Hun-Young So

Mass spectrometric studies of alkali metal ion binding on thrombin-binding aptamer DNA.

The binding sites and consecutive binding constants of alkali metal ions, (M+ = Na+, K+, Rb+, and Cs+), to thrombin-binding aptamer (TBA) DNA were studied by Fourier-transform ion cyclotron resonance spectrometry. TBA-metal complexes were produced by electrospray ionization (ESI) and the ions of interest were mass-selected for further characterization. The structural motif of TBA in an ESI solution was checked by circular dichroism. The metal-binding constants and sites were determined by the titration method and infrared multiphoton dissociation (IRMPD), respectively. The binding constant of potassium is 5–8 times greater than those of other alkali metal ions, and the potassium binding site is different from other metal binding sites. In the 1:1 TBA-metal complex, potassium is coordinated between the bottom G-quartet and two adjacent TT loops of TBA. In the 1:2 TBA—metal complex, the second potassium ion binds at the TGT loop of TBA, which is in line with the antiparallel G-quadruplex structure of TBA. On the other hand, other alkali metal ions bind at the lateral TGT loop in both 1:1 and 1:2 complexes, presumably due to the formation of ion-pair adducts. IRMPD studies of the binding sites in combination with measurements of the consecutive binding constants help elucidate the binding modes of alkali metal ions on DNA aptamer at the molecular level.

A sphingosine kinase activity assay using direct infusion electrospray ionization tandem mass spectrometry

D-erythro-Sphingosine is known to be phosphorylated by sphingosine kinase to yield sphingosine-1-phosphate. With the importance of sphingosine-1-phosphate in biological functions being made evident by recent research, a selective and convenient method of assay to measure sphingosine kinase activity is required. Here we developed a new sphingosine kinase assay using murine teratocarcinoma mutant F9-12 cells and electrospray ionization tandem mass spectrometry (ESI-MS/MS) with direct infusion. Sphingosine-1-phosphate in the crude extract of enzyme reaction mixture was selectively characterized and quantitated using precursor ion scanning for [PO(3)](-) in the negative electrospray ionization mode. The method was successfully validated for an activator and an inhibitor of sphingosine kinase. Direct quantitation of S1P without the use of radioactive reagents, chemical derivatization, and extensive chromatographic separation enables simplified assay for sphingosine kinase activity at the cellular system level, and the use of a structural analog as an internal standard provides robustness to the assay.

Preparative Liquid Chromatographic Separation for Accurate Determination of p , p′ -DDE, p , p′ -DDT, and 𝛄-HCH in Fish Oil by Isotope Dilution Mass Spectrometry

Abstract Isotope dilution mass spectrometry (IDMS) has been applied for the accurate determinations of trace p,p′-DDE, p,p′-DDT, and γ-HCH in fish oil reference materials. A preparative LC with aminopropyl normal phase column was employed at the final stage of sample clean up processes to separate the three analytes and their isomers so that the thermal decompositions of coexisting isomers of DDT and DDD in GC elution processes do not interfere the accurate analysis of p,p′-DDE and p,p′-DDT. Fractions of the LC eluent containing the target analytes were collected and separately analyzed by GC/MS. Compared to chromatograms obtained from the extract without the LC separation, the chromatograms of the fractions containing each of the three analytes showed lower interference from the other target analytes, their isomers, the thermal decomposition products, and matrix residues. By the significant reduction of interference, the accuracy of the isotope ratio measurements by GC/MS has been greatly enhanced.