Recently, we introduced the CLIR-MS method . This technique employs isotope-labelled RNAs to resolve amino acid/ribonucleotide cross-links in a single protocol, whereby segments of labelled RNA are produced by in vitro transcription prior to ligation-assembly into a full length RNA. In this new study, we have broadened the application of CLIR-MS through the use of chemically synthesized Clabelled RNAs. This enables site-specific incorporation of labelled nucleotides into the RNA. After irradiation, sample work-up and controlled degradation to nucleotide-peptide adducts, the locations of cross-linked nucleotides in the RNA sequence are pinpointed site-specifically. We demonstrated this methodological advance with a study of the interaction of the RRM domain of the RBFOX family bound to its consensus binding element (U)GCAUGU, for which we have previously determined an NMR structure and studied cross-linking (Götze et al. submitted).