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

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.