Recently we (RA, AL, FA) introduced the CLIR-MS technique which identifies RNA-protein cross-links on both protein and RNA (Fig. ). The original CLIR-MS protocol employs RNAs with contiguous regions of differentially isotope-labelled nucleotides in the cross-linking step. After partial RNA and protein digestion, peptide-oligonucleotide conjugates are identified as matched signal pairs in the precursor ion mass-spectrum. This facilitates localisation of the cross-linked nucleotide to the labelled RNA segment. One drawback of the orignial implementation of CLIR-MS is the inherent requirement for enzymatic C/ 15 N-labelled RNA synthesis (i.e. in vitro transcription). This limits the minimal length and the number and positions of labelled segments that can be included within an RNA of interest. A second limitation is the nuclease digestion step, which typically results in short oligonucleotides (i.e., 1-4 nt). This means that the specific base involved in the cross-link usually cannot be called with confidence, although longer overlapping partial sequences facilitate the localization of the interacting regions on the RNA. In this study, we sought chemical solutions to both of these problems. First, we switched from RNase digestion to alkaline hydrolysis of RNA (Fig. ), while exercising care not to degrade the FOXRRM. Hence, the mass analysis of the product mixtures detected a much greater fraction of mono-nucleotide adducts, allowing us to define ambiguously nucleotides that are crosslinked (Fig. ). Second, we employed C-labelled phosphoramidites during solid-phase synthesis of RNAs to incorporate C-labelled nucleotides site-specifically (Fig. ).