Fig. 1.

Crosslinking reagent and experimental workflow. A, CBDPS molecular diagram showing NH₂ reactive groups, CID-cleavable bonds, isotopic-coding positions, and biotin affinity-tag. Short and long crosslinker-cleavage portions are also indicated. B, General experimental workflow for in-organello crosslinking. The affinity enrichment, LCMS analysis, and data analysis steps all take advantage of the various features of the CBPDS crosslinker shown in (A). Specifically, the biotin-tag of the crosslinker allows the enrichment of crosslinker-modified peptides prior to MS analysis, the isotopic-labeling allows the use of targeted MS acquisition methods, and the mass spectral; features relating to both the isotopic-labeling and crosslinker-cleavage result in improved confidence in peptide-spectrum match identifications.