6 research outputs found

    Argon Direct Analysis in Real Time Mass Spectrometry in Conjunction with Makeup Solvents: A Method for Analysis of Labile Compounds

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    Helium direct analysis in real time (He-DART) mass spectrometry (MS) analysis of labile compounds usually tends to be challenging because of the occurrence of prominent fragmentation, which obscures the assigning of an ion to an independent species or merely a fragment in a mixture. In the present work, argon DART (Ar-DART) MS in conjunction with makeup solvents has been demonstrated to analyze a variety of labile compounds including nucleosides, alkaloids, glucose, and other small molecules. The results presented here confirm that Ar-DART can generate significantly less energetic ions than conventional He-DART and is able to produce the intact molecular ions with little or no fragmentation in both positive and negative ion modes. Adding a makeup solvent (absolute ethyl alcohol, methanol, fluorobenzene, or acetone) to the argon gas stream at the exit of the DART ion source can result in 1–2 orders of magnitude increase in detection signals. The sensitivity attainable by Ar-DART was found to be comparable to that by He-DART. The investigation of influence of solvents improves our understanding of the fundamental desorption and ionization processes in DART. The practical application of this rapid and high throughput method is demonstrated by the successful analysis of a natural product (Crude Kusnezoff Monkshood) extract, demonstrating the great potential in mixture research

    Identification of Unfolding and Dissociation Pathways of Superoxide Dismutase in the Gas Phase by Ion-Mobility Separation and Tandem Mass Spectrometry

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    Cu, Zn-superoxide dismutase (SOD1) is a homodimeric enzyme of approximately 32 kDa. Each monomer contains one Cu<sup>2+</sup> and one Zn<sup>2+</sup> ion, which play catalytic and structural roles in the enzyme. Dimer formation is also essential to its functionality. The spatial structure of this metalloenzyme is also closely related to its bioactivities. Here we investigate the structural and conformational changes of SOD1 in the gas phase by electrospray ionization mass spectrometry (ESI-MS) and ion-mobility (IM) separation combined with tandem mass spectrometry (MS/MS). First, the composition and forms of SOD1 were analyzed by ESI-MS. The dimer, monomer, and apomonomer were observed under different solvent conditions. The dimer was found to be stable, and could retain its native structure in neutral buffer. Ion-mobility separation combined with MS/MS was used to reveal the conformational changes and dissociation process of SOD1 when it was activated in the gas phase. Three different dimeric and two monomeric conformers were observed; three unfolding and dissociation pathways were also identified. The results from this study demonstrate that IM-MS/MS could be used to obtain spatial structural information on SOD1 and that the technique could therefore be employed to investigate the conformational changes in mutant SOD1, which is related to amyotrophic lateral sclerosis and other neurodegenerative disorders

    Targeted Screening Approach to Systematically Identify the Absorbed Effect Substances of <i>Poria cocos</i> <i>in Vivo</i> Using Ultrahigh Performance Liquid Chromatography Tandem Mass Spectrometry

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    <i>Poria cocos</i> are extensively used as nutritious food, dietary supplements, and oriental medicine in Asia. However, the effect substances are still not very clear. In this study, a targeted screening approach was developed to systematically identify absorbed constituents of <i>Poria cocos in vivo</i> using ultrahigh performance liquid chromatography tandem mass spectrometry combined with UNIFI software. First, incubation reactions <i>in vitro</i> with rat intestinal microflora and rat liver microsomes were conducted to sum up metabolic rules of main constituents. Second, the absorbed constituents <i>in vivo</i> were picked out and identified based on the results of metabolic study <i>in vitro</i>. Finally, the absorbed active constituents in the treatment of Alzheimer’s disease were screened by targeted network pharmacology analysis. A total of 62 absorbed prototypes and 59 metabolites were identified and characterized in dosed plasma. Thirty potential active constituents were screened, and 86 drug-targets shared by absorbed constituents and Alzheimer’s disease were discovered by targeted network pharmacology analysis. In general, this proposed targeted strategy comprehensively provides new insight for active ingredients of <i>Poria cocos</i>

    Noncovalent Interactions between Superoxide Dismutase and Flavonoids Studied by Native Mass Spectrometry Combined with Molecular Simulations

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    Misfolding and aggregation of Cu, Zn superoxide dismutase (SOD1) is implicated in the etiology of amyotrophic lateral sclerosis (ALS). The use of small molecules may stabilize the spatial structure of SOD1 dimer, thus, preventing its dissociation and aggregation. In this study, “native” mass spectrometry (MS) was used to study the noncovalent interactions between SOD1 and flavonoid compounds. MS experiments were performed on a quadruple time-of-flight (Q-ToF) mass spectrometer with an electrospray ionization (ESI) source and T-wave ion mobility. ESI-MS was used to detect the SOD1–flavonoid complexes and compare their relative binding strengths. The complement of ion mobility separation allowed comparison in the binding affinities between flavonoid isomers and provided information on the conformational changes. Molecular docking together with molecular dynamics simulations and MM/PBSA methods were applied to gain insights into the binding modes and free energies of SOD1–flavonoid complexes at the molecule level. Among all the flavonoids investigated, flavonoid glycosides preferentially bind to SOD1 than their aglycone counterparts. Naringin, one of the compounds that has the strongest binding affinity to SOD1, was subjected to further characterization. Experiment results show that the binding of naringin can stabilize SOD1 dimer and inhibit the aggregation of SOD1. Molecular simulation results suggest that naringin could reduce the dissociation of SOD1 dimers through direct interaction with the dimer interface. This developed analytical strategy could also be applied to study the interactions between SOD1 and other drug-like molecules, which may have the effect to reduce the aggregation