Kandioller, W. | bioRxiv Science

bioRxiv2026

A DIA-based quantitative crosslinking mass spectrometryframework for dynamic structural proteomics

Proteins undergo dynamic conformational rearrangements and interactions that are central to their biological functions. Quantitative crosslinking mass spectrometry enables the analysis of those dynamics and molecular interactions, but rigorous confidence assessment and empirical validation strategies for quantitative measurements remain underdeveloped, and integrated analysis of complementary structural features, including monolinks and protein-RNA adducts, remains limited. Here we present a data-independent acquisition (DIA)-based framework for quantitative crosslinking mass spectrometry (DIA-QCLMS) that combines optimized acquisition strategies, crosslink-aware spectral libraries and empirical false-discovery-rate (FDR) validation. The workflow supports crosslinks, monolinks and protein-RNA adducts and integrates spectral-library generation from two crosslinking search engines (xiSEARCH and MS Annika). To enable robust confidence assessment in DIA data, we developed a four-state target-decoy spectral library strategy that explicitly models target-target, target-decoy, decoy-target and decoy-decoy crosslink spectra. Experimental entrapment datasets enabled empirical validation of confidence estimation, whereas benchmarking with PhoX-crosslinked Cas9 demonstrated improved quantitative completeness and reproducibility compared with data-dependent acquisition. Application of the workflow to the ATP-dependent RNA helicase UAP56 (DDX39B) resolved ligand-dependent changes in intramolecular restraints, residue accessibility and candidate RNA-contact sites associated with the transition from an open to a clamped conformation. These results establish DIA-QCLMS as a scalable framework for quantitative structural proteomics and provide practical strategies for confidence-controlled analysis of dynamic protein interactions and conformational states.

biochemistry↗

bioRxiv2019

Fast and highly efficient affinity enrichment of Azide-A-DSBSO cross-linked peptides.

Cross-linking mass spectrometry is an increasingly used, powerful technique to study protein-protein interactions or to provide structural information. Due to sub-stochiometric reaction efficiencies, cross-linked peptides are usually low abundant. 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(R) 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 maintained high at ~100. The estimated enrichment efficiency was improved by factor 4 -5 (based on XL numbers) compared to enrichment via biotin and streptavidin. We were still able to detect cross-links from 0.25 g cross-linked E. coli ribosome in a background of 100 g 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 map protein-protein interactions, investigate protein architectures in more depth and help to understand complex biological processes.

biochemistry↗