Fast and highly efficient affinity enrichment of Azide-A-DSBSO cross-linked peptides

Abstract

ABSTRACTCross-linking mass spectrometry is an increasingly used, powerful technique to study protein-protein interactions or to provide structural information. Due to sub-stochiometric reaction efficiencies, cross-linked peptides are usually low abundant. This results in challenging data evaluation and the need for an effective enrichment.Here we describe an improved, easy to implement, one-step method to enrich azide-tagged, acid-cleavable disuccinimidyl bis-sulfoxide (DSBSO) cross-linked peptides using dibenzocyclooctyne (DBCO) coupled Sepharose® beads. We probed this method using recombinant Cas9 and E. coli ribosome. For Cas9, the number of detectable cross-links was increased from ~100 before enrichment to 580 cross-links after enrichment. To mimic a cellular lysate, E. coli ribosome was spiked into a tryptic HEK background at a ratio of 1:2 – 1:100. The number of detectable unique cross-links maintained high at ~100. The estimated enrichment efficiency was improved by factor 4 −5 (based on XL numbers) compared to enrichment via biotin and streptavidin. We were still able to detect cross-links from 0.25 μg cross-linked E. coli ribosome in a background of 100 μg tryptic HEK peptides, indicating a high enrichment sensitivity. In contrast to conventional enrichment techniques, like SEC, the time needed for preparation and MS measurement is significantly reduced.This robust, fast and selective enrichment method for azide-tagged linkers will contribute to map protein-protein interactions, investigate protein architectures in more depth and help to understand complex biological processes.