biostudies-literatureWelp LMGermany's Excellence StrategyMinistry of Science, Research and Arts Baden-WürttembergDeutsche ForschungsgemeinschaftInternational Max Planck Research School for Genome SciencesEuropean Research CouncilAlex's Lemonade Foundation Crazy 8 InitiativeSmith Family FoundationMarie Skłodowska-CurieMax Planck SocietyFederal Ministry of Education and ResearchNIGMS NIH HHSEuropean Union's Horizon 2020NIH HHSgkaf727https://www.ebi.ac.uk/biostudies/studies/S-EPMC12359043biostudies-literatureUnknown53(15)Ultraviolet (UV) crosslinking with mass spectrometry (XL-MS) has been established for identifying RNA- and DNA-binding proteins along with their domains and amino acids involved. Here, we explore chemical XL-MS for RNA-protein, DNA-protein, and nucleotide-protein complexes in vitro and in vivo. We introduce a specialized nucleotide-protein-crosslink search engine, NuXL, for robust and fast identification of such crosslinks at amino acid resolution. Chemical XL-MS complements UV XL-MS by generating different crosslink species, increasing crosslinked protein yields in vivo almost four-fold, and thus it expands the structural information accessible via XL-MS. Our workflow facilitates integrative structural modelling of nucleic acid-protein complexes and adds spatial information to the describNucleic acids researchChemical crosslinking extends and complements UV crosslinking in analysis of RNA/DNA nucleic acid-protein interaction sites by mass spectrometry.PMC12359043SFB1565 P12SFB1565SFB1565 P152067/1-390 729 940DP2 GM146254SFB1565 P18956148SFB860945528ERC-StG 945528 IMAPDP2GM146254469281184FOR1193Siraj Ade A P Schwarzer ACWulf ABohnsack KEFicner RUrlaub HSachsenberg TWohlgemuth IInfante DABohnsack MTLiepe JDickmanns AVos SMWelp LMViner RHorokhovskyi YSchmitzova JNetz EDybkov ODelanghe BPasen MRaabe MFritzemeier KJohannsson SWalshe JLChernev ACramer PHe YMoshkovskii SNeumann POberbeckmann EKohlbacher OGothert HfalseChemical crosslinking extends and complements UV crosslinking in analysis of RNA/DNA nucleic acid-protein interaction sites by mass spectrometry.Ultraviolet (UV) crosslinking with mass spectrometry (XL-MS) has been established for identifying RNA- and DNA-binding proteins along with their domains and amino acids involved. Here, we explore chemical XL-MS for RNA-protein, DNA-protein, and nucleotide-protein complexes in vitro and in vivo. We introduce a specialized nucleotide-protein-crosslink search engine, NuXL, for robust and fast identification of such crosslinks at amino acid resolution. Chemical XL-MS complements UV XL-MS by generating different crosslink species, increasing crosslinked protein yields in vivo almost four-fold, and thus it expands the structural information accessible via XL-MS. Our workflow facilitates integrative structural modelling of nucleic acid-protein complexes and adds spatial information to the describ2025-01-01T00:00:00Z2025 Aug2026-04-08T15:34:48.344Z2026-04-08T05:05:09.711ZS-EPMC123590434082381410.1093/nar/gkaf727