Teaching Editorial - Bioanalytical Mass Spectrometry: Many Flavors to Choose

Loo, Joseph A.

doi:10.1021/bc00036a600

Cited by 81 publications

(56 citation statements)

References 240 publications

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“…Under similar conditions, modifications at guanine residues 21, 26, 28, and 32-34, or at C 24 in the bulge region, were not affected by the presence of 3. At higher concentrations, e.g., [3] ) 500 µM, some (∼30%) secondary protection was found for G 26 and G 28 , both located in the upper stem region. Interestingly, guanine residues in the loop region were not protected by 3 under any condition.…”

Section: (3) Inhibitor 3 Inhibits the Tat-tar Interaction By Binding mentioning

confidence: 97%

Inhibitors of Protein−RNA Complexation That Target the RNA: Specific Recognition of Human Immunodeficiency Virus Type 1 TAR RNA by Small Organic Molecules

Mei¹,

Cui²,

Heldsinger³

et al. 1998

Biochemistry

159

140

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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. Drug discovery has traditionally involved a search for inhibitors of protein complexation to small molecules (e.g., substrates or ligands) or macromolecules (e.g., other proteins, nucleic acids, or polysaccharides). While agonists or antagonists of macromolecular receptors that become useful drugs must display a wide range of other attributes, including the appropriate physicochemical properties, pharmacokinetic and pharmacodynamic properties, stability, etc., they must first be protein ligands. The vast majority of available drugs act by binding noncovalently to a protein, preventing or (less often) stimulating that protein's complexation to a complement (1).Complexes of nucleic acid and proteins are key intermediates in all transcriptional and translational processes. Some nucleic acids (e.g., ribozymes) even form functional complexes with small molecules (2). Nonetheless, nucleic acids are widely viewed as ineffective targets for the discovery of low-molecular weight inhibitors. One reason is that the linear motif in single-stranded DNA and the repetitive motif in double-stranded DNA provide attractive targets for large, linear binding molecules (3), but unattractive targets for the small molecules that lead to orally available medications. Another reason is that the lack of tertiary structure in DNA does not afford the diverse topology associated with folded proteins.However, single-stranded RNA often folds into welldefined tertiary structures (4). Furthermore, such structures serve as docking sites for transcriptional activators (5) and substrates for self-splicing reactions (6, 7). Can such structured nucleic acids display sufficient shape diversity to permit the complexation of a small molecule at one site in a virtual sea of nucleic acid material? This is the essential question whose presumed negative answer hinders drug discovery at the nucleic acid level.Certain cis-acting RNA elements are essential for the gene expression of human immunodeficiency virus type 1 (HIV-1) (8). The functions and sequences of these RNA molecules have been well characterized (9-11). A segment of HIV-1 mRNA (residues 1-59), identified as the transactivation responsive element (TAR), adopts a stem-loop secondary structure consisting of a highly cons...

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Section: (3) Inhibitor 3 Inhibits the Tat-tar Interaction By Binding mentioning

confidence: 97%

Inhibitors of Protein−RNA Complexation That Target the RNA: Specific Recognition of Human Immunodeficiency Virus Type 1 TAR RNA by Small Organic Molecules

Mei¹,

Cui²,

Heldsinger³

et al. 1998

Biochemistry

159

140

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“…Under gentle conditions, both electrospray ionization [2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] and matrix-assisted laser desorption [17][18][19][20] can produce intact gas-phase ions of a variety of noncovalent biomolecule complexes. Recent studies indicate that information about the relative binding affinities of noncovalent complexes in solution can be obtained from the relative abundances of the corresponding ions observed in a mass spectrum [12][13][14][15]21].…”

Section: Introductionmentioning

confidence: 99%

“…Noncovalent heme-binding proteins such as myoglobin (Mb) and hemoglobin (Hb) have been extensively studied in solution [30][31][32][33][34][35][36][37][38][39][40][41][42] as well as in the gas phase [3][4][5][6][7][8]10,11,43]. In solution, the binding of ligands such as O 2 and CO [30,38,39], and the affinity of the protein for the heme group, have been studied [30][31][32][33][34][35][40][41][42] In gas-phase studies, McLuckey and Ramsey [7] reported that holo-Mb ions could be trapped for 1 s in a quadrupole ion trap mass spectrometer.…”

Section: Introductionmentioning

confidence: 99%

“…Recent studies, which probe the gas-phase conformation of ions by using H/D exchange [23,24], collisional cross section [5,25] and ion mobility [26,27], proton transfer reactivity [28,29], and high-energy surface-impact collisions [6], show that many proteins can have a highly compact conformation in the gas phase, although the exact nature of these conformations is not known. These results suggest the possibility that ions can retain some conformational attributes of their solution structure.Noncovalent heme-binding proteins such as myoglobin (Mb) and hemoglobin (Hb) have been extensively studied in solution [30][31][32][33][34][35][36][37][38][39][40][41][42] as well as in the gas phase [3][4][5][6][7][8]10,11,43]. In solution, the binding of ligands such as O 2 and CO [30,38,39], and the affinity of the protein for the heme group, have been studied [30][31][32][33][34][35][40][41][42] In gas-phase studies, McLuckey and Ramsey …”

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confidence: 99%

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Dissociation of heme-globin complexes by blackbody infrared radiative dissociation: Molecular specificity in the gas phase?

Gross

Zhao

Williams

1997

J. Am. Soc. Mass Spectrom.

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The temperature dependence of the unimolecular kinetics for dissociation of the heme group from holo-myoglobin (Mb) and holo-hemoglobin α-chain (Hb-α) was investigated with blackbody infrared radiative dissociation (BIRD). The rate constant for dissociation of the 9 + charge state of Mb formed by electrospray ionization from a "pseudo-native" solution is 60% lower than that of Hb-α at each of the temperatures investigated. In solutions of pH 5.5-8.0, the thermal dissociation rate for Mb is also lower than that of HB-α (Hargrove, M. S. et al. J. Biol. Chem. 1994, 269, 4207-4214). Thus, Mb is thermally more stable with respect to heme loss than Hb-α both in the gas phase and in solution. The Arrhenius activation parameters for both dissociation processes are indistinguishable within the current experimental error (activation energy 0.9 eV and pre-exponential factor of 10 8-10 s −1 ). The 9+ to 12+ charge states of Mb have similar Arrhenius parameters when these ions are formed from pseudo-native solutions. In contrast, the activation energies and pre-exponential factors decrease from 0.8 to 0.3 eV and 10 7 to 10 2 s −1 , respectively, for the 9 + to 12 + charge states formed from acidified solutions in which at least 50% of the secondary structure is lost. These results demonstrate that gas-phase Mb ions retain clear memory of the composition of the solution from which they are formed and that these differences can be probed by BIRD.Noncovalent interactions play a critical role in the function of biomolecules in solution [1]. Under gentle conditions, both electrospray ionization [2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] and matrix-assisted laser desorption [17][18][19][20] can produce intact gas-phase ions of a variety of noncovalent biomolecule complexes. Recent studies indicate that information about the relative binding affinities of noncovalent complexes in solution can be obtained from the relative abundances of the corresponding ions observed in a mass spectrum [12][13][14][15]21]. Careful control experiments are critical [22], because other factors, such as ion source conditions and gas-phase chemistry, can influence these abundances. An important question is what structural changes occur when noncovalent complexes enter the gas phase, that is, are specific interactions retained in the absence of solvent, and if so, do their bindings strengths reflect those in solution? Recent studies, which probe the gas-phase conformation of ions by using H/D exchange [23,24], collisional cross section [5,25] and ion mobility [26,27], proton transfer reactivity [28,29], and high-energy surface-impact collisions [6], show that many proteins can have a highly compact conformation in the gas phase, although the exact nature of these conformations is not known. These results suggest the possibility that ions can retain some conformational attributes of their solution structure.Noncovalent heme-binding proteins such as myoglobin (Mb) and hemoglobin (Hb) have been extensively studied in solution [30][31][32][...

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“…Micelle solutions were prepared by dissolving octyltrimethylammonium bromide [OTAB: C 8 H 17 N(CH 3 ) 3 Br] in water alone, without other additives. From the measurement of the m/z of the doubly or triply charged ions, it was revealed that the micelle of OTAB exhibited an almost symmetrical and polydisperse distribution about the mean molecular weight of 5000-5500.…”

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confidence: 99%

The possibility of the direct measurement of micelle weight by electrospray ionization mass spectrometry

Nohara

Ohkoshi

Sakai

1998

Rapid Commun. Mass Spectrom.

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Measurement of the molecular weight of micelles, which are characteristic aggregates of molecules, was conducted using electrospray ionization mass spectrometry (ESI-MS). Micelle solutions were prepared by dissolving octyltrimethylammonium bromide [OTAB: C 8 H 17 N(CH 3 ) 3 Br] in water alone, without other additives. From the measurement of the m/z of the doubly or triply charged ions, it was revealed that the micelle of OTAB exhibited an almost symmetrical and polydisperse distribution about the mean molecular weight of 5000-5500. This molecular weight information is in good accordance with other estimated values. This result demonstrated the possibility of a direct measurement of micelle weight by mass spectrometry. # 1998 John Wiley & Sons, Ltd. Received 17 August 1998; Revised 5 October 1998; Accepted 7 October 1998 Recently, molecular aggregates associated by noncovalent interactions have been actively studied regarding their function, structure, and the interaction associating them. Electrospray ionization (ESI)1,2 has made it possible to directly measure the molecular weight of biopolymer accurately by producing multiply charged ions from the solution phase.3,4 Furthermore, molecular aggregates 5 due to noncovalent interactions can be detected under the prevailing conditions of ESI because it is a soft ionization technique. A variety of investigations using ESI have reported aggregates such as protein-ligand, 6-9 enzymesubstrate or enzyme-inhibitor, 10,11 protein-protein, protein-peptide, or peptide-peptide 12-15 and antibioticsligand. 16 Various methods have been employed in the measurement of the molecular weight of the micelle that is produced by these characteristic aggregations of relatively small molecules, e.g., light-scattering spectrometry, gel-filtration liquid chromatography and viscosity measurement. These can offer the mean molecular weight and the weight distribution, calculated on the basis of an hypothesis or the rough molecular weight by adaptation of a suitable calibration curve prepared from standard samples. This communication demonstrates the possibility of the measurement of micelle weight by ESI-MS. The successful application of the mass spectrometer to the direct measurement of micelle weight may open up a new field in the research of molecular aggregates. EXPERIMENTAL MaterialsThe micelle solution was prepared by dissolving octyltrimethylammonium bromide (OTAB) in distilled deionized water, without any additives. The concentration of OTAB was 0.56 M which was four times the critical micelle concentration (CMC) of this surfactant. 17 Mass spectrometryThe mass spectrometer used was of sector type (JMS-AX505HA, JEOL, Tokyo) equipped with an ESI ion source (Analytica of Branford, Branford, CA). The ionization voltage of 3.5 kV was applied to the outlet of the stainless steel nozzle. The micelle solution was infused by a syringe pump at the constant flow rate of 1 mL min À1 . RESULTS AND DISCUSSIONThe change in bias imposed between capillary and skimmer (DCS), in the ion source, can...

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Teaching Editorial - Bioanalytical Mass Spectrometry: Many Flavors to Choose

Cited by 81 publications

References 240 publications

Inhibitors of Protein−RNA Complexation That Target the RNA: Specific Recognition of Human Immunodeficiency Virus Type 1 TAR RNA by Small Organic Molecules

Inhibitors of Protein−RNA Complexation That Target the RNA: Specific Recognition of Human Immunodeficiency Virus Type 1 TAR RNA by Small Organic Molecules

Dissociation of heme-globin complexes by blackbody infrared radiative dissociation: Molecular specificity in the gas phase?

The possibility of the direct measurement of micelle weight by electrospray ionization mass spectrometry

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