Nucleic acids (NA) form an essential class of macromolecules for storage, transmission and expression of the genetic information of a cell. While deoxyribonucleic acid (DNA) encodes the information cells need to make proteins, ribonucleic acid (RNA), conformationally more variable, play multiple cellular roles, among which protein synthesis is the main one. Protein-nucleic acids (P-NA) interactions are thus involved in many biological processes. For instance, protein-RNA interactions form ribonucleoprotein complexes that mediate translation, intracellular transport, stress response, virulence of pathogens and are involved in different diseases (cancers, neurodegenerative or infectious diseases). Protein-DNA interactions are key for the functionality (replication, transcription, repair and recombination) and stability of the genome. The identification of P-NA interaction sites at the residue level (nucleotide and amino acid) is therefore of great biological and clinical significance. However, while high throughput methods for individual DNA/RNA/proteins identification are widespread (genomics/transcriptomics/proteomics), there are only few approaches that afford precise and systematic identification of RNA/DNA interaction sites at the single residue level. A variety of biophysical techniques has been developed for structural characterization of P-NA interactions: i) atomic-resolution techniques (crystallography, NMR or single-particle electron microscopy) that face typical challenges like sample requirements, size limitations and/or unfavourable crystallization/solubility ; ii) lower resolution techniques (e.g. small-angle X-ray scattering) that still fall short in elucidating P-NA conformational changes at the single residue level. Structural mass spectrometry (MS) approaches, including native MS, ion mobility and labelling (H/D exchange) or cross-linking MS (XL-MS) tools, have emerged as key techniques for the in vitro characterization of P-NA complexes, providing information on stoichiometries, dynamics and regions of close proximities. XL-MS appears as the most potent method to gain information on both protein and nucleotide sides. Photochemical XL using UV-light (UV-XL-MS) is the most popular method to study P-NA complexes in vitro, generating nearly ‘zero-length’ cross-linked peptide-oligonucleotide conjugates. However, while XL-MS has gained popularity in protein-protein interaction studies, it is still scarcely used for providing information on P-NA complexes, mostly because of the complexity of both analytical and data processing workflows. Indeed, all steps of the P-NA XL-MS workflow present limitations, explaining why XL-MS for P-NA stays the prerogative of a handful of experts worldwide. The aim of the NAProt-XLMS project is to provide new chemical cross-linking reagents and analytical MS-based proteomic workflows for the study of P-NA complexes through the : 1) synthesis of new chemical XL reagents based on the repurposing of drugs used in chemotherapy as P-NA cross-linking multifunctional probes; 2) development of sensitive, robust analytical LC-MS/MS proteomic workflows for in vitro and in vivo P-NA cross-linking; and 3) application of the new P-NA XL-MS methods on two well-characterized systems for proof-of-concept (nuclear receptor/DNA and ribonucleoproteins/RNA complexes). Altogether, the NAProt-XLMS project will provide new chemical tools along with improved proteomic analytics and bioinformatics for more accurate and straightforward characterization of P-NA machineries. This project will thus benefit to the whole community of biologists by providing tools for a more exhaustive and comprehensive characterization of the role of P-NA interaction in biological processes, opening new perspectives for large scale system wide P-NA interaction mapping.