ArticlebioRxiv : the preprint server for biology2026
Evaluation and application of chemical decrosslinking in the context of histopathological spatial proteomics.
Article in bioRxiv : the preprint server for biology, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
Laser capture microdissection (LCM) - based spatial mass spectrometry proteomics is a rapidly emerging technique with strong potential for use in formalin-fixed, paraffin-embedded (FFPE) tissues. Several sample-preparation methods have been developed to decrosslink FFPE proteins for spatial proteomics; however, residual crosslinks often remain, and depth can remain impaired relative to fresh frozen tissue samples. To increase proteome coverage in spatially resolved LCM-FFPE samples, we investigated a panel of chemical compounds with the potential to catalyze the decrosslinking of nucleophilic functional groups on proteins. Systematic screening and optimization of temperature, incubation time, and reagent concentration led to the identification of 3,4-diaminobenzoic acid as an effective agent for improving proteome coverage in FFPE pancreatic tissue. This compound could boost precursor identifications by more than 10% at both reduced (70 °C) and high (90 °C) temperatures. Application of this chemical-decrosslinking strategy to a pancreatic ductal adenocarcinoma tissue section enabled the identification of numerous cell-type-enriched proteins with clinical and therapeutic relevance. Taken together, our findings show that chemical decrosslinking can increase proteome coverage in FFPE tissues, thereby advancing our understanding of tissue microenvironments in physiological and pathological contexts.
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