title: Liquid chromatography and mass spectrometry with post-column partial reduction for the analysis of native and scrambled disulfide bonds.
abstract: A method capable of detecting both native and scrambled disulfide bonds has been established. Nonreduced protein digests were separated using a reversed-phase C18 column, partially reduced by post-column addition of a reducing reagent, and then analyzed by mass spectrometry. Disulfide bond linkage was established by matching the retention times of cysteine-containing peptides and confirmed by the detection of the molecular weight of the disulfide-linked peptides. The application of this method was demonstrated by determination of the disulfide bond structures of an immunoglobulin G1 (IgG1) molecule and lysozyme and by the detection of four scrambled disulfide bonds in the IgG1 molecule.
authors: Merck Research Laboratories, Union, NJ 07083, USA.
authors: Xiaojuan
authors: X
authors: Li
authors: Wei
authors: W
authors: Xu
authors: Brittany
authors: B
authors: Paporello
authors: Daisy
authors: D
authors: Richardson
authors: Hongcheng
authors: H
authors: Liu
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D004220:Disulfides / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D010084:Oxidation-Reduction
chemical_list: D004220:Disulfides
keywords: Antibody; Disulfide bonds; Liquid chromatography and mass spectrometry; Lysozyme; Tris(2-carboxyethyl)phosphine hydrochloride