Publication date: Mar 23, 2025

The dual-cleavable nature of the cross-linking technology (DUCCT) enhances the reliable identification of cross-linked peptides via mass spectrometry. The DUCCT approach uses a cross-linking agent that can be selectively cleaved by two different tandem mass spectrometry techniques: collision-induced dissociation (CID) and electron transfer dissociation (ETD). This results in distinct signatures in two independent mass spectra for the same cross-linked precursor, leading to unambiguous identification and the validation of the spectra. In this study, we expanded the application of the DUCCT cross-linker to evaluate the binding domains of a specific cat dander allergen, Fel d 1, which exists as the Fel d 1 A and B protein complex, and a viral spike protein from SARS-CoV-2, which invades host cells. To assess the cross-linked products obtained by DUCCT, we utilized a software tool called Cleave-XL, which effectively identified cross-linked sites using data from CID and ETD. Dual cleavable cross-linking studies identified cross-linked peptides in these complexes, which have been reported in bioinformatics analysis and proposed for immunotherapy using synthetic peptides. A benchmark study was also conducted using a commercial cross-linker disuccinimidyl suberate (DSS). Overall, we expect that DUCCT cross-linking technology will greatly facilitate the rapid screening of binding interfaces, thereby advancing structural biology and cell signaling investigations.

Semantics