A journey towards developing a new cleavable crosslinker reagent for in-cell crosslinking

ABSTRACT: Cross-linking mass spectrometry (XL-MS) is a powerful technology that recently emerged as an essential complementary tool for elucidating protein structures and mapping interactions within a protein network. Crosslinkers which are amenable to post-linking backbone cleavage simplify peptide identification, aid in 3D structure determination and enable system-wide studies of protein-protein interactions (PPIs) in cellular environments. However, state-of-the-art cleavable linkers are fraught with practical limitations, including incomplete and harsh fragmentation operations. We herein introduce DiSPASO as a lysine-selective, MS-cleavable cross-linker with an alkynyl handle for affinity enrichment. DiSPASO was designed and developed for efficient cell membrane permeability and cross-linking while securing low cellular perturbation. We tested DiSPASO employing three different copper-based enrichment strategies using model systems with increasing complexity (Cas9-Halo, purified ribosomes, live cells). Fluorescence microscopy in-cell cross-linking experiments revealed a rapid uptake of DiSPASO into HEK 293 cells within 5 minutes. While DiSPASO represents progress in cellular PPI analysis, its limitations require a careful strategy, highlighting the complexity of developing effective XL-MS tools and the importance of continuous innovation in accurately mapping PPI networks within dynamic cellular environments.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell

SUBMITTER: Fränze Müller  

LAB HEAD: Karl Mechtler

PROVIDER: PXD056091 | Pride | 2026-10-27

REPOSITORIES: Pride