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Site-Specific Profiling of RNA-Binding Proteins Enabled by Isotopic Signature-Enhanced Mass Spectrometry.

Yicheng Liu·Guogeng Jia·Chengxi Li·Tianyu Feng·Weidi Xiao·Chu Wang
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Abstract

RNA-binding proteins (RBPs) ubiquitously regulate RNA throughout their lifespan, being extensively involved in cellular metabolism and genetic evolution. Therefore, comprehensive identification of the RNA-protein interactions, especially their interfaces with site-specific resolution, is significant to elucidate the intricate biological activities governed by RNA. Nevertheless, it remains challenging for data-dependent acquisition (DDA)-based proteomics to identify the RNA-cross-linked peptides in depth due to the low abundance and negative charge of modified peptides. To address such limitations, we developed an innovative method named "isoRIC" for profiling RNA-binding proteomes with site-specific resolution, which combines the metabolic labeling of isotopic nucleotides for photo-cross-linking of RNA-binding proteins and the real-time targeted LC-MS/MS analysis of RNA-cross-linked peptides. This method shows a dramatic improvement of sensitivity in identifying RNA-cross-linked peptides with low abundance as compared to the DDA-based proteomic approaches, enabling the discovery of novel RNA-binding proteins and precise mapping of RNA-protein binding interfaces at single amino acid resolution. We applied isoRIC in the context of pathogenic mutations and post-translational modifications to highlight the critical role of RNA-binding sites in modulating the RNA-binding ability.

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RNA-Binding ProteinsTandem Mass SpectrometryHumansProteomicsRNAPeptidesIsotope LabelingCross-Linking ReagentsJournal ArticleResearch Support, Non-U.S. Gov't

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Yicheng Liu, Guogeng Jia, Chengxi Li, Tianyu Feng, Weidi Xiao, Chu Wang. 2025-10-02. Site-Specific Profiling of RNA-Binding Proteins Enabled by Isotopic Signature-Enhanced Mass Spectrometry.. https://doi.org/10.1021/jacs.5c11224

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