Comprehensive identification of protein disulfide bonds with pepsin/trypsin digestion, Orbitrap HCD and Spectrum Identification Machine.

ABSTRACT: Disulfide bonds (SS) are post-translational modifications important for the proper folding and stabilization of many cellular proteins with therapeutic uses, including antibodies and other biologics. With budding advances of biologics and biosimilars, there is a mounting need for a robust method for accurate identification of SS. Even though several mass spectrometry methods have emerged for this task, their practical use rests on the broad effectiveness of both sample preparation methods and bioinformatics tools. Here we present a new protocol tailored toward mapping SS; it uses readily available reagents, instruments, and software. For sample preparation, a 4-h pepsin digestion at pH 1.3 followed by an overnight trypsin digestion at pH 6.5 can maximize the release of SS-containing peptid

SUBMITTER: Cui C 

PROVIDER: S-EPMC6414265 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature