Gas-phase Foot-printing of Nucleic Acids and Protein-Nucleic Acids by EDD and IRMPD

摘要

Fourier transform ion cyclotron resonance (FTICR) mass spectrometry arguably enjoys the largest selection of gas-phase activation techniques available for the tandem mass spectrometric (MS/MS) analysis of gaseous multiply-charged biomolecules. The list comprises ergodic as well as non-ergodic methods, which include sustained off-resonance irradiation collision induced dissociation (SORI-CID) and infrared multi-photon dissociation (IRMPD) among the former, and electron capture dissociation (ECD), electron transfer dissociation (ETD), and electron detachment dissociation (EDD) among the latter. These activations methods have been employed individually or in combination to carry out the characterization of complex biomolecules, including proteins, nucleic acids, polysaccharides, and lipids. The realization that non-ergodic methods are capable of inducing the fragmentation of covalent backbones without producing dissociation of non-covalent assemblies has prompted our investigation of the complexes formed by the nucleocapsid (NC) protein of HIV-1 and its cognate stemloop domains (SL1 through 4) of the genome packaging signal (PSI-RNA). While the position of the NC binding sites onto the individual stemloop has been unambiguously identified, their location onto the full-length PSI-RNA has not. For this reason, we have investigated the possibility of employing FTICR to characterize the RNA structures that are in direct contact with the viral protein. A better understanding of the specific interactions between NC and PSI-RNA is necessary to elucidate the biological functions mediated by this highly conserved region of viral genome, which could lead to the development of novel therapeutic strategies for alleviating the daunting problem of drug-resistance.