Modified Peptides as Indicators for Thermal and Nonthermal Reactions in Processed Milk

Abstract

Site-specific relative quantification of β-lactoglobulin modifications in heated milk and dairy products was performed to determine their thermal and nonthermal origins and to evaluate marker candidates for milk processing. Therefore, formation kinetics of 19 different structures at 26 binding sites were analyzed by ultrahigh-performance liquid chromatography-tandem mass spectrometry with multiple reaction monitoring (UHPLC-MS/MS/MRM) after specific protein hydrolysis. The results indicate that (i) site-specific analysis of lactulosyllysine may be a more sensitive marker for mild heat treatment than its overall content; (ii) N(ε)-carboxymethyllysine, N-terminal ketoamide, and asparagine deamidation are of thermal origin and may be good markers for rather intensive heat treatment, whereas N(ε)-carboxyethyllysine reflects thermal and nonthermal processes; (iii) the relevance of methylglyoxal-derived arginine modifications is low compared to that of other modifications; (iv) oxidation of methionine and cysteine is a rather weak indicator of thermal impact; and (v) the tryptophan modifications formylkynurenine and kynurenine are of nonthermal origin and further degraded during processing.

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].…”

“…Although lacking quantitative information, these data will help in making comprehensive milk-focused database, which may ultimately allow food manufacturers to thermally process their products as careful as possible in order to obtain products with controlled amounts of AGEs. To reach this ultimate goal, future studies applying quantitative MS procedures on isolated protein components modified in vitro with unlabelled/isotopically-labeled lactose mixtures or quantitative proteomic approaches (based on labeled proteotypic peptides or AGEs-oriented derivatizing reagents) will be advisable to gain quantifiable data on the amino acids more prone to generate adducts in various milk proteins [1,9,20,61]. These information are important points of interest to connect the extent of the Maillard reaction occurring in different commercial samples with potential nutritional implications.…”

“…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].…”

The Chemistry of Protein Oxidation in Food

“…Mass spectroscopy of partially hydrolyzed proteins may give information about the abundance of individual oxidized species and help to decide which individual products can be chosen for absolute quantification . Relative quantification of individual peptides can be performed in order to compare the effect of processing conditions on protein oxidation . Tryptic marker peptides resulting from oxidation may be employed for quantitative expression of the oxidation of individual proteins .…”

MALDI-TOF-MS Platform for Integrated Proteomic and Peptidomic Profiling of Milk Samples Allows Rapid Detection of Food Adulterations

“…In particular, 540 mass spectra were acquired for the corresponding PR-P (5 spectral replicas of the 9 percentage values assayed for each kind of adulteration; experiments were performed on triplicate samples) (data not shown); 1080 mass spectra were acquired for the corresponding PE-P of milk filtrates (10 spectral replicas of the 9 percentage values assayed for each kind of adulteration; experiments were performed on triplicate samples) either in LM and RM (data not shown); 540 mass spectra were acquired for the corresponding PE-P of milk filtrates subjected to C 18 . The latter result is probably dependent on the higher complexity of the protein and peptide mixtures present in commercial milk samples 5,15,17,19,20,[23][24][25][26] ; it also underlines that the method used for spectral acquisition can highly affect the recognition capability of the whole platform. On this basis, particular care is necessary for the initial set up of the PR-P and PE-P experimental conditions to guarantee the maximum number of signals present in the mass spectra and of the corresponding signal to noise ratios.…”