Massive deciphering of the tissue nucleic acid-binding proteome via affinity chromatography integrated with data-independent acquisition-based proteomics.

The development of inflammatory bowel disease is driven by transcriptional and epigenetic regulators of the nucleic acid-binding proteome (NABPome). However, comprehensive analysis of NABPome composition and dynamics remains challenging due to the lack of an in-depth proteomics strategy. Here, we developed an NABP-DIA methodology that integrates NABPome capture with data-independent acquisition (DIA)-based proteomics. Using NABP-DIA with an experimentally generated spectral library, we achieved a 2.1-fold increase in NABP identification, with improved quantification accuracy and specificity. Applying this method to a colitis model, we identified 118 and 25 differentially expressed NABPs in the mouse spleen and thymus, respectively. Among them, spleen proteins associated with histone methylation were significantly elevated in the colitis group. Further experiments demonstrated that inhibition of H3K27me3 histone methylation reduced the release of inflammatory factors, suggesting that epigenetic modulation may represent a promising therapeutic approach for colitis.