2 research outputs found
Comprehensive Analysis of Nonenzymatic Post-Translational β‑Lactoglobulin Modifications in Processed Milk by Ultrahigh-Performance Liquid Chromatography–Tandem Mass Spectrometry
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
Modified Peptides as Indicators for Thermal and Nonthermal Reactions in Processed Milk
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) <i>N</i><sup>ε</sup>-carboxymethyllysine,
N-terminal ketoamide, and asparagine deamidation are of thermal origin
and may be good markers for rather intensive heat treatment, whereas <i>N</i><sup>ε</sup>-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