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...
Partial-, double-enzymatic dephosphorylation and endoGluC hydrolysis as an original approach to enhancing identification of casein phosphopeptides (CPPs) by mass spectrometry.
UMR Transfrontaliere BioEcoAgro No 1158, Univ. Lille, INRAe, Univ. Liege, UPJV, YNCREA, Univ. Artois, Univ. Littoral Cote d'Opale, ICV-Institut Charles Viollette, 59000 Lille, France. firstname.lastname@example.org
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Deracinois, B., Mateos, A., Romelard, A., Boulier, A., Auger, J., Baniel, A., Ravallec, R., Flahaut, C.; Partial-, double-enzymatic dephosphorylation and endoGluC hydrolysis as an original approach to enhancing identification of casein phosphopeptides (CPPs) by mass spectrometry.. IFIS Food and Health Sciences Database 2022; doi:
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