An ion-pair free LC-MS/MS method for quantitative metabolite profiling of microbial bioproduction systems

Takenaka, Musashi; Yoshida, Takeshi; Hori, Yutaka; Bamba, Takahiro; Mochizuki, M; Vavricka, Christopher J.; Hattori, Tomohiro; Hayakawa, Yoshihiro; Hasunuma, Tomohisa; Kondo, Akihiko

doi:10.1016/j.talanta.2020.121625

Cited by 12 publications

(5 citation statements)

References 82 publications

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“…This column provides unique retention characterized by various interactions such as electrostatic dipole-dipole interactions, pi-pi interactions, and hydrophobic forces. The potential of such approach has been reported for simultaneous analysis of a broad range of metabolites [7].…”

Section: Targeted Methodology (Hplc-qqq Ms)mentioning

confidence: 99%

“…Metabolomics approach enables the comprehensive profiling and identification of cellular metabolites that are able to reflect ongoing biological processes, and/or altered states compared to normal conditions. Mass spectrometry (MS)-based metabolomics hyphenated with gas chromatography (GC-MS) or liquid chromatography (LC-MS) has been successfully used for the fingerprinting of various bacteria [5][6][7]. However, only few studies focused on the characterization of bacterial species under stress environments [8,9].…”

Section: Introductionmentioning

confidence: 99%

“…LC coupled to triple quadrupole MS in multiple reaction monitoring (MRM) mode enables sensitive and specific quantitative analysis of targeted metabolites with high reproducibility [10]. Recently, the potential of such LC-MS/MS approach has been demonstrated for the simultaneous targeted analysis of 113 primary metabolites [7]. Since primary metabolites, such as amino acids, organic acids, nucleotides, and nucleosides, are essential for energy production, normal growth, and development of living organisms, their analysis could provide valuable insights into the metabolism of numerous bacterial species.…”

Section: Introductionmentioning

confidence: 99%

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Exploiting targeted and untargeted approaches for the analysis of bacterial metabolites under altered growth conditions

et al. 2021

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In the host, pathogenic microorganisms have developed stress responses to cope with constantly changing environments. Stress responses are directly related to changes in several metabolomic pathways, which could hamper microorganisms' unequivocal identification. We evaluated the effect of various in vitro stress conditions (acidic, basic, oxidative, ethanolic, and saline conditions) on the metabolism of Staphylococcus aureus, Bacillus cereus, and Pseudomonas aeruginosa, which are common lung pathogens. The metabolite profiles of the bacteria were analyzed using liquid chromatography coupled to triple quadrupole and quadrupole time-of-flight mass spectrometry. The advantages of targeted and untargeted analysis combined with univariate and multivariate statistical analysis (principal component analysis, hierarchical cluster analysis, partial least square discriminant analysis, random forest) were combined to unequivocally identify bacterial species. In normal in vitro conditions, the targeted methodology, based on the analysis of primary metabolites, enabled the rapid and efficient discrimination of the three bacteria. In changing in vitro conditions and specifically in presence of the various stressors, the untargeted methodology proved to be more valuable for the global and accurate differentiation of the three bacteria, also considering the type of stress environment within each species. In addition, species-specific metabolites (i.e., fatty acids, polysaccharides, peptides, and nucleotide bases derivatives) were putatively identified. Good intra-day repeatability and inter-day repeatability (< 10% RSD and < 15% RSD, respectively) were obtained for the targeted and the untargeted methods. This untargeted approach highlights its importance in unusual (and less known) bacterial growth environments, being a powerful tool for infectious disease diagnosis, where the accurate classification of microorganisms is sought.

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Section: Targeted Methodology (Hplc-qqq Ms)mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Exploiting targeted and untargeted approaches for the analysis of bacterial metabolites under altered growth conditions

et al. 2021

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“…Synechocystis was transferred to the modified BG11 medium containing 5 mM Na 15 NO 3 or 15 NH 4 Cl 24 h after inoculation and cultivated under 1% (v/v) CO 2 and 100 µ mol photons m −2 s −1 at 30 • C. The culture medium corresponding to 5 mg of dry cell weight was recovered at 0, 4, and 24 h, and the intracellular metabolites were analyzed by CE-MS as described above or by LC-MS/MS MRM. The procedure of sample preparation was the same as described above Section 4.2 (measurement of the intracellular metabolite concentration) and LC-MS/MS MRM analysis was performed by employing Nexera X2 high-performance liquid chromatography system and a LCMS-8060 triple quadrupole mass spectrometer (Shimadzu Corporation, Kyoto, Japan), as described previously [28]. The 15 N labeling rate was calculated as performed in 13 C labeling experiments in previous reports [18].…”

Section: N-metabolic Turnover Analysismentioning

confidence: 99%

Nitrogen Availability Affects the Metabolic Profile in Cyanobacteria

et al. 2021

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Nitrogen is essential for the biosynthesis of various molecules in cells, such as amino acids and nucleotides, as well as several types of lipids and sugars. Cyanobacteria can assimilate several forms of nitrogen, including nitrate, ammonium, and urea, and the physiological and genetic responses to these nitrogen sources have been studied previously. However, the metabolic changes in cyanobacteria caused by different nitrogen sources have not yet been characterized. This study aimed to elucidate the influence of nitrate and ammonium on the metabolic profiles of the cyanobacterium Synechocystis sp. strain PCC 6803. When supplemented with NaNO3 or NH4Cl as the nitrogen source, Synechocystis sp. PCC 6803 grew faster in NH4Cl medium than in NaNO3 medium. Metabolome analysis indicated that some metabolites in the CBB cycle, glycolysis, and TCA cycle, and amino acids were more abundant when grown in NH4Cl medium than NaNO3 medium. 15N turnover rate analysis revealed that the nitrogen assimilation rate in NH4Cl medium was higher than in NaNO3 medium. These results indicate that the mechanism of nitrogen assimilation in the GS-GOGAT cycle differs between NaNO3 and NH4Cl. We conclude that the amounts and biosynthetic rate of cyanobacterial metabolites varies depending on the type of nitrogen.

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“…LC-MS/MS is widely used in the detection and analysis of drugs [9][10][11], pharmaceutical impurities [12,13], degradation products [14,15], and metabolites [16,17] because it has the advantages of high sensitivity, low detection limit, strong anti-interference ability, and accurate qualitative and quantitative measurability. Specifically, ultrahighperformance liquid chromatography (UPLC) and a reliable internal standard can be used to reduce the effect of matrix effect on quantitative results.…”

Section: Introductionmentioning

confidence: 99%

Pharmacokinetics of 10-Hydroxy Mesaconitine in Rat Plasma by Ultra-Performance Liquid Chromatography-Tandem Quadrupole Mass Spectrometry

Yang

Zeng

et al. 2021

Journal of Analytical Methods in Chemistry

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Mesaconitine is the predominant active ingredient in Aconitum carmichaelii Debx. The compound 10-hydroxy mesaconitine is one known metabolite of mesaconitine and is toxic. In order to better understand its pharmacokinetics, UPLC-MS/MS was used in this paper to measure the concentration of 10-hydroxy mesaconitine in the plasma of rats after oral (5 mg/kg) and intravenous (0.1 mg/kg) administration of 10-hydroxy mesaconitine. The concentrations of 10-hydroxy mesaconitine in rat plasma measured in the standard curve covered the range of 0.3–60 ng/mL. The intraday and interday precisions of the samples of 10-hydroxy mesaconitine in rat plasma were lower than 15%. In addition, the accuracies ranged between 96.0% and 109.3%, the matrix effects ranged between 88.9% and 98.1%, and the recoveries were all higher than 79.1%. The AUC(0 − t) values were 23.6 ± 5.9 and 207.6 ± 72.9 ng/mL·h for intravenous and oral administration, respectively, and the bioavailability of 10-hydroxy mesaconitine was 17.6%. Lastly, t1/2 was 1.3 ± 0.6 h and 3.1 ± 0.4 h for intravenous and oral administration, respectively.

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An ion-pair free LC-MS/MS method for quantitative metabolite profiling of microbial bioproduction systems

Cited by 12 publications

References 82 publications

Exploiting targeted and untargeted approaches for the analysis of bacterial metabolites under altered growth conditions

Exploiting targeted and untargeted approaches for the analysis of bacterial metabolites under altered growth conditions

Nitrogen Availability Affects the Metabolic Profile in Cyanobacteria

Pharmacokinetics of 10-Hydroxy Mesaconitine in Rat Plasma by Ultra-Performance Liquid Chromatography-Tandem Quadrupole Mass Spectrometry

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