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1
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Zhao Y, Fan R, Wang C, Xu S, Xie L, Hou J, Lei W, Liu J. Quantification and isotope abundance determination of 13C labeled intracellular sugar metabolites with hydrophilic interaction liquid chromatography. ANALYTICAL METHODS : ADVANCING METHODS AND APPLICATIONS 2023; 15:5666-5673. [PMID: 37855701 DOI: 10.1039/d3ay01178j] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/20/2023]
Abstract
Metabolic flux analysis (MFA) using stable isotope labeled tracers is a powerful tool to estimate fluxes through metabolic pathways. It finds applications in studying metabolic changes in diseases, regulation of cellular energetics, and novel strategies for metabolic engineering. Accurate and precise quantification of the concentration of metabolites and their labeling states is critical for correct MFA results. Utilizing an ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) system, an analytical method for simultaneously quantifying the concentration of sugar metabolites and their mass isotopologue distribution (MID) was developed. The method performs with good linearity and coefficient of determination (R2) > 0.99, while the detection limit ranged from 0.1 to 50 mg L-1. Seven sugar metabolites were detected in a labeled Brevibacterium flavum sample using the method. The detected quantities ranged from 6.15 to 3704.21 mg L-1, and 13C abundance was between 12.77% and 66.67% in the fermentation fluid and 16.28% and 91.93% in the bacterial body. Overall, the method is efficient, accurate, and suitable for analysis of labeled sugar metabolites in 13C MFA studies.
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Affiliation(s)
- Yameng Zhao
- Department of Nuclear Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.
- Shanghai Research Institute of Chemical Industry Co., Ltd, Shanghai, China
| | - Ruoning Fan
- Shanghai Research Institute of Chemical Industry Co., Ltd, Shanghai, China
| | - Chuyao Wang
- Shanghai Research Institute of Chemical Industry Co., Ltd, Shanghai, China
| | - Sen Xu
- Shanghai Research Institute of Chemical Industry Co., Ltd, Shanghai, China
| | - Long Xie
- Shanghai Research Institute of Chemical Industry Co., Ltd, Shanghai, China
| | - Jinghua Hou
- Shanghai Research Institute of Chemical Industry Co., Ltd, Shanghai, China
| | - Wen Lei
- Shanghai Research Institute of Chemical Industry Co., Ltd, Shanghai, China
| | - Jianjun Liu
- Department of Nuclear Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.
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2
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Qi G, Zou H, Peng X, He S, Zhang Q, Ye W, Jiang Y, Wang W, Ren G, Qu X. Metabolic Footprinting-Based DNA-AuNP Encoders for Extracellular Metabolic Response Profiling. Anal Chem 2023; 95:8088-8096. [PMID: 37155931 DOI: 10.1021/acs.analchem.3c01109] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/10/2023]
Abstract
Metabolic footprinting as a convenient and non-invasive cell metabolomics strategy relies on monitoring the whole extracellular metabolic process. It covers nutrient consumption and metabolite secretion of in vitro cell culture, which is hindered by low universality owing to pre-treatment of the cell medium and special equipment. Here, we report the design and a variety of applicability, for quantifying extracellular metabolism, of fluorescently labeled single-stranded DNA (ssDNA)-AuNP encoders, whose multi-modal signal response is triggered by extracellular metabolites. We constructed metabolic response profiling of cells by detecting extracellular metabolites in different tumor cells and drug-induced extracellular metabolites. We further assessed the extracellular metabolism differences using a machine learning algorithm. This metabolic response profiling based on the DNA-AuNP encoder strategy is a powerful complement to metabolic footprinting, which significantly applies potential non-invasive identification of tumor cell heterogeneity.
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Affiliation(s)
- Guangpei Qi
- Key Laboratory of Sensing Technology and Biomedical Instruments of Guangdong Province and School of Biomedical Engineering, Sun Yat-Sen University, Shenzhen 518107, China
| | - Haixia Zou
- Key Laboratory of Sensing Technology and Biomedical Instruments of Guangdong Province and School of Biomedical Engineering, Sun Yat-Sen University, Shenzhen 518107, China
| | | | - Shiliang He
- College of Health Science and Environmental Engineering, Shenzhen Technology University, Shenzhen 518118, China
| | - Qiqi Zhang
- Key Laboratory of Sensing Technology and Biomedical Instruments of Guangdong Province and School of Biomedical Engineering, Sun Yat-Sen University, Shenzhen 518107, China
| | - Wei Ye
- Key Laboratory of Sensing Technology and Biomedical Instruments of Guangdong Province and School of Biomedical Engineering, Sun Yat-Sen University, Shenzhen 518107, China
| | - Yizhou Jiang
- Key Laboratory of Sensing Technology and Biomedical Instruments of Guangdong Province and School of Biomedical Engineering, Sun Yat-Sen University, Shenzhen 518107, China
| | - Wentao Wang
- Key Laboratory of Sensing Technology and Biomedical Instruments of Guangdong Province and School of Biomedical Engineering, Sun Yat-Sen University, Shenzhen 518107, China
| | - Guangli Ren
- Department of Pediatrics, General Hospital of Southern Theater Command of PLA, Guangzhou 510010, China
| | - Xiangmeng Qu
- Key Laboratory of Sensing Technology and Biomedical Instruments of Guangdong Province and School of Biomedical Engineering, Sun Yat-Sen University, Shenzhen 518107, China
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3
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Su M, Serafimov K, Li P, Knappe C, Lämmerhofer M. Isomer selectivity of one- and two-dimensional approaches of mixed-mode and hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry for sugar phosphates of glycolysis and pentose phosphate pathways. J Chromatogr A 2023; 1688:463727. [PMID: 36566570 DOI: 10.1016/j.chroma.2022.463727] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2022] [Revised: 12/16/2022] [Accepted: 12/17/2022] [Indexed: 12/23/2022]
Abstract
In this study, the chromatographic behavior of mixed-mode and hydrophilic interaction liquid chromatography (HILIC) with the mixed-mode HILIC/strong anion-exchange (SAX) column HILICpak VT-50 2D and the two HILIC columns Atlantis Premier BEH Z-HILIC and Acquity Premier BEH Amide was assessed with regard to their separation capability of the metabolites from the glycolysis and pentose phosphate pathways. Chromatographic conditions were evaluated with the aim of achieving separation of the isomeric glycolytic phosphorylated carbohydrate metabolites free from isomeric interferences and thus allowing for selective targeted analysis by liquid chromatography with tandem mass spectrometry (MS/MS) using multiple reaction monitoring acquisition. The effects of pH values (8.0/9.0/10.0) of the ammonium bicarbonate buffer and gradient time were investigated during HILIC-MS/MS analysis, with the optimal conditions found at pH = 10.0. Separation of the pentose phosphate isomers (ribose 5- and 1-phosphate, xylulose 5-phosphate and ribulose 5-phosphate) was achieved on the mixed-mode HILIC/SAX (HILICpak VT-50 2D) column and HILIC BEH Amide column. Column performance was evaluated based on the direct comparison of chromatographic parameters, i.e. peak width at 50% and peak tailing factors of the individual metabolites. Parity plots were generated allowing a direct comparison between the normalized retention times and assessment of orthogonality of all 3 stationary phases evaluated. Separation of 7 biologically relevant hexose monophosphates metabolites turned out to be challenging by HILIC-MS/MS, with the BEH Amide providing the best individual results for such a separation. However, fructose 6-phosphate and glucose 1-phosphate co-eluted. Therefore, an on-line heart-cutting HILIC-Mixed Mode 2D-LC-QToF experiment was conducted, allowing the separation of this critical isomer pair. In this setup, the BEH Amide column in the 1D separated the majority of target metabolites, while a heart-cut of the peak from totally coeluted fructose 6-phosphate and glucose 1-phosphate was separated in the 2D with HILICpak VT50-2D column, thus allowing undisturbed determination of the glycolytic phosphorylated carbohydrate metabolites due to their chromatographic separation from hexose monophosphate metabolites. The assay specificity towards 7 common hexose monophosphates was characterized (glucose 1- and 6-phosphate, galactose 1- and 6-phosphate, fructose 6-phosphate, mannose 1- and 6-phosphate). The selectivity of some rare hexose monophosphates (allose 6-phosphate, tagatose 6-phosphate, sorbose 1-phosphate) was also tested.
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Affiliation(s)
- Min Su
- Pharmaceutical (Bio-)Analysis, Institute of Pharmaceutical Sciences, University of Tübingen, Auf der Morgenstelle 8, Tübingen 72076, Germany
| | - Kristian Serafimov
- Pharmaceutical (Bio-)Analysis, Institute of Pharmaceutical Sciences, University of Tübingen, Auf der Morgenstelle 8, Tübingen 72076, Germany
| | - Peng Li
- Pharmaceutical (Bio-)Analysis, Institute of Pharmaceutical Sciences, University of Tübingen, Auf der Morgenstelle 8, Tübingen 72076, Germany
| | - Cornelius Knappe
- Pharmaceutical (Bio-)Analysis, Institute of Pharmaceutical Sciences, University of Tübingen, Auf der Morgenstelle 8, Tübingen 72076, Germany
| | - Michael Lämmerhofer
- Pharmaceutical (Bio-)Analysis, Institute of Pharmaceutical Sciences, University of Tübingen, Auf der Morgenstelle 8, Tübingen 72076, Germany.
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4
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Abstract
Metabolomics is a powerful tool that can systematically describe global changes in the metabolome of microbes, thus improving our understanding of the mechanisms of action of antibiotics and facilitating the development of next-generation antibacterial therapies. However, current sample preparation methods are not efficient or reliable for studying the effects of antibiotics on microbes. In the present study, we reported a novel sample preparation approach using cold methanol/ethylene glycol for quenching Escherichia coli, thus overcoming the loss of intracellular metabolites caused by cell membrane damage. After evaluating the extraction efficiency of several extraction methods, we employed the optimized workflow to profile the metabolome of E. coli exposed to cephalexin. In doing so, we proved the utility of the proposed approach and provided insights into the comprehensive metabolic alterations associated with antibiotic treatment. IMPORTANCE The emergence and global spread of multidrug-resistant bacteria and genes are a global problem. It is critical to understand the interactions between antibiotics and bacteria and find alternative treatments for infections when we are moving closer to a postantibiotic era. It has been demonstrated that the bacterial metabolic environment plays an important role in the modulation of antibiotic susceptibility and efficacy. In the present study, we proposed a novel metabolomic approach for intracellular metabolite profiling of E. coli, which can be used to investigate the metabolite alterations of bacteria caused by antibiotic treatment. Further understanding of antibiotic-induced perturbations of bacterial metabolism would facilitate the discovery of new therapeutic targets and pathways.
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5
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Butman HS, Kotzé TJ, Dowd CS, Strauss E. Vitamin in the Crosshairs: Targeting Pantothenate and Coenzyme A Biosynthesis for New Antituberculosis Agents. Front Cell Infect Microbiol 2020; 10:605662. [PMID: 33384970 PMCID: PMC7770189 DOI: 10.3389/fcimb.2020.605662] [Citation(s) in RCA: 21] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/12/2020] [Accepted: 10/23/2020] [Indexed: 01/05/2023] Open
Abstract
Despite decades of dedicated research, there remains a dire need for new drugs against tuberculosis (TB). Current therapies are generations old and problematic. Resistance to these existing therapies results in an ever-increasing burden of patients with disease that is difficult or impossible to treat. Novel chemical entities with new mechanisms of action are therefore earnestly required. The biosynthesis of coenzyme A (CoA) has long been known to be essential in Mycobacterium tuberculosis (Mtb), the causative agent of TB. The pathway has been genetically validated by seminal studies in vitro and in vivo. In Mtb, the CoA biosynthetic pathway is comprised of nine enzymes: four to synthesize pantothenate (Pan) from l-aspartate and α-ketoisovalerate; five to synthesize CoA from Pan and pantetheine (PantSH). This review gathers literature reports on the structure/mechanism, inhibitors, and vulnerability of each enzyme in the CoA pathway. In addition to traditional inhibition of a single enzyme, the CoA pathway offers an antimetabolite strategy as a promising alternative. In this review, we provide our assessment of what appear to be the best targets, and, thus, which CoA pathway enzymes present the best opportunities for antitubercular drug discovery moving forward.
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Affiliation(s)
- Hailey S. Butman
- Department of Chemistry, George Washington University, Washington, DC, United States
| | - Timothy J. Kotzé
- Department of Biochemistry, Stellenbosch University, Stellenbosch, South Africa
| | - Cynthia S. Dowd
- Department of Chemistry, George Washington University, Washington, DC, United States
| | - Erick Strauss
- Department of Biochemistry, Stellenbosch University, Stellenbosch, South Africa
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6
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Gelain L, Geraldo da Cruz Pradella J, Carvalho da Costa A, van der Wielen L, van Gulik WM. A possible influence of extracellular polysaccharides on the analysis of intracellular metabolites from Trichoderma harzianum grown under carbon-limited conditions. Fungal Biol 2020; 125:368-377. [PMID: 33910678 DOI: 10.1016/j.funbio.2020.12.002] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2020] [Revised: 10/30/2020] [Accepted: 12/08/2020] [Indexed: 11/19/2022]
Abstract
Intracellular metabolites were evaluated during the continuous growth of Trichoderma harzianum P49P11 under carbon-limited conditions. Four different conditions in duplicate were investigated (10 and 20 g/L of glucose, 5.26/5.26 g/L of fructose/glucose and 10 g/L of sucrose in the feed). Differences in the values of some specific concentrations of intracellular metabolites were observed at steady-state for the duplicates. The presence of extracellular polysaccharide was confirmed in the supernatant of all conditions based on FT-IR and proton NMR. Fragments of polysaccharides from the cell wall could be released due to the shear stress and since the cells can consume them under carbon-limited conditions, this could create an unpredictable carbon flow rate into the cells. According to the values of the metabolite concentrations, it was considered that the consumption of those fragments was interfering with the analysis.
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Affiliation(s)
- Lucas Gelain
- Delft University of Technology, Department of Biotechnology, Van der Maasweg 9, 2629HZ, Delft, the Netherlands; University of Campinas, School of Chemical Engineering, Av. Albert Einstein, 500, Campinas, Brazil.
| | - José Geraldo da Cruz Pradella
- Federal University of São Paulo, Institute of Science and Technology, Av. Cesare Mansueto Giulio Lattes, 1201, S. J. Campos, Brazil
| | - Aline Carvalho da Costa
- University of Campinas, School of Chemical Engineering, Av. Albert Einstein, 500, Campinas, Brazil
| | - Luuk van der Wielen
- Delft University of Technology, Department of Biotechnology, Van der Maasweg 9, 2629HZ, Delft, the Netherlands; University of Limerick, Bernal Institute, V94 T9PX, Limerick, Ireland
| | - Walter M van Gulik
- Delft University of Technology, Department of Biotechnology, Van der Maasweg 9, 2629HZ, Delft, the Netherlands
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7
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Barsottini MRO, Copsey A, Young L, Baroni RM, Cordeiro AT, Pereira GAG, Moore AL. Biochemical characterization and inhibition of the alternative oxidase enzyme from the fungal phytopathogen Moniliophthora perniciosa. Commun Biol 2020; 3:263. [PMID: 32451394 PMCID: PMC7248098 DOI: 10.1038/s42003-020-0981-6] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2019] [Accepted: 04/30/2020] [Indexed: 01/27/2023] Open
Abstract
Moniliophthora perniciosa is a fungal pathogen and causal agent of the witches' broom disease of cocoa, a threat to the chocolate industry and to the economic and social security in cocoa-planting countries. The membrane-bound enzyme alternative oxidase (MpAOX) is crucial for pathogen survival; however a lack of information on the biochemical properties of MpAOX hinders the development of novel fungicides. In this study, we purified and characterised recombinant MpAOX in dose-response assays with activators and inhibitors, followed by a kinetic characterization both in an aqueous environment and in physiologically-relevant proteoliposomes. We present structure-activity relationships of AOX inhibitors such as colletochlorin B and analogues which, aided by an MpAOX structural model, indicates key residues for protein-inhibitor interaction. We also discuss the importance of the correct hydrophobic environment for MpAOX enzymatic activity. We envisage that such results will guide the future development of AOX-targeting antifungal agents against M. perniciosa, an important outcome for the chocolate industry.
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Affiliation(s)
- Mario R O Barsottini
- Genomics and bioEnergy Laboratory, Institute of Biology, University of Campinas, Campinas, Brazil.,Biochemistry & Biomedicine, School of Life Sciences, University of Sussex, Brighton, BN1 9QG, UK
| | - Alice Copsey
- Biochemistry & Biomedicine, School of Life Sciences, University of Sussex, Brighton, BN1 9QG, UK
| | - Luke Young
- Biochemistry & Biomedicine, School of Life Sciences, University of Sussex, Brighton, BN1 9QG, UK
| | - Renata M Baroni
- Genomics and bioEnergy Laboratory, Institute of Biology, University of Campinas, Campinas, Brazil
| | - Artur T Cordeiro
- Brazilian Biosciences National Laboratory, Brazilian Center for Research in Energy and Materials, Campinas, Brazil
| | - Gonçalo A G Pereira
- Genomics and bioEnergy Laboratory, Institute of Biology, University of Campinas, Campinas, Brazil.
| | - Anthony L Moore
- Biochemistry & Biomedicine, School of Life Sciences, University of Sussex, Brighton, BN1 9QG, UK.
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8
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Cifuente JO, Comino N, D'Angelo C, Marina A, Gil-Carton D, Albesa-Jové D, Guerin ME. The allosteric control mechanism of bacterial glycogen biosynthesis disclosed by cryoEM. Curr Res Struct Biol 2020; 2:89-103. [PMID: 34235472 PMCID: PMC8244506 DOI: 10.1016/j.crstbi.2020.04.005] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2020] [Revised: 04/12/2020] [Accepted: 04/20/2020] [Indexed: 11/10/2022] Open
Abstract
Glycogen and starch are the major carbon and energy reserve polysaccharides in nature, providing living organisms with a survival advantage. The evolution of the enzymatic machinery responsible for the biosynthesis and degradation of such polysaccharides, led the development of mechanisms to control the assembly and disassembly rate, to store and recover glucose according to cell energy demands. The tetrameric enzyme ADP-glucose pyrophosphorylase (AGPase) catalyzes and regulates the initial step in the biosynthesis of both α-polyglucans. AGPase displays cooperativity and allosteric regulation by sensing metabolites from the cell energy flux. The understanding of the allosteric signal transduction mechanisms in AGPase arises as a long-standing challenge. In this work, we disclose the cryoEM structures of the paradigmatic homotetrameric AGPase from Escherichia coli (EcAGPase), in complex with either positive or negative physiological allosteric regulators, fructose-1,6-bisphosphate (FBP) and AMP respectively, both at 3.0 Å resolution. Strikingly, the structures reveal that FBP binds deeply into the allosteric cleft and overlaps the AMP site. As a consequence, FBP promotes a concerted conformational switch of a regulatory loop, RL2, from a "locked" to a "free" state, modulating ATP binding and activating the enzyme. This notion is strongly supported by our complementary biophysical and bioinformatics evidence, and a careful analysis of vast enzyme kinetics data on single-point mutants of EcAGPase. The cryoEM structures uncover the residue interaction networks (RIN) between the allosteric and the catalytic components of the enzyme, providing unique details on how the signaling information is transmitted across the tetramer, from which cooperativity emerges. Altogether, the conformational states visualized by cryoEM reveal the regulatory mechanism of EcAGPase, laying the foundations to understand the allosteric control of bacterial glycogen biosynthesis at the molecular level of detail.
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Key Words
- AGPase, ADP-glucose pyrophosphorylase
- AMP, adenosine 5′-monophosphate
- ATP, adenosine 5′-triphosphate
- EcAGPase, AGPase from E. coli
- Enzyme allosterism
- FBP, fructose 1,6-bisphosphate
- G1P, α-d-glucose-1-phosphate
- GBE, glycogen branching enzyme
- GDE, glycogen debranching enzyme
- GP, glycogen phosphorylase
- GS, glycogen synthase
- GTA-like, glycosyltransferase-A like domain
- Glycogen biosynthesis
- Glycogen regulation
- LβH, left-handed β-helix domain
- Nucleotide sugar biosynthesis
- PPi, pyrophosphate
- RIN, residue interaction network
- SM, sensory motif
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Affiliation(s)
- Javier O. Cifuente
- Structural Biology Unit, CIC BioGUNE, Bizkaia Technology Park, 48160, Derio, Spain
| | - Natalia Comino
- Structural Biology Unit, CIC BioGUNE, Bizkaia Technology Park, 48160, Derio, Spain
| | - Cecilia D'Angelo
- Structural Biology Unit, CIC BioGUNE, Bizkaia Technology Park, 48160, Derio, Spain
| | - Alberto Marina
- Structural Biology Unit, CIC BioGUNE, Bizkaia Technology Park, 48160, Derio, Spain
| | - David Gil-Carton
- Structural Biology Unit, CIC BioGUNE, Bizkaia Technology Park, 48160, Derio, Spain
| | - David Albesa-Jové
- Structural Biology Unit, CIC BioGUNE, Bizkaia Technology Park, 48160, Derio, Spain
| | - Marcelo E. Guerin
- Structural Biology Unit, CIC BioGUNE, Bizkaia Technology Park, 48160, Derio, Spain
- IKERBASQUE, Basque Foundation for Science, 48013, Bilbao, Spain
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9
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Horsefield S, Burdett H, Zhang X, Manik MK, Shi Y, Chen J, Qi T, Gilley J, Lai JS, Rank MX, Casey LW, Gu W, Ericsson DJ, Foley G, Hughes RO, Bosanac T, von Itzstein M, Rathjen JP, Nanson JD, Boden M, Dry IB, Williams SJ, Staskawicz BJ, Coleman MP, Ve T, Dodds PN, Kobe B. NAD + cleavage activity by animal and plant TIR domains in cell death pathways. Science 2019; 365:793-799. [PMID: 31439792 DOI: 10.1126/science.aax1911] [Citation(s) in RCA: 334] [Impact Index Per Article: 55.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2019] [Accepted: 07/23/2019] [Indexed: 02/02/2023]
Abstract
SARM1 (sterile alpha and TIR motif containing 1) is responsible for depletion of nicotinamide adenine dinucleotide in its oxidized form (NAD+) during Wallerian degeneration associated with neuropathies. Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors recognize pathogen effector proteins and trigger localized cell death to restrict pathogen infection. Both processes depend on closely related Toll/interleukin-1 receptor (TIR) domains in these proteins, which, as we show, feature self-association-dependent NAD+ cleavage activity associated with cell death signaling. We further show that SARM1 SAM (sterile alpha motif) domains form an octamer essential for axon degeneration that contributes to TIR domain enzymatic activity. The crystal structures of ribose and NADP+ (the oxidized form of nicotinamide adenine dinucleotide phosphate) complexes of SARM1 and plant NLR RUN1 TIR domains, respectively, reveal a conserved substrate binding site. NAD+ cleavage by TIR domains is therefore a conserved feature of animal and plant cell death signaling pathways.
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Affiliation(s)
- Shane Horsefield
- School of Chemistry and Molecular Biosciences, Institute for Molecular Bioscience and Australian Infectious Diseases Research Centre, University of Queensland, Brisbane, QLD 4072, Australia
| | - Hayden Burdett
- School of Chemistry and Molecular Biosciences, Institute for Molecular Bioscience and Australian Infectious Diseases Research Centre, University of Queensland, Brisbane, QLD 4072, Australia
| | - Xiaoxiao Zhang
- Agriculture and Food, Commonwealth Scientific and Industrial Research Organisation, Canberra, ACT 2601, Australia.,Plant Sciences Division, Research School of Biology, The Australian National University, Canberra ACT 2601, Australia
| | - Mohammad K Manik
- School of Chemistry and Molecular Biosciences, Institute for Molecular Bioscience and Australian Infectious Diseases Research Centre, University of Queensland, Brisbane, QLD 4072, Australia
| | - Yun Shi
- Institute for Glycomics, Griffith University, Southport, QLD 4222, Australia
| | - Jian Chen
- Agriculture and Food, Commonwealth Scientific and Industrial Research Organisation, Canberra, ACT 2601, Australia.,Plant Sciences Division, Research School of Biology, The Australian National University, Canberra ACT 2601, Australia
| | - Tiancong Qi
- Department of Plant and Microbial Biology, University of California Berkeley, Berkeley, CA 94720, USA
| | - Jonathan Gilley
- John van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, ED Adrian Building, Forvie Site, Robinson Way, Cambridge CB2 0PY, UK.,Babraham Institute, Babraham, Cambridge CB22 3AT, UK
| | - Jhih-Siang Lai
- School of Chemistry and Molecular Biosciences, Institute for Molecular Bioscience and Australian Infectious Diseases Research Centre, University of Queensland, Brisbane, QLD 4072, Australia
| | - Maxwell X Rank
- School of Chemistry and Molecular Biosciences, Institute for Molecular Bioscience and Australian Infectious Diseases Research Centre, University of Queensland, Brisbane, QLD 4072, Australia
| | - Lachlan W Casey
- School of Chemistry and Molecular Biosciences, Institute for Molecular Bioscience and Australian Infectious Diseases Research Centre, University of Queensland, Brisbane, QLD 4072, Australia.,Centre for Microscopy and Microanalysis, University of Queensland, Brisbane, QLD 4072, Australia
| | - Weixi Gu
- School of Chemistry and Molecular Biosciences, Institute for Molecular Bioscience and Australian Infectious Diseases Research Centre, University of Queensland, Brisbane, QLD 4072, Australia
| | - Daniel J Ericsson
- Macromolecular Crystallography (MX) Beamlines, Australian Synchrotron, Melbourne, VIC 3168, Australia
| | - Gabriel Foley
- School of Chemistry and Molecular Biosciences, Institute for Molecular Bioscience and Australian Infectious Diseases Research Centre, University of Queensland, Brisbane, QLD 4072, Australia
| | - Robert O Hughes
- Disarm Therapeutics, 400 Technology Square, Cambridge, MA 02139, USA
| | - Todd Bosanac
- Disarm Therapeutics, 400 Technology Square, Cambridge, MA 02139, USA
| | - Mark von Itzstein
- Institute for Glycomics, Griffith University, Southport, QLD 4222, Australia
| | - John P Rathjen
- Plant Sciences Division, Research School of Biology, The Australian National University, Canberra ACT 2601, Australia
| | - Jeffrey D Nanson
- School of Chemistry and Molecular Biosciences, Institute for Molecular Bioscience and Australian Infectious Diseases Research Centre, University of Queensland, Brisbane, QLD 4072, Australia
| | - Mikael Boden
- School of Chemistry and Molecular Biosciences, Institute for Molecular Bioscience and Australian Infectious Diseases Research Centre, University of Queensland, Brisbane, QLD 4072, Australia
| | - Ian B Dry
- Agriculture and Food, Commonwealth Scientific and Industrial Research Organisation, Urrbrae, SA 5064, Australia
| | - Simon J Williams
- Plant Sciences Division, Research School of Biology, The Australian National University, Canberra ACT 2601, Australia
| | - Brian J Staskawicz
- Department of Plant and Microbial Biology, University of California Berkeley, Berkeley, CA 94720, USA
| | - Michael P Coleman
- John van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, ED Adrian Building, Forvie Site, Robinson Way, Cambridge CB2 0PY, UK.,Babraham Institute, Babraham, Cambridge CB22 3AT, UK
| | - Thomas Ve
- School of Chemistry and Molecular Biosciences, Institute for Molecular Bioscience and Australian Infectious Diseases Research Centre, University of Queensland, Brisbane, QLD 4072, Australia. .,Institute for Glycomics, Griffith University, Southport, QLD 4222, Australia
| | - Peter N Dodds
- Agriculture and Food, Commonwealth Scientific and Industrial Research Organisation, Canberra, ACT 2601, Australia.
| | - Bostjan Kobe
- School of Chemistry and Molecular Biosciences, Institute for Molecular Bioscience and Australian Infectious Diseases Research Centre, University of Queensland, Brisbane, QLD 4072, Australia.
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10
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Tian S, Wang C, Yang L, Zhang Y, Tang T. Comparison of Five Extraction Methods for Intracellular Metabolites of Salmonella typhimurium. Curr Microbiol 2019; 76:1247-1255. [PMID: 31375861 DOI: 10.1007/s00284-019-01750-4] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2019] [Revised: 06/18/2019] [Accepted: 07/24/2019] [Indexed: 11/25/2022]
Abstract
Salmonella enterica serovar typhimurium (S. typhimurium) causes food poisoning in human and animals. Its infection rate is the highest among all salmonella serotypes. Metabolomics is a potential way to study the pathogenesis of S. typhimurium via analysis of various small molecular substances. Due to the lack of a uniform protocol for the extraction of metabolites, we evaluated five commonly used extraction methods including cold methanol (CM), hot ethanol (HE), chloroform-methanol cocktail (CMC), perchloric acid (PCA), and alkali (AL) for their efficacy in extracting the intracellular metabolites of S. typhimurium. Samples were quenched in 60% methanol at - 40 °C, and then the five methods were used to extract the metabolites. After derivatization, all samples were analyzed on a gas chromatography-triple quadrupole mass spectrometry (GC-MS/MS). Our results suggest that CM and HE extraction methods provide the best compromise allowing identification of 98 and 95 metabolites in a single analysis. For targeted metabolome analysis, the optimal extraction method for alcohols and organic acids is HE. CMC preferentially extracted lipid metabolites. PCA is suitable for extraction of small molecular carbohydrates. The optimal extraction method for macromolecular carbohydrates is the CM method.
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Affiliation(s)
- Sicheng Tian
- Department of Laboratory Sciences of Public Health, West China School of Public Health and West China Fourth Hospital, Sichuan University, 16#, Section 3, South Renmin Road, West China, Chengdu, People's Republic of China
| | - Chuan Wang
- Department of Laboratory Sciences of Public Health, West China School of Public Health and West China Fourth Hospital, Sichuan University, 16#, Section 3, South Renmin Road, West China, Chengdu, People's Republic of China
| | - Le Yang
- Department of Laboratory Sciences of Public Health, West China School of Public Health and West China Fourth Hospital, Sichuan University, 16#, Section 3, South Renmin Road, West China, Chengdu, People's Republic of China
| | - Yunwen Zhang
- Department of Laboratory Sciences of Public Health, West China School of Public Health and West China Fourth Hospital, Sichuan University, 16#, Section 3, South Renmin Road, West China, Chengdu, People's Republic of China
| | - Tian Tang
- Department of Laboratory Sciences of Public Health, West China School of Public Health and West China Fourth Hospital, Sichuan University, 16#, Section 3, South Renmin Road, West China, Chengdu, People's Republic of China.
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Quantitative Profiling of Endogenous Metabolites Using Hydrophilic Interaction Liquid Chromatography-Tandem Mass Spectrometry (HILIC-MS/MS). Methods Mol Biol 2019; 1859:185-207. [PMID: 30421230 DOI: 10.1007/978-1-4939-8757-3_10] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
Abstract
Dynamic modeling of metabolic reaction networks requires absolute quantification of intracellular and extracellular metabolite concentrations with high precision and accuracy. This chapter presents a robust HILIC-ESI-MS/MS procedure for targeted quantitative profiling of more than 50 polar key metabolites in multicomponent endogenous extracts. Without using ion-pairing-agents or prior derivatization protocols, organic acids, amino acids, sugar phosphates, coenzymes, and nucleotides are measured on a triple quadrupole platform in positive and negative electrospray ionization modes with preoptimized MRM transitions. Robust polymer-based zwitterionic stationary phases (ZIC®-pHILIC) support alkaline mobile phase conditions (pH 9.2) for enhancing retention and chromatographic performance of polar analytes in bicratic elution mode without unfavourable column bleed. The quality of the method was extensively validated and demonstrated by absolute metabolite quantification in endogenous Escherichia coli extracts by comparative use of standard-based external calibration, isotope dilution, and standard addition as quantification strategies. In sum, alkaline ZIC®-pHILIC chromatography emerged as an efficient approach providing high selectivity and sensitivity for comprehensive metabolic studies.
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12
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Luo XT, Cai BD, Jiang HP, Xiao HM, Yuan BF, Feng YQ. Sensitive analysis of trehalose-6-phosphate and related sugar phosphates in plant tissues by chemical derivatization combined with hydrophilic interaction liquid chromatography-tandem mass spectrometry. J Chromatogr A 2019; 1592:82-90. [PMID: 30679043 DOI: 10.1016/j.chroma.2019.01.040] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2018] [Revised: 01/10/2019] [Accepted: 01/13/2019] [Indexed: 12/14/2022]
Abstract
Trehalose-6-phosphate (T6P) is an important signaling metabolite that is involved in many physiological processes. However, the mechanism of the biological functions of T6P is not fully understood. Quantification of T6P in plants will be beneficial to elucidate the mechanism. However, it is still a challenge to chromatographically separate and sensitively detect T6P and related sugar phosphates. In the current study, we developed a method for effective separation and sensitive detection of glucose-1-phosphate (G1P), glucose-6-phosphate (G6P), sucrose-6-phosphate (S6P) and T6P in plant tissues by chemical derivatization combined with hydrophilic interaction liquid chromatography-tandem mass spectrometry (ChD-HILIC-MS/MS). With this method, two pairs of isomers (G1P/G6P and S6P/T6P) could be well separated on a HILIC column and sensitively detected by MS with limits of detection (LODs) ranging from 0.1 to 0.6 ng mL-1. The developed method was successfully applied to the detection of endogenous G1P, G6P, S6P and T6P in small amounts of plant tissues, such as 1 mg fresh weight of Oryza sativa shoot.
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Affiliation(s)
- Xiao-Tong Luo
- Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan, 430072, China
| | - Bao-Dong Cai
- Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan, 430072, China
| | - Han-Peng Jiang
- Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan, 430072, China
| | - Hua-Ming Xiao
- Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan, 430072, China
| | - Bi-Feng Yuan
- Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan, 430072, China
| | - Yu-Qi Feng
- Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan, 430072, China.
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13
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Ji F, Shen Y, Tang L, Cai Z. Determination of intracellular metabolites concentrations in Escherichia coli under nutrition stress using liquid chromatography-tandem mass spectrometry. Talanta 2018; 189:1-7. [DOI: 10.1016/j.talanta.2018.06.050] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2018] [Revised: 06/07/2018] [Accepted: 06/13/2018] [Indexed: 11/26/2022]
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Quenching for Microalgal Metabolomics: A Case Study on the Unicellular Eukaryotic Green Alga Chlamydomonas reinhardtii. Metabolites 2018; 8:metabo8040072. [PMID: 30384421 PMCID: PMC6315863 DOI: 10.3390/metabo8040072] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2018] [Revised: 10/25/2018] [Accepted: 10/29/2018] [Indexed: 11/30/2022] Open
Abstract
Capturing a valid snapshot of the metabolome requires rapid quenching of enzyme activities. This is a crucial step in order to halt the constant flux of metabolism and high turnover rate of metabolites. Quenching with cold aqueous methanol is treated as a gold standard so far, however, reliability of metabolomics data obtained is in question due to potential problems connected to leakage of intracellular metabolites. Therefore, we investigated the influence of various parameters such as quenching solvents, methanol concentration, inclusion of buffer additives, quenching time and solvent to sample ratio on intracellular metabolite leakage from Chlamydomonas reinhardtii. We measured the recovery of twelve metabolite classes using gas chromatography mass spectrometry (GC-MS) in all possible fractions and established mass balance to trace the fate of metabolites during quenching treatments. Our data demonstrate significant loss of intracellular metabolites with the use of the conventional 60% methanol, and that an increase in methanol concentration or quenching time also resulted in higher leakage. Inclusion of various buffer additives showed 70 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) to be suitable. In summary, we recommend quenching with 60% aqueous methanol supplemented with 70 mM HEPES (−40 °C) at 1:1 sample to quenching solvent ratio, as it resulted in higher recoveries for intracellular metabolites with subsequent reduction in the metabolite leakage for all metabolite classes.
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15
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Optimization of the quenching and extraction procedures for a metabolomic analysis of Lactobacillus plantarum. Anal Biochem 2018; 557:62-68. [DOI: 10.1016/j.ab.2017.12.005] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2017] [Revised: 11/22/2017] [Accepted: 12/06/2017] [Indexed: 12/21/2022]
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16
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MOU H, HONG M, LIU XY, LI MC, HUANG MZ, CHU J, ZHUANG YP, ZHANG SL. Accurate Determination of Isotopic Abundance of Intracellular Metabolites of Saccharopolysporaerythraea Based on Ultra Performance Liquid Chromatography-Triple Quadrupole Mass Spectrometry. CHINESE JOURNAL OF ANALYTICAL CHEMISTRY 2017. [DOI: 10.1016/s1872-2040(17)61036-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
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17
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Busse C, Biechele P, de Vries I, Reardon KF, Solle D, Scheper T. Sensors for disposable bioreactors. Eng Life Sci 2017; 17:940-952. [PMID: 32624843 DOI: 10.1002/elsc.201700049] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2017] [Revised: 05/24/2017] [Accepted: 07/14/2017] [Indexed: 12/23/2022] Open
Abstract
Modern bioprocess monitoring demands sensors that provide on-line information about the process state. In particular, sensors for monitoring bioprocesses carried out in single-use bioreactors are needed because disposable systems are becoming increasingly important for biotechnological applications. Requirements for the sensors used in these single-use bioreactors are different than those used in classical reusable bioreactors. For example, long lifetime or resistance to steam and cleaning procedures are less crucial factors, while a requirement of sensors for disposable bioreactors is a cost that is reasonable on a per-use basis. Here, we present an overview of current and emerging sensors for single-use bioreactors, organized by the type of interface of the sensor systems to the bioreactor. A major focus is on non-invasive, in-situ sensors that are based on electromagnetic, semiconducting, optical, or ultrasonic measurements. In addition, new technologies like radio-frequency identification sensors or free-floating sensor spheres are presented. Notably, at this time there is no standard interface between single-use bioreactors and the sensors discussed here. In the future, manufacturers should address this shortcoming to promote single-use bioprocess monitoring and control.
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Affiliation(s)
- Christoph Busse
- Institute of Technical Chemistry Leibniz University Hannover Germany
| | - Philipp Biechele
- Institute of Technical Chemistry Leibniz University Hannover Germany
| | - Ingo de Vries
- Institute of Technical Chemistry Leibniz University Hannover Germany
| | - Kenneth F Reardon
- Department of Chemical and Biological Engineering Colorado State University USA
| | - Dörte Solle
- Institute of Technical Chemistry Leibniz University Hannover Germany
| | - Thomas Scheper
- Institute of Technical Chemistry Leibniz University Hannover Germany
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18
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Wordofa GG, Kristensen M, Schrübbers L, McCloskey D, Forster J, Schneider K. Quantifying the Metabolome of Pseudomonas taiwanensis VLB120: Evaluation of Hot and Cold Combined Quenching/Extraction Approaches. Anal Chem 2017; 89:8738-8747. [PMID: 28727413 DOI: 10.1021/acs.analchem.7b00793] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
Absolute quantification of free intracellular metabolites is a valuable tool in both pathway discovery and metabolic engineering. In this study, we conducted a comprehensive examination of different hot and cold combined quenching/extraction approaches to extract and quantify intracellular metabolites of Pseudomonas taiwanensis (P. taiwanensis) VLB120 to provide a useful reference data set of absolute intracellular metabolite concentrations. The suitability of commonly used metabolomics tools including a pressure driven fast filtration system followed by combined quenching/extraction techniques (such as cold methanol/acetonitrile/water, hot water, and boiling ethanol/water, as well as cold ethanol/water) were tested and evaluated for P. taiwanensis VLB120 metabolome analysis. In total 94 out of 107 detected intracellular metabolites were quantified using an isotope-ratio-based approach. The quantified metabolites include amino acids, nucleotides, central carbon metabolism intermediates, redox cofactors, and others. The acquired data demonstrate that the pressure driven fast filtration approach followed by boiling ethanol quenching/extraction is the most adequate technique for P. taiwanensis VLB120 metabolome analysis based on quenching efficiency, extraction yields of metabolites, and experimental reproducibility.
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Affiliation(s)
- Gossa G Wordofa
- Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , DK-2800 Lyngby, Denmark
| | - Mette Kristensen
- Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , DK-2800 Lyngby, Denmark
| | - Lars Schrübbers
- Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , DK-2800 Lyngby, Denmark
| | - Douglas McCloskey
- Department of Bioengineering, University of California, San Diego , 9500 Gilman Drive, La Jolla, California 92093-0412, United States
| | - Jochen Forster
- Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , DK-2800 Lyngby, Denmark
| | - Konstantin Schneider
- Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , DK-2800 Lyngby, Denmark
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Chung H, Lee N, Seo JA, Kim YS. Comparative analysis of nonvolatile and volatile metabolites in Lichtheimia ramosa cultivated in different growth media. Biosci Biotechnol Biochem 2017; 81:565-572. [DOI: 10.1080/09168451.2016.1256756] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/20/2022]
Abstract
Abstract
Lichtheimia ramosa is one of the predominant filamentous fungi in Korean traditional nuruk. The nonvolatile and volatile metabolites of L. ramosa cultivated in three growth media: complete medium (CM), potato dextrose broth (PDB), and sabouraud dextrose broth (SDB), were investigated and compared. Among nonvolatile metabolites, serine, lysine, and ornithine increased in CM and PDB cultivated with L. ramosa during the exponential phase. In addition, glucose level increased in CM whereas decreased in PDB and SDB. The major volatile metabolites in the extract samples were acetic acid, ethanol, 3-methyl-2-buten-1-ol, 2-phenylethanol, ethylacetate, 2-furaldehyde, 5-(hydroxymethyl)-2-furaldehyde, 2,3-dihydro-3,5,-dihydroxy-6-methyl-4H-pyran-4-one, and α-humulene. In particular, the levels of volatile metabolites related to makgeolli (e.g., acetic acid, ethanol, and ethyl acetate) were highest in extracts cultivated in CM. On the other hand, the level of 2-phenylethanol was relatively higher in PDB and SDB, possibly due to there being more phenylalanine present in the biomass sample in media.
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Affiliation(s)
- Hyun Chung
- Department of Food Science and Engineering, Ewha Womans University, Seoul, Korea
| | - NaKyeom Lee
- Department of Food Science and Engineering, Ewha Womans University, Seoul, Korea
| | - Jeong-Ah Seo
- School of Systems Biomedical Science, Soongsil University, Seoul, Korea
| | - Young-Suk Kim
- Department of Food Science and Engineering, Ewha Womans University, Seoul, Korea
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20
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Jorge TF, Rodrigues JA, Caldana C, Schmidt R, van Dongen JT, Thomas-Oates J, António C. Mass spectrometry-based plant metabolomics: Metabolite responses to abiotic stress. MASS SPECTROMETRY REVIEWS 2016; 35:620-49. [PMID: 25589422 DOI: 10.1002/mas.21449] [Citation(s) in RCA: 163] [Impact Index Per Article: 18.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/22/2014] [Revised: 10/02/2014] [Accepted: 10/14/2014] [Indexed: 05/08/2023]
Abstract
Metabolomics is one omics approach that can be used to acquire comprehensive information on the composition of a metabolite pool to provide a functional screen of the cellular state. Studies of the plant metabolome include analysis of a wide range of chemical species with diverse physical properties, from ionic inorganic compounds to biochemically derived hydrophilic carbohydrates, organic and amino acids, and a range of hydrophobic lipid-related compounds. This complexitiy brings huge challenges to the analytical technologies employed in current plant metabolomics programs, and powerful analytical tools are required for the separation and characterization of this extremely high compound diversity present in biological sample matrices. The use of mass spectrometry (MS)-based analytical platforms to profile stress-responsive metabolites that allow some plants to adapt to adverse environmental conditions is fundamental in current plant biotechnology research programs for the understanding and development of stress-tolerant plants. In this review, we describe recent applications of metabolomics and emphasize its increasing application to study plant responses to environmental (stress-) factors, including drought, salt, low oxygen caused by waterlogging or flooding of the soil, temperature, light and oxidative stress (or a combination of them). Advances in understanding the global changes occurring in plant metabolism under specific abiotic stress conditions are fundamental to enhance plant fitness and increase stress tolerance. © 2015 Wiley Periodicals, Inc. Mass Spec Rev 35:620-649, 2016.
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Affiliation(s)
- Tiago F Jorge
- Plant Metabolomics Laboratory, Instituto de Tecnologia Química e Biológica António Xavier-Universidade Nova de Lisboa (ITQB-UNL), Avenida República, 2780-157, Oeiras, Portugal
| | - João A Rodrigues
- Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, Avenida Professor Egas Moniz, 1649-028, Lisboa, Portugal
| | - Camila Caldana
- Max-Planck-partner group at the Brazilian Bioethanol Science and Technology Laboratory/CNPEM, 13083-970, Campinas-SP, Brazil
| | - Romy Schmidt
- Institute of Biology I, RWTH Aachen University, Worringerweg 1, 52074, Aachen, Germany
| | - Joost T van Dongen
- Institute of Biology I, RWTH Aachen University, Worringerweg 1, 52074, Aachen, Germany
| | - Jane Thomas-Oates
- Jane Thomas-Oates, Centre of Excellence in Mass Spectrometry, and Department of Chemistry, University of York, Heslington, York, YO10 5DD, UK
| | - Carla António
- Plant Metabolomics Laboratory, Instituto de Tecnologia Química e Biológica António Xavier-Universidade Nova de Lisboa (ITQB-UNL), Avenida República, 2780-157, Oeiras, Portugal
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21
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Gautam CK, Madhav M, Sinha A, Jabez Osborne W. VIT-CMJ2: Endophyte of Agaricus bisporus in Production of Bioactive Compounds. IRANIAN JOURNAL OF BIOTECHNOLOGY 2016; 14:19-24. [PMID: 28959322 PMCID: PMC5435028 DOI: 10.15171/ijb.1287] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/08/2015] [Revised: 12/05/2015] [Accepted: 02/15/2016] [Indexed: 12/29/2022]
Abstract
BACKGROUND Agaricus bisporus is an edible basidiomycete fungus. Both the body and the mycelium contain compounds comprising a wide range of antimicrobial molecules, contributing in improvement of immunity and tumor-retardation. OBJECTIVES The presence of endophytes capable of producing bioactive compounds was investigated in Agaricus bisporus. MATERIALS AND METHODS Endophytes from Agaricus bisporus was isolated on LB agar. The obtained isolates were characterized morphologically and biochemically. Further 16S rRNA sequencing was implemented for molecular analysis of isolates. The isolate was mass produced and the bioactive compounds were extracted using ethyl acetate, chloroform and hexane. Agar well diffusion method was carried out to seek the potential of any antimicrobial activity of the crude bioactive compounds against known pathogens. GC-MS and FT-IR analysis were performed for the identification of bioactive compounds. RESULTS VIT-CMJ2 was identified as Enterobacter sp. as revealed by 16S rRNA sequencing. Chloroform extract of VIT-CMJ2 showed a maximum zone of inhibition of 19 mm against Salmonella typhi followed by hexane and ethyl acetate extracts. The GC-MS analysis revealed the presence of several bioactive compounds having effective antimicrobial activity like butyl ester, Behenicalcohol, S , S-dioxide derivatives and some others which were later confirmed by FT-IR spectral stretches. CONCLUSIONS The present study shows the insight on the way endophytes interact with Agaricus bisporus; thereby improving the nutritional profile.
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Affiliation(s)
| | | | | | - William Jabez Osborne
- Department of Biomolecules Lab, School of Bio Sciences and Technology, VIT University, Vellore, India
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22
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Feng B, Guo Z, Zhang W, Pan Y, Zhao Y. Metabolome response to temperature-induced virulence gene expression in two genotypes of pathogenic Vibrio parahaemolyticus. BMC Microbiol 2016; 16:75. [PMID: 27113578 PMCID: PMC4845332 DOI: 10.1186/s12866-016-0688-5] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2015] [Accepted: 04/14/2016] [Indexed: 04/03/2023] Open
Abstract
BACKGROUND Vibrio parahaemolyticus is a main causative agent of serious human seafood-borne gastroenteritis disease. Many researchers have investigated its pathogenesis by observing the alteration of its virulence factors in different conditions. It was previously known that culture conditions will influence the gene expression and the metabolic profile of V. parahaemolyticus, but little attention has been paid on the relationship between them. In this study, for the first time, the metabolomics response in relation to the expression of two major virulence genes, tdh and trh, induced at three temperatures (4, 25 and 37 °C) was examined in two genotypes of pathogenic Vibrio parahaemolyticus (ATCC33846 (tdh+/trh-/tlh+) and ATCC17802 (tdh-/trh+/tlh+)). RESULTS Reverse transcription real-time PCR (RT-qPCR) analysis illustrated that the expression levels of tdh and trh induced at 25 °C in V. parahaemolyticus were significantly higher than those induced at 4 and 37 °C. Principal components analysis (PCA) based on the UPLC & Q-TOF MS data presented clearly distinct groups among the samples treated by different temperatures. Metabolic profiling demonstrated that 179 of 1,033 kinds of identified metabolites in ATCC33846 changed significantly (p <0.01) upon culturing at different temperatures, meanwhile 101 of 930 kinds of metabolites changed (p <0.01) in ATCC17802. Pearson's correlation analysis highlighted the correlation between metabolites and virulence gene expression levels. At the threshold of | r | = 1, p <0.01, 12 kinds of metabolites showed extremely significant correlations with tdh expression, and 4 kinds of metabolites significantly correlated with trh expression. It is interesting that 3D, 7D, 11D-Phytanic acid showed the same trend with pyrophosphate, whose derivative could activate the degradation of phytanic acid. Several metabolites could be sorted into the same class by the method of chemical taxonomy, by assuming that they are involved in the same metabolic pathways. CONCLUSIONS This research can help to find biomarkers to monitor virulence gene expression, and can further help laboratory and clinical research of V. parahaemolyticus from the perspective of metabolomics.
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Affiliation(s)
- Bo Feng
- College of Food Science and Technology, Shanghai Ocean University, No. 999 Hu Cheng Huan Road, Shanghai, China
| | - Zhuoran Guo
- College of Food Science and Technology, Shanghai Ocean University, No. 999 Hu Cheng Huan Road, Shanghai, China
| | - Weijia Zhang
- College of Food Science and Technology, Shanghai Ocean University, No. 999 Hu Cheng Huan Road, Shanghai, China
- Shanghai Engineering Research Center of Aquatic-Product Processing & Preservation, Shanghai, China
- Laboratory of Quality & Safety Risk Assessment for Aquatic Products on Storage and Preservation (Shanghai), Ministry of Agriculture, Shanghai, China
| | - Yingjie Pan
- College of Food Science and Technology, Shanghai Ocean University, No. 999 Hu Cheng Huan Road, Shanghai, China
- Shanghai Engineering Research Center of Aquatic-Product Processing & Preservation, Shanghai, China
- Laboratory of Quality & Safety Risk Assessment for Aquatic Products on Storage and Preservation (Shanghai), Ministry of Agriculture, Shanghai, China
| | - Yong Zhao
- College of Food Science and Technology, Shanghai Ocean University, No. 999 Hu Cheng Huan Road, Shanghai, China.
- Shanghai Engineering Research Center of Aquatic-Product Processing & Preservation, Shanghai, China.
- Laboratory of Quality & Safety Risk Assessment for Aquatic Products on Storage and Preservation (Shanghai), Ministry of Agriculture, Shanghai, China.
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Beri D, Olson DG, Holwerda EK, Lynd LR. Nicotinamide cofactor ratios in engineered strains of Clostridium thermocellum and Thermoanaerobacterium saccharolyticum. FEMS Microbiol Lett 2016; 363:fnw091. [PMID: 27190292 DOI: 10.1093/femsle/fnw091] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 04/07/2016] [Indexed: 12/30/2022] Open
Abstract
Clostridium thermocellum and Thermoanaerobacterium saccharolyticum are bacteria under investigation for production of biofuels from plant biomass. Thermoanaerobacterium saccharolyticum has been engineered to produce ethanol at high yield (>90% of theoretical) and titer (>70 g/l). Efforts to engineer C. thermocellum have not, to date, been as successful, and efforts are underway to transfer the ethanol production pathway from T. saccharolyticum to C. thermocellum One potential challenge in transferring metabolic pathways is the possibility of incompatible levels of nicotinamide cofactors. These cofactors (NAD(+), NADH, NADP(+) and NADPH) and their oxidation state are important in the context of microbial redox metabolism. In this study we directly measured the concentrations and reduced oxidized ratios of these cofactors in a number of strains of C. thermocellum and T. saccharolyticum by using acid/base extraction and enzymatic assays. We found that cofactor ratios are maintained in a fairly narrow range, regardless of the metabolic network modifications considered. We have found that the ratios are similar in both organisms, which is a relevant observation in the context of transferring the T. saccharolyticum ethanol production pathway to C. thermocellum.
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Affiliation(s)
- Dhananjay Beri
- Thayer School of Engineering, Dartmouth College, 14 Engineering Drive, Hanover, NH 03755, USA BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN 37830, USA
| | - Daniel G Olson
- Thayer School of Engineering, Dartmouth College, 14 Engineering Drive, Hanover, NH 03755, USA BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN 37830, USA
| | - Evert K Holwerda
- Thayer School of Engineering, Dartmouth College, 14 Engineering Drive, Hanover, NH 03755, USA BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN 37830, USA
| | - Lee R Lynd
- Thayer School of Engineering, Dartmouth College, 14 Engineering Drive, Hanover, NH 03755, USA BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN 37830, USA
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Kim SJ, Kim SH, Kim JH, Hwang S, Yoo HJ. Understanding Metabolomics in Biomedical Research. Endocrinol Metab (Seoul) 2016; 31:7-16. [PMID: 26676338 PMCID: PMC4803564 DOI: 10.3803/enm.2016.31.1.7] [Citation(s) in RCA: 46] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/27/2015] [Revised: 11/03/2015] [Accepted: 11/10/2015] [Indexed: 02/06/2023] Open
Abstract
The term "omics" refers to any type of specific study that provides collective information on a biological system. Representative omics includes genomics, proteomics, and metabolomics, and new omics is constantly being added, such as lipidomics or glycomics. Each omics technique is crucial to the understanding of various biological systems and complements the information provided by the other approaches. The main strengths of metabolomics are that metabolites are closely related to the phenotypes of living organisms and provide information on biochemical activities by reflecting the substrates and products of cellular metabolism. The transcriptome does not always correlate with the proteome, and the translated proteome might not be functionally active. Therefore, their changes do not always result in phenotypic alterations. Unlike the genome or proteome, the metabolome is often called the molecular phenotype of living organisms and is easily translated into biological conditions and disease states. Here, we review the general strategies of mass spectrometry-based metabolomics. Targeted metabolome or lipidome analysis is discussed, as well as nontargeted approaches, with a brief explanation of the advantages and disadvantages of each platform. Biomedical applications that use mass spectrometry-based metabolomics are briefly introduced.
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Affiliation(s)
- Su Jung Kim
- Biomedical Research Center, Department of Convergence Medicine, Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
| | - Su Hee Kim
- Biomedical Research Center, Department of Convergence Medicine, Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
| | - Ji Hyun Kim
- Biomedical Research Center, Department of Convergence Medicine, Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
| | - Shin Hwang
- Division of Liver Transplantation and Hepatobiliary Surgery, Department of Surgery, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
| | - Hyun Ju Yoo
- Biomedical Research Center, Department of Convergence Medicine, Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea.
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GUO ML, LIU XY, HUANG MZ, LI MC, CHU J, ZHUANG YP, ZHANG SL. 13C-assisted Ultra-High Performance Liquid Chromatography-Triple Quadrupole Mass Spectrometry Method for Precise Determination of Intracellular Metabolites in Pichia pastoris. CHINESE JOURNAL OF ANALYTICAL CHEMISTRY 2016. [DOI: 10.1016/s1872-2040(16)60906-4] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
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Shen Y, Fatemeh T, Tang L, Cai Z. Quantitative metabolic network profiling of Escherichia coli: An overview of analytical methods for measurement of intracellular metabolites. Trends Analyt Chem 2016. [DOI: 10.1016/j.trac.2015.07.006] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/22/2022]
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Nishino S, Okahashi N, Matsuda F, Shimizu H. Absolute quantitation of glycolytic intermediates reveals thermodynamic shifts in Saccharomyces cerevisiae strains lacking PFK1 or ZWF1 genes. J Biosci Bioeng 2015; 120:280-6. [DOI: 10.1016/j.jbiosc.2015.01.012] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2014] [Revised: 12/18/2014] [Accepted: 01/09/2015] [Indexed: 10/23/2022]
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Biechele P, Busse C, Solle D, Scheper T, Reardon K. Sensor systems for bioprocess monitoring. Eng Life Sci 2015. [DOI: 10.1002/elsc.201500014] [Citation(s) in RCA: 120] [Impact Index Per Article: 12.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Affiliation(s)
- Philipp Biechele
- Institute of Technical Chemistry; Leibniz University; Hannover Germany
| | - Christoph Busse
- Institute of Technical Chemistry; Leibniz University; Hannover Germany
| | - Dörte Solle
- Institute of Technical Chemistry; Leibniz University; Hannover Germany
| | - Thomas Scheper
- Institute of Technical Chemistry; Leibniz University; Hannover Germany
| | - Kenneth Reardon
- Department of Chemical and Biological Engineering; Colorado State University; Fort Collins CO USA
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Recent advances in targeting coenzyme A biosynthesis and utilization for antimicrobial drug development. Biochem Soc Trans 2015; 42:1080-6. [PMID: 25110006 DOI: 10.1042/bst20140131] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
The biosynthesis and utilization of CoA (coenzyme A), the ubiquitous and essential acyl carrier in all organisms, have long been regarded as excellent targets for the development of new antimicrobial drugs. Moreover, bioinformatics and biochemical studies have highlighted significant differences between several of the bacterial enzyme targets and their human counterparts, indicating that selective inhibition of the former should be possible. Over the past decade, a large amount of structural and mechanistic data has been gathered on CoA metabolism and the CoA biosynthetic enzymes, and this has facilitated the discovery and development of several promising candidate antimicrobial agents. These compounds include both target-specific inhibitors, as well as CoA antimetabolite precursors that can reduce CoA levels and interfere with processes that are dependent on this cofactor. In the present mini-review we provide an overview of the most recent of these studies that, taken together, have also provided chemical validation of CoA biosynthesis and utilization as viable targets for antimicrobial drug development.
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Marriott AS, António C, Thomas-Oates J. Application of Carbonaceous Materials in Separation Science. POROUS CARBON MATERIALS FROM SUSTAINABLE PRECURSORS 2015. [DOI: 10.1039/9781782622277-00103] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/29/2023]
Abstract
Porous carbons in the separation sciences occupy an important niche owing to their unique retention characteristics, chemical stability and the ability to control pore structure through template strategies. However, these same synthetic processes utilise oil-based carbonising resins and high temperature, energy-intensive pyrolysis steps to ensure the carbon product has pore-size regularity, minimal micropore content and homogeneous surface chemistry. This chapter will primarily focus on the development of porous carbons for application as chromatographic stationary phases. Discussion will cover the unique characteristics of the porous carbon retention mechanism and its application in separating a broad range of analyte classes. The chapter then moves on to describe the current disadvantages in the manufacture of commercially available carbon phase and then highlight recent efforts aimed at the development of alternative porous carbon stationary phases derived from sustainable carbon precursors.
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Affiliation(s)
| | - Carla António
- Plant Metabolomics Laboratory, Instituto de Tecnologia Química e Biológica António Xavier-Universidade Nova de Lisboa (ITQB-UNL) Av. República 2780-157 Oeiras Portugal
| | - Jane Thomas-Oates
- Department of Chemistry, University of York York YO10 5DD UK
- Centre of Excellence in Mass Spectrometry, University of York York YO10 5DD UK
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Chu DB, Troyer C, Mairinger T, Ortmayr K, Neubauer S, Koellensperger G, Hann S. Isotopologue analysis of sugar phosphates in yeast cell extracts by gas chromatography chemical ionization time-of-flight mass spectrometry. Anal Bioanal Chem 2015; 407:2865-75. [PMID: 25673246 DOI: 10.1007/s00216-015-8521-9] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2014] [Revised: 01/17/2015] [Accepted: 01/27/2015] [Indexed: 01/05/2023]
Abstract
Metabolic flux analysis is based on the measurement of isotopologue ratios. In this work, a new GC-MS-based method was introduced enabling accurate determination of isotopologue distributions of sugar phosphates in cell extracts. A GC-TOFMS procedure was developed involving a two-step online derivatization (ethoximation followed by trimethylsilylation) offering high mass resolution, high mass accuracy and the potential of retrospective data analysis typical for TOFMS. The information loss due to fragmentation intrinsic for isotopologue analysis by electron ionization could be overcome by chemical ionization with methane. A thorough optimization regarding pressure of the reaction gas, emission current, electron energy and temperature of the ion source was carried out. For a substantial panel of sugar phosphates both of the glycolysis and the pentose phosphate pathway, sensitive determination of the protonated intact molecular ions together with low abundance fragment ions was successfully achieved. The developed method was evaluated for analysis of Pichia pastoris cell extracts. The measured isotopologue ratios were in the range of 55:1-2:1. The comparison of the experimental isotopologue fractions with the theoretical fractions was excellent, revealing a maximum bias of 4.6% and an average bias of 1.4%.
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Affiliation(s)
- Dinh Binh Chu
- Division of Analytical Chemistry, Department of Chemistry, University of Natural Resources and Life Sciences, BOKU Vienna, Muthgasse 18, 1190, Vienna, Austria
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Alkaline conditions in hydrophilic interaction liquid chromatography for intracellular metabolite quantification using tandem mass spectrometry. Anal Biochem 2015; 475:4-13. [PMID: 25600449 DOI: 10.1016/j.ab.2015.01.002] [Citation(s) in RCA: 67] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2014] [Revised: 12/08/2014] [Accepted: 01/05/2015] [Indexed: 11/22/2022]
Abstract
Modeling of metabolic networks as part of systems metabolic engineering requires reliable quantitative experimental data of intracellular concentrations. The hydrophilic interaction liquid chromatography-electrospray ionization-tandem mass spectrometry (HILIC-ESI-MS/MS) method was used for quantitative profiling of more than 50 hydrophilic key metabolites of cellular metabolism. Without prior derivatization, sugar phosphates, organic acids, nucleotides, and amino acids were measured under alkaline and acidic mobile phase conditions with pre-optimized multiple reaction monitoring (MRM) transitions. Irrespective of the polarity mode of the acquisition method used, alkaline conditions achieved the best quantification limits and linear dynamic ranges. Fully 90% of the analyzed metabolites presented detection limits better than 0.5pmol (on column), and 70% presented 1.5-fold higher signal intensities under alkaline mobile phase conditions. The quality of the method was further demonstrated by absolute quantification of selected metabolites in intracellular extracts of Escherichia coli. In addition, quantification bias caused by matrix effects was investigated by comparison of calibration strategies: standard-based external calibration, isotope dilution, and standard addition with internal standards. Here, we recommend the use of alkaline mobile phase with polymer-based zwitterionic hydrophilic interaction chromatography (ZIC-pHILIC) as the most sensitive scenario for absolute quantification for a broad range of metabolites.
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Lieder S, Jahn M, Seifert J, von Bergen M, Müller S, Takors R. Subpopulation-proteomics reveal growth rate, but not cell cycling, as a major impact on protein composition in Pseudomonas putida KT2440. AMB Express 2014; 4:71. [PMID: 25401072 PMCID: PMC4230896 DOI: 10.1186/s13568-014-0071-6] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2014] [Accepted: 08/18/2014] [Indexed: 12/14/2022] Open
Abstract
Population heterogeneity occurring in industrial microbial bioprocesses is regarded as a putative effector causing performance loss in large scale. While the existence of subpopulations is a commonly accepted fact, their appearance and impact on process performance still remains rather unclear. During cell cycling, distinct subpopulations differing in cell division state and DNA content appear which contribute individually to the efficiency of the bioprocess. To identify stressed or impaired subpopulations, we analyzed the interplay of growth rate, cell cycle and phenotypic profile of subpopulations by using flow cytometry and cell sorting in conjunction with mass spectrometry based global proteomics. Adjusting distinct growth rates in chemostats with the model strain Pseudomonas putida KT2440, cells were differentiated by DNA content reflecting different cell cycle stages. The proteome of separated subpopulations at given growth rates was found to be highly similar, while different growth rates caused major changes of the protein inventory with respect to e.g. carbon storage, motility, lipid metabolism and the translational machinery. In conclusion, cells in various cell cycle stages at the same growth rate were found to have similar to identical proteome profiles showing no significant population heterogeneity on the proteome level. In contrast, the growth rate clearly determines the protein composition and therefore the metabolic strategy of the cells.
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Affiliation(s)
- Sarah Lieder
- Institute for Biochemical Engineering, University of Stuttgart, Allmandring 31, Stuttgart, Germany
| | - Michael Jahn
- Department of Environmental Microbiology, Helmholtz Centre for Environmental Research—UFZ, Permoserstr. 15, Leipzig, 04318, Germany
| | - Jana Seifert
- Department of Proteomics, Helmholtz Centre for Environmental Research—UFZ, Permoserstr. 15, Leipzig, 04318, Germany
- Institute of Animal Nutrition, University of Hohenheim, Emil-Wolff-Straße 8 and 10, Stuttgart, 70599, Germany
| | - Martin von Bergen
- Department of Proteomics, Helmholtz Centre for Environmental Research—UFZ, Permoserstr. 15, Leipzig, 04318, Germany
- Department of Metabolomics, Helmholtz Centre for Environmental Research—UFZ, Permoserstr. 15, Leipzig, 04318, Germany
- Department of Biotechnology, Chemistry and Environmental Engineering, University of Aalborg, Sohngaardsholmsvej 49, Aalborg, 9000, Denmark
| | - Susann Müller
- Department of Environmental Microbiology, Helmholtz Centre for Environmental Research—UFZ, Permoserstr. 15, Leipzig, 04318, Germany
| | - Ralf Takors
- Institute for Biochemical Engineering, University of Stuttgart, Allmandring 31, Stuttgart, Germany
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Jayavelu ND, Bar NS. Metabolomic studies of human gastric cancer: Review. World J Gastroenterol 2014; 20:8092-8101. [PMID: 25009381 PMCID: PMC4081680 DOI: 10.3748/wjg.v20.i25.8092] [Citation(s) in RCA: 50] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/19/2013] [Revised: 07/20/2013] [Accepted: 08/06/2013] [Indexed: 02/06/2023] Open
Abstract
Metabolomics is a field of study in systems biology that involves the identification and quantification of metabolites present in a biological system. Analyzing metabolic differences between unperturbed and perturbed networks, such as cancerous and non-cancerous samples, can provide insight into underlying disease pathology, disease prognosis and diagnosis. Despite the large number of review articles concerning metabolomics and its application in cancer research, biomarker and drug discovery, these reviews do not focus on a specific type of cancer. Metabolomics may provide biomarkers useful for identification of early stage gastric cancer, potentially addressing an important clinical need. Here, we present a short review on metabolomics as a tool for biomarker discovery in human gastric cancer, with a primary focus on its use as a predictor of anticancer drug chemosensitivity, diagnosis, prognosis, and metastasis.
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Mushtaq MY, Choi YH, Verpoorte R, Wilson EG. Extraction for metabolomics: access to the metabolome. PHYTOCHEMICAL ANALYSIS : PCA 2014; 25:291-306. [PMID: 24523261 DOI: 10.1002/pca.2505] [Citation(s) in RCA: 103] [Impact Index Per Article: 9.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/19/2013] [Revised: 12/21/2013] [Accepted: 12/26/2013] [Indexed: 05/24/2023]
Abstract
INTRODUCTION The value of information obtained from a metabolomic study depends on how much of the metabolome is present in analysed samples. Thus, only a comprehensive and reproducible extraction method will provide reliable data because the metabolites that will be measured are those that were extracted and all conclusions will be built around this information. OBJECTIVE To discuss the efficiency and reliability of available sample pre-treatment methods and their application in different fields of metabolomics. METHODS The review has three sections: the first deals with pre-extraction techniques, the second discusses the choice of extraction solvents and their main features and the third includes a brief description of the most used extraction techniques: microwave-assisted extraction, solid-phase extraction, supercritical fluid extraction, Soxhlet and a new method developed in our laboratory--the comprehensive extraction method. RESULTS Examination of over 200 studies showed that sample collection, homogenisation, grinding and storage could affect the yield and reproducibility of results. They also revealed that apart from the solvent used for extraction, the extraction techniques have a decisive role on the metabolites available for analysis. CONCLUSION It is essential to evaluate efficacy and reproducibility of sample pre-treatment as a first step to ensure the reliability of a metabolomic study. Among the reviewed methods, the comprehensive extraction method appears to provide a promising approach for extracting diverse types of metabolites.
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Affiliation(s)
- Mian Yahya Mushtaq
- Natural Products Laboratory, Institute of Biology, Leiden University, 2300 RA, Leiden, The Netherlands
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36
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Achbergerová L, Nahálka J. Degradation of polyphosphates by polyphosphate kinases from Ruegeria pomeroyi. Biotechnol Lett 2014; 36:2029-35. [DOI: 10.1007/s10529-014-1566-6] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2014] [Accepted: 05/19/2014] [Indexed: 11/25/2022]
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Hernández Bort JA, Shanmukam V, Pabst M, Windwarder M, Neumann L, Alchalabi A, Krebiehl G, Koellensperger G, Hann S, Sonntag D, Altmann F, Heel C, Borth N. Reduced quenching and extraction time for mammalian cells using filtration and syringe extraction. J Biotechnol 2014; 182-183:97-103. [PMID: 24794799 PMCID: PMC4071440 DOI: 10.1016/j.jbiotec.2014.04.014] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2013] [Revised: 04/08/2014] [Accepted: 04/11/2014] [Indexed: 10/30/2022]
Abstract
In order to preserve the in vivo metabolite levels of cells, a quenching protocol must be quickly executed to avoid degradation of labile metabolites either chemically or biologically. In the case of mammalian cell cultures cultivated in complex media, a wash step previous to quenching is necessary to avoid contamination of the cell pellet with extracellular metabolites, which could distort the real intracellular concentration of metabolites. This is typically achieved either by one or multiple centrifugation/wash steps which delay the time until quenching (even harsh centrifugation requires several minutes for processing until the cells are quenched) or filtration. In this article, we describe and evaluate a two-step optimized protocol based on fast filtration by use of a vacuum pump for quenching and subsequent extraction of intracellular metabolites from CHO (Chinese hamster ovary) suspension cells, which uses commercially available components. The method allows transfer of washed cells into liquid nitrogen within 10-15s of sampling and recovers the entire extraction solution volume. It also has the advantage to remove residual filter filaments in the final sample, thus preventing damage to separation columns during subsequent MS analysis. Relative to other methods currently used in the literature, the resulting energy charge of intracellular adenosine nucleotides was increased to 0.94 compared to 0.90 with cold PBS quenching or 0.82 with cold methanol/AMBIC quenching.
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Affiliation(s)
| | - Vinoth Shanmukam
- ACIB GmbH, Austrian Centre of Industrial Biotechnology, Vienna, Austria; Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria
| | - Martin Pabst
- Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Austria
| | - Markus Windwarder
- Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Austria
| | - Laura Neumann
- Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Austria
| | | | | | - Gunda Koellensperger
- ACIB GmbH, Austrian Centre of Industrial Biotechnology, Vienna, Austria; Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Austria
| | - Stephan Hann
- ACIB GmbH, Austrian Centre of Industrial Biotechnology, Vienna, Austria; Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Austria
| | | | - Friedrich Altmann
- ACIB GmbH, Austrian Centre of Industrial Biotechnology, Vienna, Austria; Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Austria
| | | | - Nicole Borth
- ACIB GmbH, Austrian Centre of Industrial Biotechnology, Vienna, Austria; Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria.
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39
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Assessment of Mycobacterium tuberculosis pantothenate kinase vulnerability through target knockdown and mechanistically diverse inhibitors. Antimicrob Agents Chemother 2014; 58:3312-26. [PMID: 24687493 DOI: 10.1128/aac.00140-14] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Pantothenate kinase (PanK) catalyzes the phosphorylation of pantothenate, the first committed and rate-limiting step toward coenzyme A (CoA) biosynthesis. In our earlier reports, we had established that the type I isoform encoded by the coaA gene is an essential pantothenate kinase in Mycobacterium tuberculosis, and this vital information was then exploited to screen large libraries for identification of mechanistically different classes of PanK inhibitors. The present report summarizes the synthesis and expansion efforts to understand the structure-activity relationships leading to the optimization of enzyme inhibition along with antimycobacterial activity. Additionally, we report the progression of two distinct classes of inhibitors, the triazoles, which are ATP competitors, and the biaryl acetic acids, with a mixed mode of inhibition. Cocrystallization studies provided evidence of these inhibitors binding to the enzyme. This was further substantiated with the biaryl acids having MIC against the wild-type M. tuberculosis strain and the subsequent establishment of a target link with an upshift in MIC in a strain overexpressing PanK. On the other hand, the ATP competitors had cellular activity only in a M. tuberculosis knockdown strain with reduced PanK expression levels. Additionally, in vitro and in vivo survival kinetic studies performed with a M. tuberculosis PanK (MtPanK) knockdown strain indicated that the target levels have to be significantly reduced to bring in growth inhibition. The dual approaches employed here thus established the poor vulnerability of PanK in M. tuberculosis.
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Wang JM, Chu Y, Li W, Wang XY, Guo JH, Yan LL, Ma XH, Ma YL, Yin QH, Liu CX. Simultaneous determination of creatine phosphate, creatine and 12 nucleotides in rat heart by LC-MS/MS. J Chromatogr B Analyt Technol Biomed Life Sci 2014; 958:96-101. [PMID: 24705537 DOI: 10.1016/j.jchromb.2014.03.008] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2013] [Revised: 01/27/2014] [Accepted: 03/05/2014] [Indexed: 11/18/2022]
Abstract
A simple, rapid and sensitive LC-MS/MS method was developed and validated for simultaneous determination of creatine phosphate (CP), creatine (Cr) and 12 nucleotides in rat heart. The analytes, ATP, ADP, AMP, GTP, GDP, GMP, CTP, CDP, CMP, UTP, UDP, UMP, CP, Cr, were extracted from heart tissue with pre-cooled (0°C) methanol/water (1:1, v/v) and separated on a Hypersil Gold AQ C18 column (150mm×4.6mm, 3μm) using an isocratic elution with a mobile phase consisting of 2mmol/L ammonium acetate in water (pH 10.0, adjusted with ammonia). The detection was performed by negative ion electrospray ionization in selective reaction monitoring mode (SRM). In the assay, all the analytes showed good linearity over the investigated concentration range (r>0.99). The accuracy was between 80.7% and 120.6% and the precision expressed in RSD was less than 15.6%. This method was successfully applied to measure the concentrations of the 12 nucleotides, creatine phosphate and creatine in rat heart for the first time.
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Affiliation(s)
- Jun-mei Wang
- Department of Pharmacology and Toxicology, Tasly R&D Institute, Tianjin Tasly Group Co., Ltd, Tianjin 300410, China; Heilongjiang University of Traditional Chinese Medicine, Harbin 150040, China
| | - Yang Chu
- Department of Pharmacology and Toxicology, Tasly R&D Institute, Tianjin Tasly Group Co., Ltd, Tianjin 300410, China
| | - Wei Li
- Department of Pharmacology and Toxicology, Tasly R&D Institute, Tianjin Tasly Group Co., Ltd, Tianjin 300410, China.
| | - Xiang-yang Wang
- Department of Pharmacology and Toxicology, Tasly R&D Institute, Tianjin Tasly Group Co., Ltd, Tianjin 300410, China
| | - Jia-hua Guo
- Department of Pharmacology and Toxicology, Tasly R&D Institute, Tianjin Tasly Group Co., Ltd, Tianjin 300410, China
| | - Lu-lu Yan
- Department of Pharmacology and Toxicology, Tasly R&D Institute, Tianjin Tasly Group Co., Ltd, Tianjin 300410, China
| | - Xiao-hui Ma
- Department of Pharmacology and Toxicology, Tasly R&D Institute, Tianjin Tasly Group Co., Ltd, Tianjin 300410, China
| | - Ying-li Ma
- Heilongjiang University of Traditional Chinese Medicine, Harbin 150040, China
| | - Qi-hui Yin
- Department of Pharmacology and Toxicology, Tasly R&D Institute, Tianjin Tasly Group Co., Ltd, Tianjin 300410, China; Heilongjiang University of Traditional Chinese Medicine, Harbin 150040, China
| | - Chang-xiao Liu
- Tianjin State Key Laboratory of Pharmacokinetics and Pharmacodynamics, Tianjin Institute of Pharmaceutical Research, Tianjin 300193, China
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Szoboszlai N, Guo X, Ozohanics O, Oláh J, Gömöry Á, Mihucz VG, Jeney A, Vékey K. Determination of energy metabolites in cancer cells by porous graphitic carbon liquid chromatography electrospray ionization mass spectrometry for the assessment of energy metabolism. Anal Chim Acta 2014; 819:108-15. [DOI: 10.1016/j.aca.2014.01.055] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2013] [Revised: 01/21/2014] [Accepted: 01/27/2014] [Indexed: 11/29/2022]
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Seifar RM, Ras C, Deshmukh AT, Bekers KM, Suarez-Mendez CA, da Cruz AL, van Gulik WM, Heijnen JJ. Quantitative analysis of intracellular coenzymes in Saccharomyces cerevisiae using ion pair reversed phase ultra high performance liquid chromatography tandem mass spectrometry. J Chromatogr A 2013; 1311:115-20. [DOI: 10.1016/j.chroma.2013.08.076] [Citation(s) in RCA: 46] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2013] [Revised: 08/20/2013] [Accepted: 08/21/2013] [Indexed: 11/17/2022]
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Sasidharan K, Soga T, Tomita M, Murray DB. A yeast metabolite extraction protocol optimised for time-series analyses. PLoS One 2012; 7:e44283. [PMID: 22952947 PMCID: PMC3430680 DOI: 10.1371/journal.pone.0044283] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2012] [Accepted: 07/31/2012] [Indexed: 11/19/2022] Open
Abstract
There is an increasing call for the absolute quantification of time-resolved metabolite data. However, a number of technical issues exist, such as metabolites being modified/degraded either chemically or enzymatically during the extraction process. Additionally, capillary electrophoresis mass spectrometry (CE-MS) is incompatible with high salt concentrations often used in extraction protocols. In microbial systems, metabolite yield is influenced by the extraction protocol used and the cell disruption rate. Here we present a method that rapidly quenches metabolism using dry-ice ethanol bath and methanol N-ethylmaleimide solution (thus stabilising thiols), disrupts cells efficiently using bead-beating and avoids artefacts created by live-cell pelleting. Rapid sample processing minimised metabolite leaching. Cell weight, number and size distribution was used to calculate metabolites to an attomol/cell level. We apply this method to samples obtained from the respiratory oscillation that occurs when yeast are grown continuously.
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Affiliation(s)
- Kalesh Sasidharan
- Institute for Advanced Biosciences, Keio University, Nipponkoku 403-1, Daihouji, Tsuruoka City, Yamagata, Japan
| | - Tomoyoshi Soga
- Institute for Advanced Biosciences, Keio University, Nipponkoku 403-1, Daihouji, Tsuruoka City, Yamagata, Japan
| | - Masaru Tomita
- Institute for Advanced Biosciences, Keio University, Nipponkoku 403-1, Daihouji, Tsuruoka City, Yamagata, Japan
| | - Douglas B. Murray
- Institute for Advanced Biosciences, Keio University, Nipponkoku 403-1, Daihouji, Tsuruoka City, Yamagata, Japan
- * E-mail:
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Rational engineering of enzyme allosteric regulation through sequence evolution analysis. PLoS Comput Biol 2012; 8:e1002612. [PMID: 22807670 PMCID: PMC3395594 DOI: 10.1371/journal.pcbi.1002612] [Citation(s) in RCA: 66] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2012] [Accepted: 05/29/2012] [Indexed: 12/04/2022] Open
Abstract
Control of enzyme allosteric regulation is required to drive metabolic flux toward desired levels. Although the three-dimensional (3D) structures of many enzyme-ligand complexes are available, it is still difficult to rationally engineer an allosterically regulatable enzyme without decreasing its catalytic activity. Here, we describe an effective strategy to deregulate the allosteric inhibition of enzymes based on the molecular evolution and physicochemical characteristics of allosteric ligand-binding sites. We found that allosteric sites are evolutionarily variable and comprised of more hydrophobic residues than catalytic sites. We applied our findings to design mutations in selected target residues that deregulate the allosteric activity of fructose-1,6-bisphosphatase (FBPase). Specifically, charged amino acids at less conserved positions were substituted with hydrophobic or neutral amino acids with similar sizes. The engineered proteins successfully diminished the allosteric inhibition of E. coli FBPase without affecting its catalytic efficiency. We expect that our method will aid the rational design of enzyme allosteric regulation strategies and facilitate the control of metabolic flux. Design of allosterically regulatable enzyme is essential to develop a highly efficient metabolite production. However, mutations on allosteric ligand binding sites often disrupt the catalytic activity of enzyme. To aid the design process of allosterically controllable enzymes, we develop an effective computational strategy to deregulate the allosteric inhibition of enzymes based on sequence evolution analysis of allosteric ligand-binding sites. We analyzed the molecular evolution and amino acid composition of catalytic and allosteric sites of enzymes, and discovered that allosteric sites are evolutionarily variable and comprised of more hydrophobic residues than catalytic sites. We then experimentally tested our strategy of enzyme allosteric regulation and found that the designed mutations effectively deregulated allosteric inhibition of FBPase. We believe that our method will aid the rational design of enzyme allosteric regulation and help to facilitate control of metabolic flux.
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Olavarría K, Valdés D, Cabrera R. The cofactor preference of glucose-6-phosphate dehydrogenase from Escherichia coli--modeling the physiological production of reduced cofactors. FEBS J 2012; 279:2296-309. [PMID: 22519976 DOI: 10.1111/j.1742-4658.2012.08610.x] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
In Escherichia coli, the pentose phosphate pathway is one of the main sources of NADPH. The first enzyme of the pathway, glucose-6-phosphate dehydrogenase (G6PDH), is generally considered an exclusive NADPH producer, but a rigorous assessment of cofactor preference has yet to be reported. In this work, the specificity constants for NADP and NAD for G6PDH were determined using a pure enzyme preparation. Absence of the phosphate group on the cofactor leads to a 410-fold reduction in the performance of the enzyme. Furthermore, the contribution of the phosphate group to binding of the transition state to the active site was calculated to be 3.6 kcal·mol(-1). In order to estimate the main kinetic parameters for NAD(P) and NAD(P)H, we used the classical initial-rates approach, together with an analysis of reaction time courses. To achieve this, we developed a new analytical solution to the integrated Michaelis-Menten equation by including the effect of competitive product inhibition using the ω-function. With reference to relevant kinetic parameters and intracellular metabolite concentrations reported by others, we modeled the sensitivity of reduced cofactor production by G6PDH as a function of the redox ratios of NAD/NADH (rR(NAD)) and NADP/NADPH (rR(NADP)). Our analysis shows that NADPH production sharply increases within the range of thermodynamically feasible values of rR(NADP), but NADH production remains low within the range feasible for rR(NAD). Nevertheless, we show that certain combinations of rR(NADP) and rR(NAD) sustain greater levels of NADH production over NADPH.
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Affiliation(s)
- Karel Olavarría
- Laboratorio de Bioquímica y Biología Molecular, Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Santiago, Chile
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Current trends and challenges in sample preparation for global metabolomics using liquid chromatography-mass spectrometry. Anal Bioanal Chem 2012; 403:1523-48. [PMID: 22576654 DOI: 10.1007/s00216-012-6039-y] [Citation(s) in RCA: 353] [Impact Index Per Article: 27.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2012] [Revised: 03/13/2012] [Accepted: 04/10/2012] [Indexed: 01/26/2023]
Abstract
The choice of sample-preparation method is extremely important in metabolomic studies because it affects both the observed metabolite content and biological interpretation of the data. An ideal sample-preparation method for global metabolomics should (i) be as non-selective as possible to ensure adequate depth of metabolite coverage; (ii) be simple and fast to prevent metabolite loss and/or degradation during the preparation procedure and enable high-throughput; (iii) be reproducible; and (iv) incorporate a metabolism-quenching step to represent true metabolome composition at the time of sampling. Despite its importance, sample preparation is often an overlooked aspect of metabolomics, so the focus of this review is to explore the role, challenges, and trends in sample preparation specifically within the context of global metabolomics by liquid chromatography-mass spectrometry (LC-MS). This review will cover the most common methods including solvent precipitation and extraction, solid-phase extraction and ultrafiltration, and discuss how to improve analytical quality and metabolite coverage in metabolomic studies of biofluids, tissues, and mammalian cells. Recent developments in this field will also be critically examined, including in vivo methods, turbulent-flow chromatography, and dried blood spot sampling.
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Armando JW, Boghigian BA, Pfeifer BA. LC-MS/MS quantification of short-chain acyl-CoA's in Escherichia coli demonstrates versatile propionyl-CoA synthetase substrate specificity. Lett Appl Microbiol 2011; 54:140-8. [PMID: 22118660 DOI: 10.1111/j.1472-765x.2011.03184.x] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/15/2022]
Abstract
AIMS This paper utilized quantitative LC-MS/MS to profile the short-chain acyl-CoA levels of several strains of Escherichia coli engineered for heterologous polyketide production. To further compare and potentially expand the levels of available acyl-CoA molecules, a propionyl-CoA synthetase gene from Ralstonia solanacearum (prpE-RS) was synthesized and expressed in the engineered strain BAP1. METHODS AND RESULTS Upon feeding propionate, the engineered E. coli strains had increased the levels of both propionyl- and methylmalonyl-CoA of 6- to 30-fold and 3·7- to 6·8-fold, respectively. Expression of prpE-RS resulted in no significant increases in acetyl-, butyryl- and propionyl-CoA when fed the corresponding substrates (sodium acetate, butyrate or propionate). More interesting, however, were the results from strain BAP1 engineered for native prpE overexpression, which indicated increases in the same range of acyl-CoA formation. CONCLUSIONS The increased acyl-CoA levels across the strains profiled in this study reflect the genetic modifications implemented for improved polyketide production and also indicate flexibility of the native PrpE. SIGNIFICANCE AND IMPACT OF THE STUDY The results provide direct evidence of enhanced acyl-CoA levels correlating to those strains engineered for polyketide biosynthesis. This information and the inherent flexibility of the native PrpE enzyme support future efforts to characterize, engineer and extend acyl-CoA precursor supply for additional heterologous biosynthetic attempts.
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Affiliation(s)
- J W Armando
- Department of Chemical and Biological Engineering, Tufts University, Medford, MA, USA
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Vielhauer O, Zakhartsev M, Horn T, Takors R, Reuss M. Simplified absolute metabolite quantification by gas chromatography–isotope dilution mass spectrometry on the basis of commercially available source material. J Chromatogr B Analyt Technol Biomed Life Sci 2011; 879:3859-70. [DOI: 10.1016/j.jchromb.2011.10.036] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2011] [Revised: 10/21/2011] [Accepted: 10/25/2011] [Indexed: 02/08/2023]
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Junne S, Klingner A, Kabisch J, Schweder T, Neubauer P. A two-compartment bioreactor system made of commercial parts for bioprocess scale-down studies: impact of oscillations on Bacillus subtilis fed-batch cultivations. Biotechnol J 2011; 6:1009-17. [PMID: 21751400 DOI: 10.1002/biot.201100293] [Citation(s) in RCA: 52] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
This study describes an advanced version of a two-compartment scale-down bioreactor that simulates inhomogeneities present in large-scale industrial bioreactors on the laboratory scale. The system is made of commercially available parts and is suitable for sterilization with steam. The scale-down bioreactor consists of a usual stirred tank bioreactor (STR) and a plug flow reactor (PFR) equipped with static mixer modules. The PFR module with a working volume of 1.2 L is equipped with five sample ports, and pH and dissolved oxygen (DO) sensors. The concept was applied using the non-sporulating Bacillus subtilis mutant strain AS3, characterized by a SpoIIGA gene knockout. In a fed-batch process with a constant feed rate, it is found that oscillating substrate and DO concentration led to diminished glucose uptake, ethanol formation and an altered amino acid synthesis. Sampling at the PFR module allowed the detection of dynamics at different concentrations of intermediates, such as pyruvic acid, lactic acid and amino acids. Results indicate that the carbon flux at excess glucose and low DO concentrations is shifted towards ethanol formation. As a result, the reduced carbon flux entering the tricarboxylic acid cycle is not sufficient to support amino acid synthesis following the oxaloacetic acid branch point.
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Affiliation(s)
- Stefan Junne
- Chair of Bioprocess Engineering, Department of Biotechnology, Technische Universität Berlin, Berlin, Germany.
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Sugimoto T, Bamba T, Izumi Y, Nomura H, Shiina T, Fukusaki E. Use of ultra-performance liquid chromatography/time-of-flight mass spectrometry with nozzle-skimmer fragmentation for comprehensive quantitative analysis of secondary metabolites in Arabidopsis thaliana. J Sep Sci 2011; 34:3587-96. [DOI: 10.1002/jssc.201100552] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2011] [Revised: 09/14/2011] [Accepted: 09/21/2011] [Indexed: 11/09/2022]
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