Differential Enzymatic ¹⁶O/¹⁸O Labeling for the Detection of Cross-Linked Nucleic Acid-Protein Heteroconjugates

Cross-linking of nucleic acids to proteins in combination with mass spectrometry permits the precise identification of interacting residues between nucleic acid-protein complexes. However, the mass spectrometric identification and characterization of cross-linked nucleic acid-protein heteroconjugates within a complex sample is challenging. Here we establish a novel enzymatic differential ¹⁶O/¹⁸O-labeling approach, which uniquely labels heteroconjugates. We have developed an automated data analysis workflow based on OpenMS for the identification of differentially isotopically labeled heteroconjugates against a complex background. We validated our method using synthetic model DNA oligonucleotide-peptide heteroconjugates, which were subjected to the labeling reaction and analyzed by high-resolution FTICR mass spectrometry.