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