title: Electrochemistry-assisted top-down characterization of disulfide-containing proteins.
abstract: Covalent disulfide bond linkage in a protein represents an important challenge for mass spectrometry (MS)-based top-down protein structure analysis as it reduces the backbone cleavage efficiency for MS/MS dissociation. This study presents a strategy for solving this critical issue via integrating electrochemistry (EC) online with a top-down MS approach. In this approach, proteins undergo electrolytic reduction in an electrochemical cell to break disulfide bonds and then undergo online ionization into gaseous ions for analysis by electron-capture dissociation (ECD) and collision-induced dissociation (CID). The electrochemical reduction of proteins allows one to remove disulfide bond constraints and also leads to increased charge numbers of the resulting protein ions. As a result, sequence coverage was significantly enhanced, as exemplified by β-lactoglobulin A (24 vs 75 backbone cleavages before and after electrolytic reduction, respectively) and lysozyme (5 vs 66 backbone cleavages before and after electrolytic reduction, respectively). This methodology is fast and does not need chemical reductants, which would have an important impact in high-throughput proteomics research.
authors.affiliation: Center for Intelligent Chemical Instrumentation, Department of Chemistry and Biochemistry, Ohio University, Athens, Ohio 45701, United States.
authors.forename: Yun
authors.initials: Y
authors.lastname: Zhang
authors.forename: Weidong
authors.initials: W
authors.lastname: Cui
authors.forename: Hao
authors.initials: H
authors.lastname: Zhang
authors.forename: Howard D
authors.initials: HD
authors.lastname: Dewald
authors.forename: Hao
authors.initials: H
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004220:Disulfides / Q000737:chemistry*; D004563:Electrochemistry; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry; D010084:Oxidation-Reduction; D011506:Proteins / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D004220:Disulfides; D011506:Proteins; D009113:Muramidase