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.