Fast and Highly Efficient Affinity Enrichment of Azide-A-DSBSO Cross-Linked Peptides

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, crosslinked 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, acidcleavable 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.

Reliable identification of protein-protein interactions by crosslinking mass spectrometry

“…There have been several recent advances in enrichment and detection of crosslinked peptides by Crosslinking MS that suggest it will soon be able to map large portions of the cellular interactome in a single experiment 3 , 13 , 25 . This will open the door to detecting changes in these interactomes in different cellular states by quantification of the abundances of the detected crosslinks 2 , 26 .…”

Cleavable Cross-Linkers and Mass Spectrometry for the Ultimate Task of Profiling Protein–Protein Interaction Networks in Vivo

“…In particular, for nonexperts, it is still hard to know which enrichment technique, analysis software, and software settings to choose. However, recent efforts regarding selective enrichment strategies (e.g., refs ( 44 and 45 )), the capture of lower abundant proteins, 49 the harmonization of standards, 126 clever FDR control, 86 , 88 as well as the improved sensitivity of mass spectrometers and increased computational power show that within the next 2–5 years, scientists will truly be able to dig deeper than ever before in the interactome of cells, organelles, or tissues. As mentioned in the Applications and Bottlenecks section, the combination with FAIMS 79 or ion mobility (caps-PASEF 46 ) as an additional separation dimension will further alleviate issues in the dynamic range and therefore boost sensitivity toward cross-link detection in future studies.…”

“…That is, we circumvented the need for biotin as an intermediate step. 45 Of note, affinity-based enrichment methods usually do not differentiate between monolinked peptides (= type 0 or dead-end cross-links; one side of the cross-linker is hydrolyzed and not connected to any amino acid) and cross-linked (= type 2) peptides. Because those monolinked peptides are formed in excess, the more informative cross-linked peptides are still a minority within the total peptide population.…”

“…Recently, we additionally developed a streamlined enrichment workflow with improved performance by directly coupling the linker to dibenzocyclooctyne (DBCO)-functionalized beads. 45 …”

“…Furthermore, low concentrations of DMSO (e.g., 5%) can be added to the sample tube prior to injection to minimize sample losses due to hydrophobic cross-linked peptides adhering onto the plastic material of reaction tubes. 24 , 45 …”