Disulfides are important for maintaining
the protein native structure,
but they may undergo rearrangement in the presence of free Cys residues,
especially under elevated temperatures. Disulfide rearrangement may
result in protein aggregation, which is associated with in
vivo pathologies in organisms and in vitro protein functionality in food systems. In a food context, it is
therefore important to understand the process of disulfide rearrangement
on a site-specific level in order to control aggregation. In the present
study, a liquid chromatography–mass spectrometry (LC–MS)-based
bottom-up site-specific proteomic approach was optimized to study
disulfide rearrangements in beta-lactoglobulin (β-LG) under
different heat treatments (60–90 °C). Artifactual disulfide
rearrangement observed during sample preparation using a conventional
protocol was detected and minimized by blocking the remaining free
Cys residues with iodoacetamide in the presence of urea after heat
treatment. Use of endoproteinase Glu-C for enzymatic hydrolysis allowed,
for the first time, identification and comparison of the relative
intensity of all theoretically possible β-LG disulfide cross-links
formed by the heat treatments. Non-native disulfides were formed from
heat treatment at approx. 70 °C where β-LG started to unfold,
while higher levels of inter-molecular disulfide links were formed
at ≥80 °C, in agreement with β-LG aggregation detected
by size exclusion chromatography analysis. Collectively, the Cys residues
of the surface-located native disulfide Cys66-Cys160 were proposed
to be more reactive, participating in heat-induced disulfide rearrangement,
compared to other Cys residues. The abundant signal of non-native
disulfide bonds containing Cys66, especially Cys66-Cys66, observed
after heating suggested that Cys66 is a key disulfide-linked Cys residue
in β-LG participating in heat-induced inter-molecular disulfide
bonds and the corresponding protein aggregation.
Thermal treatment is often employed in food processing to tailor product properties by manipulating the ingredient functionality, but these elevated temperatures may accelerate oxidation and nutrient loss. Here, oxidation of different whey protein systems [α-lactalbumin (α-LA), β-lactoglobulin (β-LG), a mix of α-LA and β-LG (whey model), and a commercial whey protein isolate (WPI)] was investigated during heat treatment at 60−90 °C and a UHT-like treatment by LC-MS-based proteomic analysis. The relative modification levels of each oxidation site were calculated and compared among different heat treatments and sample systems. Oxidation increased significantly in protein systems after heating at ≥90 °C but decreased in systems with higher complexity [pure protein (α-LA > β-LG) > whey model > WPI]. In α-LA, Cys, Met, and Trp residues were found to be most prone to oxidation. In β-LG-containing protein systems, Cys residues were suggested to scavenge most of the reactive oxidants and undergo an oxidation-mediated disulfide rearrangement. The rearranged disulfide bonds contributed to protein aggregation, which was suggested to provide physical protection against oxidation. Overall, limited loss of amino acid residues was detected after acidic hydrolysis followed by UHPLC analysis, which showed only a minor effect of heat treatment on protein oxidation in these protein systems.
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