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Introduction to Metabolomics
What is it and what are its applications? Dr. Matthew Davey
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Focus of the two sessions:
The aim of this course is to provide an overview of the applications, laboratory equipment and online bioinformatic portals for metabolomics research. Plant Bias – all techniques transferable to other organisms
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What is the metabolome? Total quantitative collection of chemical compounds (metabolites) present in an organism eg. sugars, amino acids, phenolics, lipids Not proteins or peptides Highly complex Thorough and unbiased assessment of all metabolites within an organism
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Complexity Physically and chemically complex
Large range of molecular weights 10’s to 100’s MW (and size) Polar and non-polar metabolites Volatiles Variation in number of known metabolites per species Yeast Saccharomyces cerevisiae (584) E. coli (436) Plant kingdom (up to ) Human (2900) Very wide concentration range mM to sub-pM Temporal changes - flux
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What is the metabolome?
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“Metabolomics” first appeared in the literature in 1998 (Fiehn et al
“Metabolomics” first appeared in the literature in 1998 (Fiehn et al Metabolomics) Measuring many metabolites is nothing new, but…the scale of the analysis is Rather than look at individual reactions to understand an organism (reductionist theory) an attempt is made to measure the whole system (systems biology)
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Environment Assign Function Response Expression
Why study the metabolome? – direct link between genetic and environmental signals Environment Assign Function Response Expression
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Why study the metabolome?
Need to understand metabolites and metabolic pathways before we can exploit them Metabolic status of cells provides a clearer indication of health than mRNA or proteins Advance systems biology Trait development in crops eg, salt and drought tolerance; defence; photoprotection Genetic engineering safety – substantial equivalences High value products – cosmetics; medicine Biofuels Plant disease biomarkers Plant population / evolutionary studies Applications Biomarkers for: Disease drug intervention environmental stress Nutrigenomics Personal health assessments Personalised medicine Metabolic engineering
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Why study the metabolome?
Examples of early applications: Diagnosis of coronary heart disease using metabonomics Brindle, J.T. et al.(2002). Rapid and noninvasive diagnosis of the presence and severity of coronary heart disease using 1H-NMR-based metabonomics. Nature Medicine, 8, Metabolite profiling for plant functional genomics Arabidopsis thaliana – model species. Quantified and identified many metabolites and related different genotypes to their metabolic profiles (by GC-MS) Fiehn et al.(2000). Metabolite profiling for plant functional genomics. Nature Biotechnology, 18,
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Why is this application important in natural systems?
Plants Abiotic Biotic Grow (morphological traits) Function (biochemical traits) Time Population spread Natural selection (traits related to fitness eg, survival, reproduction) Local adaptation (variation in traits) Very little is known about variation in such metabolic traits – how do we measure them?
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Techniques used in measuring the metabolome
Dunn et al. 2005
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Sample collection STOP metabolism
How to get metabolites out of the cell Solvent extraction and storage Methanol (hot or cold) Methanol/chloroform/water Hot ethanol Ball milling or grinding with mortar/pestle Store at -80’C Cold methanol Hot Ethanol or Methanol Freeze clamping for plants Liquid nitrogen (-196’C) Spike to check recovery
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Mass Spectrometry (MS)
Detecting metabolites – Metabolic Fingerprinting Mass Spectrometry (MS) Nuclear Magnetic Resonance (NMR) Fourier Transform Infra Red (FT-IR) High throughput screening for metabolic phenotypes
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Metabolite fingerprinting
Arabidopsis petraea Wales Arabidopsis petraea Sweden Davey et al. 2008
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Detecting metabolites – Metabolic Profiling
High Performance Liquid Chromatography (HPLC) – Photodiode array (PDA) – Mass spectrometry (MS) The mirror crack’d: the intense blue colour of Ophrys speculum is produced by both chemical and structural means Silvia Vignolini1,2, Matthew P. Davey1, Julia Tratt3, Svante Malmgren4, Richard Bateman3, Paula Rudall3, Ullrich Steiner2, and Beverley J. Glover1 New Phytologist in press HPLC PDA MS Cyanidin 3- (3-malonyl glucoside) 534.90 448.9 (-malonyl) (-hexose and malonyl) MS/MS
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Metabolic Profiling - Gas Chromatography (GC) – Flame Ionisation Detector (FID)
Triglycerides Free fatty acids Polar lipids Lipid profiling for biofuels
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Output files – can you see the difference?
Mass (Bin) (1000’s) Output files – can you see the difference? Need multivariate statistics Sample name (100’s) Intensity Total Ion Counts (1000’s)
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Multivariate Data Analysis
Unsupervised Principal Component Analysis (PCA) Supervised Partial Least Squares -Discriminant Analysis (PLS-DA) Hierarchical Cluster Analysis (HCA) Trygg et al. 2007
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Metabolite matches and mapping based on mass matching
Large number of online databases for metabolite and network mapping Updated yearly in Nucleic Acids Research Galperin and Fernández-Suárez 2012 Brown et al. 2009 KEGG is the most widely used/known site for metabolic mapping – there are errors but getting better! Another common site is MAPMAN Eg. Search mass
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Can overlay transcriptomic and proteomic data – very complex!
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MetExplore and CytoScape
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Summary Metabolomics – logical progression of genomic and post-genomic science Diverse range of applications – especially trait identification Range of fingerprinting and profiling techniques Large datasets require multivariate statistics Large number of online databases for metabolite identification and mapping
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Introduction to Metabolomics
Overview of techniques Targeted and non-targeted metabolomics (metabolite extraction procedures, equipment GC-MS, HPLC-PDA-MS)
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AIMS: 1: Experimental Design 2: Quenching and Extractions 3: Instrumentation 4: How to identify metabolites / websites
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Will this answer your hypothesis?
Experimental design: need to consider… Organism Purity of sample (eg, is it contaminated with bacteria, fungus) How much sample (weight) do you need for analysis? 1-10mg MS 50-100mg NMR How many samples do you need for correct biological interpretation? How many samples do you have access to? Cellular compartments - whole cell, ER, mito? How are you going to stop (quench) metabolism within seconds ? Location – lab, field, hospital ward How are you going to extract the metabolites? Which techniques do you have available for analysis? MS, HPLC, NMR, GC Will this answer your hypothesis? How are you going to interpret the data – uni- or multivariate statistics?
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Extractions – structural diversity
Amino acids Monosaccharides Trisaccharides Organic acids Hormones Fatty acids Lipids Sterols / Terpenes Vitamins
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Extractions – chemical and physical properties
1: Molecular weight = the sum of weights of all atoms making the molecule, H2O = 18 (18 g per mol); lipids = >1000 g per mol 2: Molecular size = the 3D size of the structure, measured as Å 3: *Polarity* = differences in electronegativity: Polar (large difference in positive and negative charges) (hydrophilic) non(a)-polar compounds (no or little difference in charge) (hydrophobic) more O and H = more polar; more N = less polar 4: Volatility = depends on boiling and melting point – liquid to gas phase (more polar = less volatile) 5: *Solubility* = related to polarity, temperature and size (like dissolves like) To dissolve - particles need to separate and fit between the solvent spaces Eg, in polar metabolites, a positive end of a metabolite attaches to a negative end of solvent – cannot happen if a positive charge has no negative charge to attach to 6: Stability = thermal or oxidative instability
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Quenching - Stop enzymatic metabolism
Turnover rate is fast: reaction half lives < 1s glucose to glucose-6-phosphate 0.3 to 1 mM per s ATP used at a rate of 1.5mM per s Cold (< -40oC) Hot (>80oC) Acid (pH <2.0) Alkaline (pH > 10) Hot or cold Ethanol/Methanol Liquid nitrogen Perchloric acid/Sodium Hydroxide Cold NaCl Freeze dry Once stopped – how do you extract the metabolites?
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Extractions – solvent choice
Polarity Highly non-polar (hydrophobic) Highly polar (hydrophilic) Extract in Solvent Heptane Ethyl acetate Acetone Ethanol Hexane Chloroform Acetonitrile Water Dichloromethane Methanol Perchloric acid NaCl Metabolites extracted Lipids Carotenoids Amino acids Sugars Fatty acids Chlorophylls Phenolics Organic acids Nucleotides Waxes Steroids Alcohols Organic amines Phosphates Terpenes Flavonoids Alkaloids Tissue disruption Pestle and mortar Sonication Ball mill Microwave Solid Phase Extractions (SPE)
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Extractions – quality control
ALWAYS validate methodologies •Pool from representative samples after extraction •Run at the start and end, an every 5 or 10 samples during data acquisition •Observe technical reproducibility Spike (add) extract with known amount of non-interfering substance – can you recover all of that spike after your analysis?
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What next – need to analyse metabolites
Very common bi-phasic metabolite extraction procedure: Solvent mixture A = MeOH/CHCl3/H2O, 2.5:1:1, v/v/v at –20 oC; Solvent mixture B = MeOH/CHCl3, 1:1, v/v at –20 oC Solvent C = deionised/distilled H2O at 4 oC What next – need to analyse metabolites
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Techniques used in measuring the metabolome
Dunn et al. 2005
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Separating metabolites Basics - Thin Layer Chromatography Paper or Silica Gel
Aim is to separate (resolve) different metabolites in a mixture Maximum number of peaks that can be resolved is called ‘peak capacity’ Can be increased by changing ratio of liquids/solvents or temperature
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Separation - HPLC – PDAD High Performance Liquid Chromatography Photodiode Array Detection
Same principle as TLC but as particles are packed tight it needs pumps to push solvents
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Separation - HPLC – PDAD
Columns are packed full of resin (STATIONARY PHASE) Solvents flow into the column and around the resin (MOBILE PHASE) Normal phase liquid chromatography -column is packed full of a polar compound (eg. alkyl nitrile) -non-polar mobile phase such as hexane -good for lipids Reverse-phase liquid chromatography -column is packed with a non-polar silica compound (eg C8 octasilane or C18 octadecylsilane) -polar mobile phases such as water/methanol/acetonitrile -changes in pH, salts, solvent affect retention times -good for phenolics, sugars, amino acids, drugs, pesticides
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Separation - HPLC – PDAD
Columns are packed full of resin (STATIONARY PHASE) Solvents flow into the column and around the resin (MOBILE PHASE) Isocratic – same solvents ratios running through column eg. 100% Methanol Gradient - change in solvent ratios over time eg. start at 80 % acetonitrile 20% 1% formic acid finish at 60% acetonitrol 40% 1% formic acid over 20 minutes Different metabolites will have different retention times Now need to detect the metabolites coming off the column
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SPECTROPHOTOMETRY - Absorption of UV and visible light
Detection - HPLC – PDAD SPECTROPHOTOMETRY - Absorption of UV and visible light Absorption of electromagnetic radiation Intensity of light passing through a sample falls off exponentially as it progresses through the sample Usually linear with concentration Beer-Lambert law Typical photo diode array detector measures absorbance from 260 to 800 nm What does a typical HPLC trace look like?
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Detection - HPLC – PDAD followed by UV-absorbance Multi-scan wavelength (200 to 500nm) HPLC separation – set wavelength in UV range (284 nm) Type of flavonoid? What does this UV spectrum tell us about the compound?
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Flavonol – eg. Quercitin
Band II Band I at nm Flavone – eg. Apigenin Band I and II close Band II Band I at nm Flavonol – eg. Quercitin Band I more defined and further away from band II
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Separation – GC-FID Gas Chromatography – Flame Ionisation Detection
Same principle as HPLC but use GAS rather than Liquid to separate metabolites chemwiki.ucdavis.edu Column diameter µm 10 – 100 metres long Typically Helium is the MOBILE phase Non-polar Fused Silica is the STATIONARY phase Hydrogen is used for flame
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Separation – GC-FID Gas Chromatography – Flame Ionisation Detection
Samples (about 1µL) are injected into a hot (250 ºC) glass tube where the sample is vapourised Vapour goes into the column Separation based on the difference in partition coefficients between the solid(liquid) stationary phase and the mobile gas phase Increasing temperature biases compounds to leave the stationary phase and enter the gas phase chemwiki.ucdavis.edu Many polar compounds are NOT volatile Sugars, amino acids, organic acids Derivatisation – make compounds volatile By making them more apolar Silylation is the most widely used technique, replaces an acidic hydrogen with an alkylsilyl group Eg. SiMe3, to form tri-methyl silyl (TMS) derivatives (MSTFA)
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Detection – GC-FID Gas Chromatography – Flame Ionisation Detection
Mixture of Hydrogen and Air is used for flame The flame jet is one electrode Another electrode near tip of flame Voltage output Connected to chart recorder or PC When sample emerges from column it ionises and increases signal voltage
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Gas Chromatography (GC) – Flame Ionisation Detector (FID)
Increasing oven temperature Triglycerides Free fatty acids Polar lipids Lipid profiling for biofuels
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Identifying metabolites Mass Spectrometry
Direct Injection Mass Spectrometry (DIMS) HPLC-PDA-MS GC-MS
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Mass Spectrometry (MS)
Detecting metabolites – Metabolic Fingerprinting High throughput screening for metabolic phenotypes Mass Spectrometry (MS)
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Metabolite fingerprinting
Arabidopsis petraea Wales Arabidopsis petraea Sweden A instrument that measures the masses of molecules that have been converted into ions - have been electrically charged (positive or negative). Measure mass over charge (m/z), not just the mass of ions. mass/1 or mass/2
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Identification – MS Ionisation
Individual metabolites in the sample are ionised and either become positively or negatively charged. Acceleration (Separation) These ions are then accelerated so that they all have the same amount of energy. Deflection (Separation) The ions are then deflected by a magnetic field according to their masses. The lighter and more charged they are, the more they are deflected. Detection The ions passing through the machine are detected electrically.
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Ionisation– MS Many different types:
Atmospheric Pressure Chemical Ionisation (APCI) (good for polar compounds) Chemical Ionisation (CI) Electron Impact (EI) (hard) Electrospray Ionisation (ESI) (good for polar compounds) (soft) Fast Atom Bombardment (FAB) Matrix Assisted Laser Desorption Ionisation (MALDI)
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Electrospray Ionisation– MS
Sample is dissolved in a polar, volatile solvent and pumped through a narrow, stainless steel capillary at a flow rate of usually <1 mL per min. A high voltage (3 or 4 kV) is applied to the tip of the capillary Sample emerging from the tip is dispersed into an aerosol of highly charged droplets, aided by a nebulising gas (usually N or He) that direct the spray towards the MS. The charged droplets diminish in size by solvent evaporation, assisted by a warm flow of N (drying gas) Eventually charged sample ions, free from solvent, are released from the droplets, some of which pass through a sampling cone and into the MS under high vacuum.
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Positive or Negative Ionisation? – MS
Positive ion mode: often by addition of H+, Na+, K+ Negative ion mode: often by loss of H+ or addition of Cl- If the sample has functional groups that readily accept a proton (H+) then positive ion detection is used e.g. amines R-NH2 + H+ = R-NH3+ as in proteins or peptides. If the sample has functional groups that readily lose a proton then negative ion detection is used e.g. carboxylic acids R-CO2H = R-CO2- and alcohols R-OH = R-O- as in saccharides or oligonucleotides
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Detection– MS The detector monitors the ion current, amplifies it and the signal is then transmitted to the data system where it is recorded in the form of mass spectra . The m/z values of the ions are plotted against their intensities to show the number of components in the sample, the molecular mass of each component, and the relative abundance of the various components in the sample. Sensitivity: The ppm (parts per million) mass accuracy is a percent error quoting the difference between the measured and calculated mass for a particular ion. 0.1% would be equivalent to 1000 ppm error. Standard 5ppm error is equivalent to % - eg: for a mass 100 m/z the error would be +/ and on mass 1000 error would be +/- 0.5.
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Detecting metabolites – Metabolic Profiling
High Performance Liquid Chromatography (HPLC) – Photodiode array (PDA) – Mass spectrometry (MS) HPLC PDA MS Cyanidin 3- (3-malonyl glucoside) 534.90 448.9 (-malonyl) (-hexose and malonyl) MS/MS
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Metabolic Profiling - Gas Chromatography (GC) – Mass spectrometry (MS)
Unique fragmentation patterns
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GC-MS NIST fragment metabolite library – eg. fatty alcohol
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Introduction to Metabolomics
Metabolic mapping - Identifying proteins and genes associated with your metabolites
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AIMS: 1: How to obtain metabolic, reaction, protein, transcript and gene data from your identified metabolite 2: How to map your metabolite list
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Genomics Post-Genomics Microarray data RNA-Seq iTRAQ GC-MS LC-MS NMR
Post-Genomics Microarray data RNA-Seq iTRAQ GC-MS LC-MS NMR
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How to obtain metabolic, reaction, protein, transcript and gene data from your identified metabolite
Very few good websites for metabolic mapping The key sites are: Reactome Human Metabolome Database Biocyc KEGG Plantcyc MetExplore
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How to obtain metabolic, reaction, protein, transcript and gene data from your identified metabolite
Human Metabolome Database – excellent pathway mapping – eg, Aspirin
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How to obtain metabolic, reaction, protein, transcript and gene data from your identified metabolite
Biocyc collection of 2038 Pathway/Genome Databases Many pathways derived from genome screen – in silico pathways
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How to obtain metabolic, reaction, protein, transcript and gene data from your identified metabolite
Biocyc – very comprehensive website for many species
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How to obtain metabolic, reaction, protein, transcript and gene data from your identified metabolite
Biocyc – Photosynthesis outline
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How to obtain metabolic, reaction, protein, transcript and gene data from your identified metabolite
Biocyc – pressing more detail adds enzyme (EC) and gene data (AGI codes)
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How to obtain metabolic, reaction, protein, transcript and gene data from your identified metabolite
Biocyc – metabolite structures can also be viewed
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How to obtain metabolic, reaction, protein, transcript and gene data from your identified metabolite
Biocyc – RuBisCO – fixes CO2 in plants
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How to obtain metabolic, reaction, protein, transcript and gene data from your identified metabolite
Biocyc – RuBisCO – enzyme – relation to genes and reactions
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How to obtain metabolic, reaction, protein, transcript and gene data from your identified metabolite
Biocyc – RuBisCO – gene location on chloroplast
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How to obtain metabolic, reaction, protein, transcript and gene data from your identified metabolite
Biocyc – RuBisCO – hyperlinks to other sites for gene and protein information
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How to obtain metabolic, reaction, protein, transcript and gene data from your identified metabolite
KEGG – TCA cycle – EC numbers for reactions – colour coded per species
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How to obtain metabolic, reaction, protein, transcript and gene data from your identified metabolite
KEGG – malate formation ( )
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How to obtain metabolic, reaction, protein, transcript and gene data from your identified metabolite
Plantcyc – similar format to Biocyc
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How to obtain metabolic, reaction, protein, transcript and gene data from your identified metabolite
Plantcyc – metabolic mapping – colour code reactions from your datasets
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How to obtain metabolic, reaction, protein, transcript and gene data from your identified metabolite
Plantcyc – zoom into individual pathways
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How to obtain metabolic, reaction, protein, transcript and gene data from your identified metabolite
Plantcyc – hyperlinks back to original pathway information
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How to obtain metabolic, reaction, protein, transcript and gene data from your identified metabolite
MetExplore
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How to obtain metabolic, reaction, protein, transcript and gene data from your identified metabolite
MetExplore – Cytoscape plugins
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