ETD-Cleavable Linker for Confident Cross-linked Peptide Identifications.

Abstract

Peptide cross-links formed using the homobifunctional-linker diethyl suberthioimidate (DEST) are shown to be ETD-cleavable. DEST has a spacer arm consisting of a 6-carbon alkyl chain and it cleaves at the amidino groups created upon reaction with primary amines. In ETD MS2 spectra, DEST cross-links can be recognized based on mass pairs consisting of peptide-NH2• and peptide+linker+NH3 ions, and backbone cleavages are more equally distributed over the two constituent peptides compared with collisional activation. Dead ends that are often challenging to distinguish from cross-links are diagnosed by intense reporter ions. ETD mass pairs can be used in MS3 experiments to confirm cross-link identifications. These features provide a simple but reliable approach to identify cross-links that should facilitate studies of protein complexes.