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.