Metabolomics-based component profiling of Halomonas sp. KM-1 during different growth phases in poly(3-hydroxybutyrate) production.

To investigate the relationship between the production of poly(3-hydroxybutyrate) (PHB) and metabolic changes during different growth phases, a non-sterile batch fermentation process involving an alkalophilic and halophilic bacterium, Halomonas sp. KM-1, was used. Intracellular metabolites were analyzed using gas chromatography-mass spectrometry to characterize the metabolic profile. Significant changes relating to PHB production were observed in the TCA cycle, lipid-synthesis and amino acid biosynthetic pathways were found to shift dramatically between the exponential growth and stationary phases. During the stationary phase, 17 metabolites were upregulated and a cell dry mass of 17.8 g/L that included 44.8% PHB was observed at 24h in 5% glucose-supplemented cultures, whereas 11 metabolites were upregulated and a cell dry mass of 38.4 g/L that included 73.7% PHB was observed at 36 h in 10% glucose-supplemented cultures. This study provides pattern analysis of metabolite regulation during PHB accumulation, indicating that multicomponent and phase-specific mechanisms are involved.

Taxonomic characterization and metabolic analysis of the Halomonas sp. KM-1, a highly bioplastic poly(3-hydroxybutyrate)-producing bacterium.

In a brief previous report, the gram-negative moderately halophilic bacterium, Halomonas sp. KM-1, that was isolated in our laboratory was shown to produce the bioplastic, poly(3-hydroxybutyrate) (PHB), using biodiesel waste glycerol (Kawata and Aiba, Biosci. Biotechnol. Biochem., 74, 175-177, 2010). Here, we further characterized this KM-1 strain and compared it to other Halomonas strains. Strain KM-1 was subjected to a polyphasic taxonomic study. Strain KM-1 was rod-shaped and formed colonies on a plate that were cream-beige in color, smooth, opaque, and circular with entire edges. KM-1 grew under environmental conditions of 0.1%-10% (w/v) NaCl, pH 6.5-10.5 and at temperatures between 10°C and 45°C. The G+C content of strain KM-1 was 63.9 mol%. Of the 16 Halomonas strains examined in this study, the strain KM-1 exhibited the highest production of PHB (63.6%, w/v) in SOT medium supplemented with 10% glycerol, 10.0 g/L sodium nitrate and 2.0 g/L dipotassium hydrogen phosphate. The intracellular structures within which PHB accumulated had the appearance of intracellular granules with a diameter of approximately 0.5 µm, as assessed by electron microscopy. The intra- and extra-cellular metabolites of strain KM-1 were analyzed by capillary electrophoresis mass spectrometry. In spite of the high amount of PHB stored intra-cellularly, as possible precursors for PHB only a small quantity of 3-hydroxybutyric acid and acetyl CoA, and no quantity of 3-hydroxybutyl CoA, acetoacetyl CoA and acetoacetate were detected either intra- or extra-cellularly, suggesting highly efficient conversion of these precursors to PHB.

Rapid and accurate determination of deoxyribonucleoside monophosphates from DNA using micellar electrokinetic chromatography with a cationic surfactant additive.

A micellar electrokinetic chromatography (MEKC) method for rapid and accurate determination of 2'-deoxyribonucleoside 5'-monophosphates (dNMPs), four structural elements of DNA, is described. MEKC separation at an optimized pH enabled complete separation of four dNMPs. The use of a cationic surfactant additive for MEKC led to the reversal of EOF, which enhanced the migration velocities of the negatively charged dNMPs. Under the optimized condition, full-baseline separation of the four dNMPs assuring accurate peak integration was obtained within 5 min. For the given separation condition, pH-mediated on-column sample stacking was optimized and applied to enhance sensitivity up to 6-fold. Analytical precision was improved by spiking iothalamate as an internal standard. The accuracy of dNMP quantitation was ensured with dNMP standard solutions determined by inductively coupled plasma-optical emission spectroscopy that measured phosphorous quantity. Performance of the proposed method was ultimately proven by accurate quantitation of a DNA oligonucleotide that was enzymatically hydrolyzed prior to dNMP analysis. The proposed MEKC method turned out to be a reliable analytical method for dNMPs that features high speed, high sensitivity, and high precision, and could be utilized for high-accuracy determination of the amount of DNA as well as the base composition of DNA.

A sheathless CE/ESI-MS interface with an ionophore membrane-packed electro-conduction channel.

A robust and convenient sheathless CE/ESI-MS interface realized with an ionophore membrane-packed electro-conduction channel is described. Sheathless interfaces that may provide higher sensitivity for MS detection than sheath flow-supported interfaces generally show instability and short lifetimes due to their imperfection in making an electrical contact with the emitter tip. In this work, we designed a sheathless interface based on an ionophore membrane-packed electro-conduction channel. At the joining point of the CE capillary and the emitter capillary, the conduction channel was implemented toward the exterior of the interface body, where a platinum wire electrode was placed. The conduction channel transferred the electric field from the external Pt electrode to the joining point, but prevented the effluent of CE from leaking. The interface body was designed to have receptacles for standard capillary tubing with finger-tight fittings, which allowed easy replacement of capillary tubing. Stable electrospray was observed for an extended time period without any signs of bubbling or damage to the emitter tip. No significant increment of dead-volume at the interface was observed for well-aligned capillaries. Sensitive and stable CE-MS detection of the model compound of creatinine and uric acid was demonstrated.

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.

Rapid quantification of DNA methylation through dNMP analysis following bisulfite-PCR.

We report a novel method for rapid quantification of the degree of DNA methylation of a specific gene. Our method combined bisulfite-mediated PCR and quantification of deoxyribonucleoside monophosphate (dNMP) contents in the PCR product through capillary electrophoresis. A specific bisulfite-PCR product was enzymatically hydrolyzed to dNMP monomers which were quantitatively analyzed through subsequent capillary electrophoresis. PCR following bisulfite treatment converts unmethylated cytosines to thymines while leaving methyl-cytosines unchanged. Then the ratio of cytosine to thymine determined by capillary electrophoresis represents the ratio of methyl-cytosine to cytosine in genomic locus of interest. Pure oligonucleotides with known sequences were processed in parallel as standards for normalization of dNMP peaks in capillary electrophoresis. Sources of quantification uncertainty such as carryovers of dNTPs or primers and incomplete hydrolysis were examined and ruled out. When the method was applied to samples with known methylation levels (by bisulfite-mediated sequencing) as a validation, deviations were within +/-5%. After bisulfite-PCR, the analytical procedure can be completed within 1.5 h.

Determination of serum cortisol using isotope dilution-liquid chromatography-mass spectrometry as a candidate reference method.

We propose isotope-dilution mass spectrometry as a candidate reference method for determination of serum cortisol. The method uses liquid chromatography-mass spectrometry (LC-MS), interfaced with electrospray ionization, and selective monitoring of the [M + H]+ ions of cortisol and isotopically labeled cortisol. The isotope-dilution-liquid chromatography-mass spectrometry (ID-LC-MS) method simplifies sample-preparation, because samples are processed by simple solvent extraction without further clean-up and derivatization. We studied the time required for complete equilibration of endogenous cortisol and labeled cortisol spiked into serum and found it to be less than 1 h. The repeatability and the reproducibility of the method were evaluated and found to be 0.55% of the measurement value. CRM 192 and 193 from the Bureau Communautaire de Reference were analyzed for verification of the method. The results obtained from the ID-LC-MS method agreed with the certified values. The relative uncertainty of measurement results for samples in the range of a few tens of micrograms per kilogram to several hundred micrograms per kilogram was evaluated and found to be 0.56%. Immunoassay carried out by three independent clinical laboratories produced results more than 15% higher than this ID-LC-MS method, suggesting the presence of bias in the immunoassay methods.