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

Abstract

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.

A Multiparticulate Delivery System for Potential Colonic Targeting Using Bovine Serum Albumin as a Model Protein

“…The 17 disulfide bridge within BSA gives it a high propensity to aggregate once BSA unfolds upon external stress and expose the disulfide bonds to the solvent (37). This fact helps to explain the existence of 10% aggregation immediately upon reconstitution of commercial lyophilized BSA powder, which was observed in this study and also in the literature (27, 38). Therefore, BSA is a good model protein to study protein degradation via aggregation during spray layering and enteric coating.…”

Differentiation of glycated residue numbers on heat‐induced structural changes of bovine serum albumin

“…During heat treatment, protein undergoes a series of structural changes, which could lead to protein unfolding and further aggregation . Heat‐induced structural changes include breakage of hydrogen bonds and disulfide bonds, and exposure of hydrophobic groups of indole, benzyl, isopropyl and isobutyl groups . Those groups could interact and form intermolecular hydrogen bonds, disulfide bonds and hydrophobic interactions, leading to protein aggregation.…”

“…[3][4][5] Heat-induced structural changes include breakage of hydrogen bonds and disulfide bonds, and exposure of hydrophobic groups of indole, benzyl, isopropyl and isobutyl groups. 6,7 Those groups could interact and form intermolecular hydrogen bonds, 8 disulfide bonds 7 and hydrophobic interactions, 6 leading to protein aggregation. Glycation with reducing sugars by Maillard reaction is a promising strategy to regulate protein aggregation.…”

Self-entanglement of bovine serum albumin in shear flow: cumulative effects and irreversibility

“…Importantly, the domains are also stabilized by 17 disulfide bonds (marked by yellow in Fig. 1) [19,20]. A large number of disulfide bonds make the topology of the protein complicated, with a number of linked loops formed by the backbones closed via disulfide bridges.…”