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

To investigate thiol-disulfide interchange reactionsin heatedmilk yielding non-native intramolecular rearranged and intermolecularcross-linked proteins, a proteomic study based on nanoLC-ESI-Q-Orbitrap-MS/MSand dedicated bioinformatics was accomplished. Raw milk samples heatedfor different times and various commercial dairy products were analyzed.Qualitative experiments on tryptic digests of resolved protein mixturesassigned the corresponding disulfide-linked peptides. Results confirmedthe limited data available on few milk proteins, generated the widestinventory of components (63 in number) involved in thiol-disulfideexchange processes, and provided novel structural information on S-S-bridgedmolecules. Quantitative experiments on unresolved protein mixturesfrom both sample typologies estimated the population of moleculesassociated with thiol-disulfide reshuffling processes. Disulfide-linkedpeptides associated with native intramolecular S-S bonds generallyshowed a progressive reduction depending on heating time/harshness,whereas those related to specific non-native intramolecular/intermolecularones showed an opposite quantitative trend. This was associated witha temperature-dependent augmented reactivity of definite native proteinthiols and S-S bridges, which determined the formation of non-nativerearranged monomers and cross-linked oligomers. Results provided novelinformation for possibly linking the nature and extent of thiol-disulfideexchange reactions in heated milk proteins to the corresponding functionaland technological characteristics, with possible implications on fooddigestibility, allergenicity, and bioactivity.