uk/sci/1.0/sci.schema.json", "id": "pmid:34574245", "kind": "science-record", "revision": 8, "content_hash": "sha256:d8fd40fe2bfca191b9dcf534df2a237748af59fc5177be3c3efbf905cb9f8c7d", "created_at": "2026-11-01T15:41:54Z", "updated_at": "2026-11-01T22:47:36Z", "generator": {"name": "corpus-assay-engine", "run_id": "reading-sci-2026-11-01"}, "paper": {"state": "complete", "title": "Partial-, Double-Enzymatic Dephosphorylation and EndoGluC Hydrolysis as an Original Approach to Enhancing Identification of Casein Phosphopeptides (CPPs) by Mass Spectrometry.", "doi": "10.3390/foods10092134", "pmid": "34574245", "authors": ["Barbara Deracinois", "Aurélie Matéos", "Audrey Romelard", "Audrey Boulier", "Julie Auger", "Alain Baniel", "Rozenn Ravallec", "Christophe Flahaut"], "journal": "Foods (Basel, Switzerland)", "year": 2021, "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.", "claims": [{"id": "claim-method", "statement": "Two-way ANOVA multiple comparisons using a p-value inferior to 0.05 (p < 0.05) were carried out from the MS data and performed in duplicates.", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:34574245", "locator": "sections[11] \"3.1.1. 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