Liquid chromatography and mass spectrometry with post-column partial reduction for the analysis of native and scrambled disulfide bonds.

TitleLiquid chromatography and mass spectrometry with post-column partial reduction for the analysis of native and scrambled disulfide bonds.
Publication TypeJournal Article
Year of Publication2013
AuthorsLi X, Xu W, Paporello B, Richardson D, Liu H
JournalAnalytical biochemistry
Volume439
Issue2
Pagination184-6
Date Published2013 Aug 15
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.

DOI10.1073/pnas.1301766110
Alternate JournalAnal. Biochem.