Measuring dissociation constants of RNA and aminoglycoside antibiotics by electrospray ionization mass spectrometry.

Electrospray ionization mass spectrometry (ESI-MS) has been used to determine the dissociation constants (K(D)s) and binding stoichiometry for tobramycin and paromomycin with a 27-nucleotide RNA construct representing the A-site of the 16S ribosomal RNA. K(D) values determined by holding the ligand concentration fixed are compared with K(D) values derived by holding the RNA target concentration fixed. Additionally, the effect of solution conditions such as the amount of organic solvent present and the amount of salt present in the solution on the K(D) measurement is investigated. It is shown that the preferred method for determining dissociation constants using ESI-MS is holding the RNA target concentration fixed below the expected K(D) and titrating the ligand. K(D) measurements should also be carried out at as high as possible salt concentration to minimize nonspecific binding due primarily to electrostatic interactions. For tobramycin, two nonequivalent binding sites were found with K(D1) = 352 nM and K(D2) = 9 microM. For paromomycin, there is only one binding site with K(D) = 52 nM.

Characterization of multipole storage assisted dissociation: implications for electrospray ionization mass spectrometry characterization of biomolecules.

Ions accumulated in an rf-only multipole for extended periods of time prior to mass analysis can experience a significant degree of fragmentation and produce mass spectra which do not reflect the true nature of the intact analyte(s). This phenomenon, termed multipole storage assisted dissociation (MSAD), places constraints on the maximum number of ions which can be accumulated in the multipole storage device as a result of its finite space charge limit. This phenomenon can be exploited to produce dissociation spectra that are dominated by fragment ions providing important sequence/structure information. In this work we further explore MSAD and characterize parameters including accumulation time, source pressure, and the electrostatic configuration of the multipole storage device, which mediate the phenomenon. Operating parameters are identified that can either enhance or eliminate the phenomenon.

Enhanced gas-phase hydrogen-deuterium exchange of oligonucleotide and protein ions stored in an external multipole ion reservoir.

Rapid gas-phase hydrogen-deuterium (H-D) exchange from D(2)O and ND(3) into oligonucleotide and protein ions was achieved during storage in a hexapole ion reservoir. Deuterated gas is introduced through a capillary line that discharges directly into the low-pressure region of the reservoir. Following exchange, the degree of H-D exchange is determined using Fourier transform ion cyclotron resonance mass spectrometry. Gas-phase H-D exchange experiments can be conducted more than 100 times faster than observed using conventional in-cell exchange protocols that require lower gas pressures and additional pump-down periods. The short experimental times facilitate the quantitation of the number of labile hydrogens for less reactive proteins and structured oligonucleotides. For ubiquitin, we observe approximately 65 H-D exchanges after 20 s. Exchange rates of > 250 hydrogens s(-1) are observed for oligonucleotide ions when D(2)O or ND(3) is admitted directly into the external ion reservoir owing to the high local pressure in the hexapole. Partially deuterated oligonucleotide ions have been fragmented in the reservoir using infrared multiphoton dissociation (IRMPD). The resulting fragment ions show that exchange predominates at charged sites on the 5'- and 3'-ends of the oligonucleotide, whereas exchange is slower in the core. This hardware configuration is independent of the mass detector and should be compatible with other mass spectrometric platforms including quadrupole ion trap and time-of-flight mass spectrometers.

A gated-beam electrospray ionization source with an external ion reservoir. A new tool for the characterization of biomolecules using electrospray ionization mass spectrometry.

In this work we describe a micro-electrospray ionization source equipped with an atmospheric pressure external ion shutter. The solenoid-activated shutter prevents the electrospray plume from entering the inlet capillary unless triggered to the 'open' position. When in the 'closed' position, a stable electrospray plume is maintained between the electrospray ionization (ESI) emitter and the electrically isolated face of the shutter. When the shutter is triggered, a 'slice' of ions is allowed to enter the inlet capillary and is subsequently accumulated in an external ion reservoir comprised of a radio frequency only (rf-only) hexapole and a pair of electrostatic elements. Following ion accumulation in the external ion reservoir, intact molecular ions of proteins, oligonucleotides, and noncovalent complexes can be stored for extended intervals (>30 minutes) prior to being transferred to the Fourier transform ion cyclotron resonance (FTICR) trapped ion cell for mass analysis. By introducing reactive gases directly into the external ion reservoir during the storage interval, ion-molecule reactions, such as H/D exchange, can be performed at high effective pressures. This scheme obviates the need for the long reaction times and delays associated with restoring base pressure in the trapped ion cell and allows H/D exchange reactions to be conducted in a fraction of the time required using conventional in-cell exchange approaches. The back face of the shutter arm contains an elastomeric material which can be positioned to seal the inlet to the mass spectrometer resulting in lower base pressure in the ion reservoir and the FTICR cell. Additionally, it is noted that blocking the ESI plume during non-accumulation events results in reduced fouling of the source electrodes and longer times between required source cleaning.

Determinants of aminoglycoside-binding specificity for rRNA by using mass spectrometry.

We have developed methods for studying the interactions between small molecules and RNA and have applied them to characterize the binding of three classes of aminoglycoside antibiotics to ribosomal RNA subdomains. High-resolution MS was used to quantitatively identify the noncovalent binding interactions between mixtures of aminoglycosides and multiple RNA targets simultaneously. Signal overlap among RNA targets was avoided by the addition of neutral mass tags that direct each RNA target to a unique region of the spectrum. In addition to determining binding affinities, the locations of the binding sites on the RNAs were identified from a protection pattern generated by fragmenting the aminoglycoside/RNA complex. Specific complexes were observed for the prokaryotic rRNA A-site subdomain with ribostamycin, paromomycin, and lividomycin, whereas apramycin preferentially formed a complex with the eukaryotic subdomain. We show that differences in binding between paromomycin and ribostamycin can be probed by using an MS-MS protection assay. We have introduced specific base substitutions in the RNA models and have measured their impact on binding affinity and selectivity. The binding of apramycin to the prokaryotic subdomain strongly depends on the identity of position 1408, as evidenced by the selective increase in affinity for an A1408G mutant. An A1409-G1491 mismatch pair in the prokaryotic subdomain enhanced the binding of tobramycin and bekanamycin. These observations demonstrate the power of MS-based methods to provide molecular insights into small molecule/RNA interactions useful in the design of selective new antimicrobial drugs.

Multiplexed screening of neutral mass-tagged RNA targets against ligand libraries with electrospray ionization FTICR MS: a paradigm for high-throughput affinity screening.

We demonstrate that binding of mixtures of aminoglycosides can be measured simultaneously against multiple RNA targets of identical length and similar (or identical) molecular weight. Addition of a neutral mass tag to one of the RNA targets shifts the detected peaks to a higher mass/charge ratio, where complexes with small molecules can be identified unambiguously. An appropriately placed neutral mass tag does not alter RNA--ligand binding. The utility of this strategy is demonstrated with model RNAs corresponding to the decoding region of the prokaryotic and eukaryotic rRNAs and a mixture of five aminoglycosides. Complexes are observed between the aminoglycoside library and the prokaryotic rRNA model, while no aminoglycoside was observed to bind to the mass-tagged eukaryotic rRNA model. The differential binding data is consistent with the eukaryotic A-site rRNA having a different conformation compared with the prokaryotic A-site that prevents entry and binding of neomycin-class aminoglycosides. Mass spectrometric analysis of neutral mass-tagged macromolecular targets represents a new high-throughput screening paradigm in which the interaction of multiple targets against a collection of small molecules can be evaluated in parallel.

Infrared multiphoton dissociation in an external ion reservoir.

In this work we present a novel scheme for performing infrared multiphoton dissociation (IRMPD) external to the mass analyzer in an external ion reservoir consisting of an rf-only multipole and a pair of electrostatic lens elements. Ions generated by electrospray ionization (ESI) are accumulated in an rf-only hexapole and dissociated by irradiation at 10.6 microns from a CW CO2 laser in the source region of the mass spectrometer. This scheme is unique from other IRMPD schemes as dissociation occurs in a spatially distinct region of the spectrometer and is independent of the mass spectrometry platform used to analyze the fragment ions. The effectiveness of the technique is demonstrated with ESI IRMPD FTICR mass spectrometry of a 20-mer phosphorothioate oligonucleotide. A comparison of the external IRMPD scheme with nozzle-skimmer dissociation and conventional in-cell IRMPD reveals a significant improvement in signal-to-noise ratio and fragment yield, particularly for larger, more highly charged fragment ions.

Inhibitors of protein-RNA complexation that target the RNA: specific recognition of human immunodeficiency virus type 1 TAR RNA by small organic molecules.

TAR RNA represents an attractive target for the intervention of human immunodeficiency virus type 1 (HIV-1) replication by small molecules. We now describe three small molecule inhibitors of the HIV-1 Tat-TAR interaction that target the RNA, not the protein. The chemical structures and RNA binding characteristics of these inhibitors are unique for each molecule. Results from various biochemical and spectroscopic methods reveal that each of the three Tat-TAR inhibitors recognizes a different structural feature at the bulge, lower stem, or loop region of TAR. Furthermore, one of these Tat-TAR inhibitors has been demonstrated, in cellular environments, to inhibit (a) a TAR-dependent, Tat-activated transcription and (b) the replication of HIV-1 in a latently infectious model.

Electrospray ionization mass spectrometric study of the multiple intracellular monomeric and polymeric hemoglobins of Glycera dibranchiata.

The intracellular hemoglobin (Hb) of the marine polychaete Glycera dibranchiata is comprised of two groups of globins differing in their primary structures and state of aggregation. About six electrophoretically and chromatographically distinct monomeric Hbs which have Leu as the distal residue, and an equal number of polymeric Hbs which have the usual distal His, have been identified to date. Deconvolution of the electrospray ionization mass spectra (ESI-MS) of the Hbs and of their carbamidomethylated, reduced, and reduced/carbamidomethylated forms, using a maximum entropy-based approach (MaxEnt), showed the presence of at least 18 peaks attributable to monomer Hbs (14,500-15,200 Da) and an approximately equal number of polymer Hb peaks (15,500-16,400 Da). Although the ratio of the monomer to polymer components in pooled Hb preparations remained constant at 60:40, Hb from individuals had generally less than 6 monomer and 6 polymer components; -2 of the 19 individuals appeared to be deficient in polymer Hbs. Taking into account possible fragmentations of the known monomeric and polymeric globin sequences, we estimate conservatively that there are 10 monomeric and an equal number of polymeric Hbs, the majority comprising a single free Cys. Surprisingly, the calculated mass of the sequence deduced from the high-resolution monomer Hb crystal structures does not correspond to any of the observed masses. ESI-MS of the monomer Hb crystal revealed 11 components, of which 5, accounting for 67% of total, were related to the three major sequences GMG2-4. These findings underline the need for routine mass spectrometric characterization of all protein preparations. The complete resolution of the Glycera Hb ESI-MS using MaxEnt processing illustrates the power of this method to resolve complex protein mixtures.

Discovery of selective, small-molecule inhibitors of RNA complexes--I. The Tat protein/TAR RNA complexes required for HIV-1 transcription.

We have developed a therapeutic program focusing on the inhibition of a human immunodeficiency virus-1 specific protein-RNA interaction. This program begins with a search for small organic molecules that would interfere with the binding of Tat protein to TAR RNA. The methodologies chosen to study the HIV-1 Tat-TAR interaction and inhibition include gel mobility shift assays, scintillation proximity assays, filtration assays, and mass spectrometry. These methods helped establish in vitro high-throughput screening assays which rapidly identified Tat-TAR inhibitors from our corporate compound library. Tat-activated reporter gene assays were then used to investigate the cellular activities of the Tat-TAR inhibitors. The cellular activity, selectivity, and toxicity data for select Tat-TAR inhibitors were determined. Evaluation of both the cellular data and the Tat-TAR inhibition results led to further testing in anti-HIV-1 infection assays.

HIV-1 Tat peptide binding to TAR RNA by electrospray ionization mass spectrometry.

Electrospray ionization mass spectrometry (ESI-MS) has been used to study the noncovalent complexes formed from the interaction between HIV-1 Tat peptide and Tat protein with TAR RNA. Both positive ion and negative ion ESI mass spectra showed a maximum stoichiometry of 3:1 between Tat peptide and TAR RNA. However, the higher order complexes only occurred at high relative concentrations of Tat peptide. The 1:1 Tat peptide-TAR RNA complex is believed to involve only specific interactions, whereas the higher order complexes involve nonspecific interactions. Relative binding affinities between Tat peptide and TAR RNA and its various mutants (TAR missing the three-nucleotide bulge, TAR with a poly(ethylene glycol) linker in the bulge region, and TAR with a poly(ethylene glycol) linker in the loop region) can be differentiated by competitive binding experiments and ESI-MS measurements. The gas phase mass spectrometry experiments are consistent with solution phase studies, as they show that mutations in the bulge region reduce TAR RNA affinity to Tat peptide.