Peptide-RNA photocrosslinks with tunable RNA chain map RNA interfaces proteome-wide

Abstract

Photocrosslinking mass spectrometry (MS) can map protein-RNA interfaces in living cells, but current isolation procedures for peptide-RNA crosslinks eliminate most RNA and therefore cannot identify the crosslinked RNA sequence. We introduce pepR-MS, a method that enriches peptide-RNA crosslinks with RNA chains of tunable length. To maximize proteomic depth, we applied pepR-MS to generate highly truncated RNA adducts, identifying over 34,000 unique crosslinks in 589 proteins of breast cancer cells and pinpointing RNA contacts in hundreds of structured domains and intrinsically disordered regions. By modulating nuclease digestion, longer RNA chains of up to five nucleotides were retained and sequenced alongside the crosslinked peptide using a specialized MS strategy. For RNA sequence assignment, we further introduce the computational tool SALT, which identified peptide-RNA sequence pairs for over 1,600 crosslinks in a single LC-MS run. Together, pepR-SALT provides a framework for mapping protein-RNA interfaces across the proteome and transcriptome at single-amino acid and nucleotide resolution.