title: Methodology for determining disulfide linkage patterns of closely spaced cysteine residues.
abstract: We report the development and application of a method for determining bonding patterns in disulfide-linked peptides containing closely spaced cysteine residues. Through the utility of classic N-terminal sequencing chemistry coupled with facile liquid chromatography and mass spectrometric analysis of the cleavage products, we report the ability to demonstrate unambiguous assignment of paired cysteine residues, using human insulin as a model protein. The conditions of the technique were selected and optimized to maintain disulfide integrity. In a forthcoming article, we will present the results of this method as applied to the complete elucidation of linkages in disulfide variants of a therapeutic monoclonal antibody of the IgG2 subclass.
authors.affiliation: Analytical Sciences, Amgen, Inc., 4000 Nelson Road, Longmont, Colorado 80503, USA.
authors.forename: Bing
authors.initials: B
authors.lastname: Zhang
authors.forename: Steven L
authors.initials: SL
authors.lastname: Cockrill
mesh_terms: D000595:Amino Acid Sequence; D002853:Chromatography, Liquid / Q000191:economics / Q000379:methods*; D003545:Cysteine / Q000032:analysis*; D004220:Disulfides / Q000032:analysis*; D006801:Humans; D007328:Insulin / Q000032:analysis*; D010455:Peptides / Q000032:analysis; D021241:Spectrometry, Mass, Electrospray Ionization / Q000191:economics / Q000379:methods*
chemical_list: D004220:Disulfides; D007328:Insulin; D010455:Peptides; D003545:Cysteine