title: Identification of reduction-susceptible disulfide bonds in transferrin by differential alkylation using O(16)/O(18) labeled iodoacetic acid.
abstract: Stabilization of native three-dimensional structure has been considered for decades to be the main function of disulfide bonds in proteins. More recently, it was becoming increasingly clear that in addition to this static role, disulfide bonds are also important for many other aspects of protein behavior, such as regulating protein function in a redox-sensitive fashion. Dynamic disulfide bonds can be taken advantage of as candidate anchor sites for site-specific modification (such as PEGylation of conjugation to a drug molecule), but are also frequently implicated in protein aggregation (through disulfide bond scrambling leading to formation of intermolecular covalent linkages). A common feature of all these labile disulfide bonds is their high susceptibility to reduction, as they need to be selectively regulated by either specific local redox conditions in vivo or well-controlled experimental conditions in vitro. The ability to identify labile disulfide bonds in a cysteine-rich protein can be extremely beneficial for a variety of tasks ranging from understanding the mechanistic aspects of protein function to identification of troublesome "hot spots" in biopharmaceutical products. Herein, we describe a mass spectrometry (MS)-based method for reliable identification of labile disulfide bonds, which consists of limited reduction, differential alkylation with an O(18)-labeled reagent, and LC-MS/MS analysis. Application of this method to a cysteine-rich protein transferrin allows the majority of its native disulfide bonds to be measured for their reduction susceptibility, which appears to reflect both solvent accessibility and bond strain energy.
authors.affiliation: Department of Chemistry, University of Massachusetts-Amherst, 140 Thatcher Drive, LSL N369, Amherst, MA, 01003, USA.
authors.forename: Shunhai
authors.initials: S
authors.lastname: Wang
authors.forename: Igor A
authors.initials: IA
authors.lastname: Kaltashov
mesh_terms: D000478:Alkylation / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000032:analysis* / Q000737:chemistry; D003553:Cystine / Q000032:analysis* / Q000737:chemistry; D004229:Dithiothreitol / Q000737:chemistry / Q000494:pharmacology; D005240:Feasibility Studies; D006801:Humans; D007202:Indicators and Reagents / Q000737:chemistry*; D019807:Iodoacetic Acid / Q000737:chemistry*; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010103:Oxygen Isotopes; D011487:Protein Conformation / Q000187:drug effects; D055550:Protein Stability / Q000187:drug effects; D019163:Reducing Agents / Q000737:chemistry* / Q000494:pharmacology; D053719:Tandem Mass Spectrometry; D014168:Transferrin / Q000737:chemistry*
chemical_list: D007202:Indicators and Reagents; D010103:Oxygen Isotopes; D019163:Reducing Agents; D014168:Transferrin; D003553:Cystine; D003545:Cysteine; D004229:Dithiothreitol; D019807:Iodoacetic Acid