Abstract
Cross-linking mass spectrometry is an increasingly used, powerful technique to study protein–protein interactions or to provide structural information. Due to substochiometric reaction efficiencies, cross-linked peptides are usually low abundance. 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 was maintained high at ∼100. The estimated enrichment efficiency was improved by a factor of 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 ribosomes 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 mapping protein–protein interactions, investigating protein architectures in more depth, and helping to understand complex biological processes.
Highlights
Cross-linking mass spectrometry (XL-MS) has emerged as a widely used tool for studying protein−protein interactions and to obtain structural information on protein complexes
Since cross-linked peptides are on average larger and higher charged, compared to linear peptides, enrichment is often done via size exclusion (SEC)[7−10] or strong cation exchange chromatography (SCX),[11−13] respectively
Further optimization and improvements must be done in future experiments, we believe that the published protocol will be of great value to other groups aiming to enrich cross-linked samples
Summary
Cross-linking mass spectrometry (XL-MS) has emerged as a widely used tool for studying protein−protein interactions and to obtain structural information on protein complexes. In the field of in vivo cross-linking, the linker molecule has to permeate the cell membrane, and until it has reached reactive amino acid residues in a close enough proximity, it is already partly hydrolyzed. This leads to low substochiometric reaction efficiencies.[4−6] In conclusion, enrichment of cross-linked peptides is crucial. Cross-linkers with an affinity tag are used, aiming to get a deeper proteome coverage.[6,15−17] As such, e.g., biotin is widely used as affinity tag, due to an effectively working enrichment via streptavidin and the commercial availability of the respective tools.[16−19]
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