title: Disulfide Linkage Characterization of Disulfide Bond-Containing Proteins and Peptides by Reducing Electrochemistry and Mass Spectrometry.
abstract: Unravelling of disulfide linkage patterns is a crucial part of protein characterization, whether it is for a previously uncharacterized protein in basic research or a recombinant pharmaceutical protein. In the biopharmaceutical industry, elucidation of the cysteine connectivities is a necessity to avoid disulfide scrambled and incorrectly folded forms in the final product. Mass spectrometry (MS) is a highly utilized analytical tool for this due to fast and accurate characterization. However, disulfide bonds being an additional covalent bond in the protein structure represent a challenge in protein sequencing by tandem MS (MS/MS). Electrochemical (EC) reduction of disulfide bonds has recently been demonstrated to provide efficient reduction efficiencies, significantly enhancing sequence coverages in online coupling with MS characterization. In this study, the potential use of EC disulfide reduction in combination with MS characterization for disulfide mapping was assessed. We employed two approaches based on (1) the high flexibility and instant information about the degree of reduction in infusion EC-MS to generate partially reduced species on the intact protein level and (2) the preserved link between parent disulfide-linked fragments and free reduced peptides in an LC-EC-MS platform of nonreduced proteolytic protein digestions. Here we report the successful use of EC as a partial reduction approach in mapping of disulfide bonds of intact human insulin (HI) and lysozyme. In addition, we established a LC-EC-MS platform advantageous in disulfide characterization of complex and highly disulfide-bonded proteins such as human serum albumin (HSA) by online EC reduction of nonreduced proteolytic digestions.
authors.affiliation: Protein Engineering, Novo Nordisk A/S , Novo Nordisk Park, 2760 Måløv, Denmark.|Department of Proteomics, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen , 2200 Copenhagen, Denmark.
authors.forename: Christian N
authors.initials: CN
authors.lastname: Cramer
authors.affiliation: Protein Engineering, Novo Nordisk A/S , Novo Nordisk Park, 2760 Måløv, Denmark.
authors.forename: Kim F
authors.initials: KF
authors.lastname: Haselmann
authors.affiliation: Department of Proteomics, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen , 2200 Copenhagen, Denmark.
authors.forename: Jesper V
authors.initials: JV
authors.lastname: Olsen
authors.affiliation: Protein Engineering, Novo Nordisk A/S , Novo Nordisk Park, 2760 Måløv, Denmark.
authors.forename: Peter Kresten
authors.initials: PK
authors.lastname: Nielsen
mesh_terms: D004220:Disulfides / Q000032:analysis* / Q000737:chemistry*; D055664:Electrochemical Techniques; D006801:Humans; D007328:Insulin / Q000737:chemistry*; D013058:Mass Spectrometry; D009113:Muramidase / Q000737:chemistry*; D010084:Oxidation-Reduction; D012709:Serum Albumin / Q000737:chemistry*
chemical_list: D004220:Disulfides; D007328:Insulin; D012709:Serum Albumin; D009113:Muramidase