title: 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.
authors.affiliation: Laboratory of Food Chemistry and Biochemistry, Leuven Food Science and Nutrition Research Centre (LFoRCe), KU Leuven, Kasteelpark Arenberg 20, box 2463, B-3001 Leuven, Belgium.
authors.forename: Ine
authors.initials: I
authors.lastname: Rombouts
authors.affiliation: Laboratory of Food Chemistry and Biochemistry, Leuven Food Science and Nutrition Research Centre (LFoRCe), KU Leuven, Kasteelpark Arenberg 20, box 2463, B-3001 Leuven, Belgium.
authors.forename: Bert
authors.initials: B
authors.lastname: Lagrain
authors.affiliation: Deutsche Forschungsanstalt für Lebensmittelchemie, Leibniz Institut, Lise-Meitner-Straβe 34, D-85354 Freising, Germany.
authors.forename: Katharina A
authors.initials: KA
authors.lastname: Scherf
authors.affiliation: Laboratory of Food Chemistry and Biochemistry, Leuven Food Science and Nutrition Research Centre (LFoRCe), KU Leuven, Kasteelpark Arenberg 20, box 2463, B-3001 Leuven, Belgium.
authors.forename: Marlies A
authors.initials: MA
authors.lastname: Lambrecht
authors.affiliation: Deutsche Forschungsanstalt für Lebensmittelchemie, Leibniz Institut, Lise-Meitner-Straβe 34, D-85354 Freising, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Koehler
authors.affiliation: Laboratory of Food Chemistry and Biochemistry, Leuven Food Science and Nutrition Research Centre (LFoRCe), KU Leuven, Kasteelpark Arenberg 20, box 2463, B-3001 Leuven, Belgium.
authors.forename: Jan A
authors.initials: JA
authors.lastname: Delcour
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D004583:Electrons; D006358:Hot Temperature; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D066329:Protein Aggregates; D012710:Serum Albumin, Bovine / Q000737:chemistry* / Q000302:isolation & purification; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D004220:Disulfides; D010455:Peptides; D066329:Protein Aggregates; D012710:Serum Albumin, Bovine; D003545:Cysteine