Comprehensive Analysis of Nonenzymatic Post-Translational β-Lactoglobulin Modifications in Processed Milk by Ultrahigh-Performance Liquid Chromatography–Tandem Mass Spectrometry

Abstract

Nonenzymatic post-translational protein modifications (nePTMs) result in changes of the protein structure that may severely influence physiological and technological protein functions. In the present study, ultrahigh-performance liquid chromatography-electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) was applied for the systematic identification and site-specific analysis of nePTMs of β-lactoglobulin in processed milk. For this purpose, β-lactoglobulin, which had been heated with lactose under conditions to force nePTM formation (7 d/60 °C), was screened for predicted modifications by using full scans and enhanced resolution scan experiments combined with enhanced product ion scans. Thus, the main glycation, glycoxidation, oxidation, and deamidation products of lysine, arginine, methionine, cysteine, tryptophan, and asparagine, as well as the N-terminus, were identified. Using these MS data, a very sensitive scheduled multiple reaction monitoring method suitable for the analysis of milk products was developed. Consequently, 14 different PTM structures on 25 binding sites of β-lactoglobulin were detected in different milk products.

Proteomic characterization of intermediate and advanced glycation end-products in commercial milk samples

“…S2) was compiled and used for MS and MS/MS data-driven database searching. Some of these amino acid modifications have already been detected in milk proteins [2][3][4][5]13,15,17,19,20]; others have been identified in other model proteins/peptides after their heating in the presence of sugars and/or sugar oxidation products [8][9][10]15,[29][30][31][32][33][34][35][36][37][38].…”

“…Supplementary Information reports the cases in which this ambiguity occurred. Nevertheless, it has to be mentioned that the Amadori compounds and CML, CEL and DH derivatives are generally more stable than the various isobaric hemiaminal counterparts [10,19].…”

“…Regarding milk proteins, peptide mapping experiments were performed on isolated model components heated in vitro with various sugars to simulate food processing conditions or on a very reduced number of proteins isolated by chromatographic or electrophoretic procedures from commercial milk samples [1,4,9,[13][14][15][16][17][18] and references therein]. Although a number of studies was realized in this context, AGEs assignment was generally restricted to the case of CML adducts, complementing data on oxidized Lys, Met, Cys, Tyr and Trp derivatives, lysinoalanine, histidinoalanine, pyroglutamate and deamidated Asn [2][3][4]13,19]. Recently, untargeted LC-ESI-MS/MS multiple reaction monitoring (MRM) procedures were used to evaluate the different AGEs present in β-lactoglobulin (β-LG) from heated milk models [19].…”

“…Although a number of studies was realized in this context, AGEs assignment was generally restricted to the case of CML adducts, complementing data on oxidized Lys, Met, Cys, Tyr and Trp derivatives, lysinoalanine, histidinoalanine, pyroglutamate and deamidated Asn [2][3][4]13,19]. Recently, untargeted LC-ESI-MS/MS multiple reaction monitoring (MRM) procedures were used to evaluate the different AGEs present in β-lactoglobulin (β-LG) from heated milk models [19]. When a derived targeted MRM procedure was applied to the analysis of β-LG from commercial milk samples, only Lact, CML, CEL, MG-H, MG-DH and 3-DG-H adducts were detected and assigned to specific residues [19,20].…”

“…Recently, untargeted LC-ESI-MS/MS multiple reaction monitoring (MRM) procedures were used to evaluate the different AGEs present in β-lactoglobulin (β-LG) from heated milk models [19]. When a derived targeted MRM procedure was applied to the analysis of β-LG from commercial milk samples, only Lact, CML, CEL, MG-H, MG-DH and 3-DG-H adducts were detected and assigned to specific residues [19,20]. Some of these derivatives were already identified in β-casein treated in vitro with glucose or MG [21].…”