Formation and reshuffling of disulfide bonds in bovine serum albumin demonstrated using tandem mass spectrometry with collision-induced and electron-transfer dissociation.

Thermolysin hydrolyzates of freshly isolated, extensively stored (6 years, 6 °C, dry) and heated (60 min, 90 °C, in excess water) bovine serum albumin (BSA) samples were analyzed with liquid chromatography (LC) electrospray ionization (ESI) tandem mass spectrometry (MS/MS) using alternating electron-transfer dissociation (ETD) and collision-induced dissociation (CID). The positions of disulfide bonds and free thiol groups in the different samples were compared to those deduced from the crystal structure of native BSA. Results revealed non-enzymatic posttranslational modifications of cysteine during isolation, extensive dry storage, and heating. Heat-induced extractability loss of BSA was linked to the impact of protein unfolding on the involvement of specific cysteine residues in intermolecular and intramolecular thiol-disulfide interchange and thiol oxidation reactions. The here developed approach holds promise for exploring disulfide bond formation and reshuffling in various proteins under conditions relevant for chemical, biochemical, pharmaceutical and food processing.