2025/09/07 by Shuyao Sha, Bernhard Küster, Jakob Trendel · 1 voice
Biochemistry, Genetics and Molecular Biology · #Isolation (microbiology) #Mass spectrometry #Molecular biophysics #Nucleic acid #Peptide #Proteome #RNA #RNA and protein synthesis mechanisms #Transfer RNA
paper · pdf · doi:10.1101/2025.09.05.674461
openalex publication_date 2025/09/07 · openalex created_date 2025/10/10 · openalex updated_date 2026/08/01
Abstract Photo-crosslinking mass spectrometry enables the identification of protein-RNA interactions in living cells, pinpointing interaction interfaces at single-amino acid resolution. However, current isolation procedures for peptide-RNA crosslinks eliminate the RNA moiety, prohibiting sequencing of the RNA alongside the crosslinked peptide. Here, we introduce peptide-RNA crosslink isolation for sequencing by mass spectrometry or pepR-MS, a method that enriches peptide-RNA crosslinks with RNA chains of tunable length. Applied to breast cancer cells, pepR-MS identifies over 21,000 unique crosslinks at 4,757 crosslinking sites in 744 proteins. Employing different nucleases, we capture crosslinks with RNA moieties up to six nucleotides, revealing RNA crosslinking preferences at domain and subdomain resolution. Finally, we demonstrate mass spectrometry-based sequential sequencing of both peptide and RNA from the same crosslink, providing a starting point for the analysis of long-chain peptide-RNA crosslinks that map interaction interfaces across the proteome and transcriptome.