Molecular characterization of asphaltenes by conventional analytical techniques is a challenge because of their compositional complexity, high heteroatom content, and asphaltene aggregate formation at low concentrations. Thus, most common characterization techniques rely on bulk properties or solution-phase behavior (solubility). Proposed over 20 years ago, the Boduszynski model proposes a continuous progression in petroleum composition (molecular weight, structure, and heteroatom content) as a function of the atmospheric equivalent boiling point. Although exhaustive detailed compositional analysis of petroleum distillates validates the continuum model, the available compositional data from asphaltene fractions supports the extension of the continuum model into the nondistillables only indirectly. Asphaltenes, defined by their insolubility in alkane solvents, accumulate in high-boiling fractions and form stable aggregate structures at low parts per billion (ppb) concentrations, far below the concentration required for most mass analyzers. Here, we present direct mass spectral detection of stable asphaltene aggregates at lower concentrations than previously published and observe the onset of asphaltene nanoaggregate formation by time-of-flight mass spectrometry (TOF−MS). We conclude that a fraction of asphaltenes must be present as nanoaggregates (not monomers) in all atmospheric pressure and laser-based ionization methods. Thus, those methods access a subset of the asphaltene continuum.

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Catalase-peroxidases (KatG) produced by Burkholderia pseudomallei, Escherichia coli, and Mycobacterium tuberculosis catalyze the oxidation of NADH to form NAD ؉ and either H 2 O 2 or superoxide radical depending on pH. The NADH oxidase reaction requires molecular oxygen, does not require hydrogen peroxide, is not inhibited by superoxide dismutase or catalase, and has a pH optimum of 8.75, clearly differentiating it from the peroxidase and catalase reactions with pH optima of 5.5 and 6.5, respectively, and from the NADH peroxidase-oxidase reaction of horseradish peroxidase. B. pseudomallei KatG has a relatively high affinity for NADH (K m ‫؍‬ 12 M), but the oxidase reaction is slow (k cat ‫؍‬ 0.54 min ؊1 ) compared with the peroxidase and catalase reactions. The catalase-peroxidases also catalyze the hydrazinolysis of isonicotinic acid hydrazide (INH) in an oxygen-and H 2 O 2 -independent reaction, and KatG-dependent radical generation from a mixture of NADH and INH is two to three times faster than the combined rates of separate reactions with NADH and INH alone. The major products from the coupled reaction, identified by high pressure liquid chromatography fractionation and mass spectrometry, are NAD ؉ and isonicotinoyl-NAD, the activated form of isoniazid that inhibits mycolic acid synthesis in M. tuberculosis. Isonicotinoyl-NAD synthesis from a mixture of NAD ؉ and INH is KatG-dependent and is activated by manganese ion. M. tuberculosis KatG catalyzes isonicotinoyl-NAD formation from NAD ؉ and INH more efficiently than B. pseudomallei KatG.

Activation of the pro-drug isoniazid (INH) as an anti-tubercular drug in Mycobacterium tuberculosis Isonicotinic acid hydrazide (isoniazid or INH)3 is a widely used anti-tubercular pro-drug that requires activation in a reaction involving the catalase-peroxidase KatG of Mycobacterium tuberculosis (MtKatG) (1) whereby the hydrazine group is removed and the isonicotinyl portion is added to NAD ϩ to generate isonicotinyl-NAD or IN⅐NAD (see Fig. 1). Once formed, IN⅐NAD inhibits the synthesis of mycolic acids, and therefore, the growth of M. tuberculosis, by binding to the longchain enoyl acyl carrier protein reductase (InhA) (2). Despite understanding the role of IN⅐NAD in the inhibition of mycolic acid synthesis and knowing that KatG is required for INH activation in vivo, uncertainties about the mechanism of its formation remain.The involvement of MtKatG in INH activation suggested that the peroxidatic process had a role, and this provided a focus for several studies employing external oxidants such as peroxyacetic acid (3), t-butyl hydroperoxide (4 -6) and low levels of H 2 O 2 (7, 6) to activate the peroxidatic pathway for INH oxidation and activation. In addition, EPR studies have demonstrated that INH can serve as an electron source to reduce peroxidatic intermediates, including specific Trp ⅐ radical species following peroxyacetic acid oxidation of MtKatG (3,8).A multiplicity of methods has been employed to directly and indirectly assay INH activation, including the determination of INH oxidation to isonicotinic acid (9, 10), the HPLC assay of INH disappearance (11), the inactivation of InhA in a mixture of InhA and KatG (7,12,13,14), the HPLC detection of IN⅐NAD (4, 15), and the direct measurement of IN⅐NAD using its characteristic absorbance at 326 nm (6,7,12,15,16). Reports of INH activation in mixtures lacking an external oxidant (4,5,6,9,12,14,15) initially suggested that the peroxidatic process may not be required, but the mixtures of INH, NADH, and KatG would have supported NADH reduction of molecular oxygen to superoxide and low levels of H 2 O 2 (15) to activate the peroxidase reaction.Despite the considerable evidence that a peroxidatic process is involved in IN⅐NAD synthesis, attempts to rationalize the reaction entirely in terms of the peroxidatic pathway generally produced models that were incomplete from the standpoint of electron balance (5,7,10) or that involved hypothetical intermediates not characterized in any other system (5, 6). For example, a common scheme shows the isonicotinyl radical, generated in a peroxidatic reaction, reacting with NAD ϩ to yield IN⅐NAD, when such a reaction would actually yield the IN⅐NAD ϩ ⅐ radical (Fig. 1). The need to reduce this radical in an oxidizing environment suggests that more than a simple peroxidatic pathway is involved in the INH activation process. This rationale leads to superoxide, O 2. , a known reducing agent with

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Electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS) has been used to study noncovalent interactions between the rrp apo-repressor (TrpR), its co-repressor tryptophan and its specific operator DNA. In 5 mM ammonium acetate, TrpR was detected as a partially unfolded monomer. In the presence of a 21-base-pair DNA possessing the two symmetrically arranged CTAG consensus sequences required for specific TrpR binding, a homodimer-dsDNA complex with a 1 : 1 stoichiometry was observed. Co-repressor was not needed for the complex to form under our experimental conditions. Collision induced dissociation (CID-MS) revealed that this complex was very stable in the gas phase since dissociation was achieved only at energies that also broke covalent bonds. We saw no evidence for the presence of the six water molecules that mediate the interaction between the protein and the DNA in the crystal structure. To check the binding specificity of the TrpR for its target DNA, a competitive experiment was undertaken: the protein was mixed with an equimolar amount of three different DNAs in which the two CTAG sequences were separated by 2, 4, and 6 bp, respectively. Only the DNA with the correct consensus spacing of 4 bp was able to form stable interactions with TrpR. This experiment demonstrates the potential of ESI-MS to test the sequence-specificity of protein-DNA complexes. The interactions between the TrpR-DNA complex and 5-methyl-, L-and D-tryptophan were also investigated. Two molecules of 5-methyl-or L-tryptophan were bound with high affhity to the TrpR-DNA complex. On the other hand, D-tryptophan appeared to bind to the complex with poor specificity and poor affinity.Keywords: electrospray ionization; noncovalent; protein-DNA interaction, time-of-flight mass spectrometry, trp repressor Over the last ten years, electrospray ionization mass spectrometry (ESI-MS) has demonstrated a high potential for the characterization of biomolecules by allowing the determination of molecular masses up to about 150 kDa with an accuracy of 0.01% (Fenn et al., 1990;Mann & Wilm, 1995 such as pH, buffers, temperature, and ion kinetic energy are carefully controlled, then noncovalent interactions between molecules can also be preserved during the ionization-desorption process. This technique has been successfully used to study protein-protein (Light-Wahl et al

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) of proteins and peptides was performed on samples deposited onto non-porous ether-type polyurethane (PU) membranes. Spectra obtained using PU membranes showed that mass resolution and accuracy were equivalent to values observed using a metal target, and superior to those obtained using poly(vinylidene difluoride) (PVDF) membranes. A small apparent increase in the mass of proteins and also loss of resolution were observed at very high laser irradiance due to charging, but were not observed under normal conditions. Analysis of NaCl-doped standards demonstrated that PU membranes yielded better results than a metallic target for salt-containing solutions. Relatively strong hydrophobic interactions between the proteins and peptides and the PU membrane allowed the incorporation of a washing step. This step allowed for the removal of salts and buffer components and thus provided an increase in resolution and mass accuracy. Digestion of citrate synthase (a protein of molecular weight 47 886) with trypsin was performed directly on the surface of the membrane for variable periods of time, and characteristic peptide fragments were observed by MALDI-TOFMS. Delayed extraction was used to increase the resolution and to permit more accurate mass assignments for those fragments. The use of PU membranes for MALDI-TOFMS analysis of proteins with higher molecular weights is also demonstrated.