Covalent protein-nucleic acid conjugates form an original class of compounds that occur in nature or can be generated in vitro through cross-linking to investigate domains involved in protein/nucleic acid interactions. Their mass spectrometry fragmentation patterns are poorly characterized. We have used electrospray-ionization mass spectrometry (ESI-MS) combined with collision-induced dissociation (CID) to characterize microcin C7-C51, an antimicrobial nucleotide peptide that targets aspartyl-tRNA synthetase and inhibits translation. The fragments of microcin C7-C51 were analyzed in positive- and negative-ion modes and compared with those of the corresponding unmodified heptapeptide and to the derived aspartyl-adenylate. The positive- and negative-ion mode fragments of microcin C7-C51 provided information on both the nucleotide and peptide moieties. Accurate mass measurement obtained using an LTQ Orbitrap instrument was a key factor for a comprehensive interpretation of the fragments. The experimental results obtained permitted the proposal of stepwise fragmentation pathways involving ion-dipole complexes. The data provide a better understanding of nucleotide peptide fragmentation in the gas phase.