Merck Research Laboratories, Union, NJ 07083, USA.
Anal Biochem. 2013 Aug 15;439(2):184-6. doi: 10.1016/j.ab.2013.04.021. Epub 2013 Apr 30.
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.
已经建立了一种能够同时检测天然和错配二硫键的方法。非还原蛋白消化物使用反相 C18 柱进行分离，通过柱后添加还原试剂部分还原，然后通过质谱分析。通过匹配含半胱氨酸肽的保留时间来确定二硫键的连接，并通过检测二硫键连接肽的分子量来确认。通过测定免疫球蛋白 G1（IgG1）分子和溶菌酶的二硫键结构以及检测 IgG1 分子中的四个错配二硫键，证明了该方法的应用。
