Proteomic Characterization of Native and Rearranged Disulfide Bonds in Proteins from Thermally Treated and Commercial Milk Samples

To investigate thiol-disulfide interchange reactions
in heated
milk yielding non-native intramolecular rearranged and intermolecular
cross-linked proteins, a proteomic study based on nanoLC-ESI-Q-Orbitrap-MS/MS
and dedicated bioinformatics was accomplished. Raw milk samples heated
for different times and various commercial dairy products were analyzed.
Qualitative experiments on tryptic digests of resolved protein mixtures
assigned the corresponding disulfide-linked peptides. Results confirmed
the limited data available on few milk proteins, generated the widest
inventory of components (63 in number) involved in thiol-disulfide
exchange processes, and provided novel structural information on S–S-bridged
molecules. Quantitative experiments on unresolved protein mixtures
from both sample typologies estimated the population of molecules
associated with thiol-disulfide reshuffling processes. Disulfide-linked
peptides associated with native intramolecular S–S bonds generally
showed a progressive reduction depending on heating time/harshness,
whereas those related to specific non-native intramolecular/intermolecular
ones showed an opposite quantitative trend. This was associated with
a temperature-dependent augmented reactivity of definite native protein
thiols and S–S bridges, which determined the formation of non-native
rearranged monomers and cross-linked oligomers. Results provided novel
information for possibly linking the nature and extent of thiol-disulfide
exchange reactions in heated milk proteins to the corresponding functional
and technological characteristics, with possible implications on food
digestibility, allergenicity, and bioactivity.