bumpus metabolomics lecture

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MetabolomicsMetabolomics

Namandjé N. BumpusDepartment of Pharmacology and Molecular SciencesDepartment of Pharmacology and Molecular Sciences

Johns Hopkins University School of Medicine

05 December 2012

Metabolomics Beginnings

• 1951 - Williams, R.J., et al., Individual metabolic patterns and human disease: An exploratory study utilizing predominantly paperstudy utilizing predominantly paper chromatographic methods. U. Texas Publication No. 5109, Univ. of Texas, Austin, 1951,204pp

• 1966 - Dalgliesh et al. publish the first GC/MS metabolomic experiments in Biochemical Journal.Journal.

• 1971 Horning publishes “Metabolic Profiles: Gas-Phase Methods for Analysis of Metabolites” in Clinical Chemistry

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• 1995 – The untargeted LC-MS metabolomicsstudy “Chemical Characterization of a Family of

Metabolomics Beginnings

study Chemical Characterization of a Family of Brain Lipids That Induce Sleep” published in Science.

• 1998 - SG Oliver et al use the term ‘metabolome’ for the first time in Trends in BiotechnologyBiotechnology.

• 2004 - METLIN, a web-based data repository of metabolites was launched.

Metabolome• Metabolome refers to the entire collection of

endogenous small molecules in an organism.Organic molecules detectable in the body with a• Organic molecules detectable in the body with a MW < 1000 Da

• Includes nucleotides, peptides, ketones, aldehydes, amines, amino acids, steroids in addition to exogenous molecules such as food additives, pollutants, drugs and drug metabolites

• In humans microbial products also have to be• In humans, microbial products also have to be considered.

• Metabolite concentrations range from abundant (> 1 uM) to relatively rare (< 1 nM).

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Metabolome

• Metabolites serve as cofactors for signaling pathwayspathways

• Vast majority of clinical diagnostic tests assay for small molecules

• 50% of currently marketed drugs are derived from previously identified metabolites

• Modulation of endogenous metabolites play a role in an approximate 30% of genetic disorders

Utility of Metabolomics

• Phenotype identification/metabolic signatures

• Measure metabolite flux through pathwaysg p y

• Monitor enzymatic activity

• Identify novel enzyme/gene functions

• Assess environmental/toxic/drug outcomes

• Determine the effects of genetic knockdowns/knockinsknockdowns/knockins

• Assess food/plant quality

• Water quality testing

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Human Metabolite Concentrations

Environmental Chemicals

Drugs

Chemicals Found in Food

Drug Metabolites

M mM M nM pM fM

Endogenous metabolites

Drugs

Metabolomics• Untargeted metabolomics - unbiased

global analysis of small molecules in a cell g ytype, tissue, biological fluid or whole organism.

• Targeted metabolomics – measurement of fi i f ll l l ia finite group of small molecules using

assays developed and optimized specifically for their detection.

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• Detection of metabolites depends largely on extraction procedure

– Nucleotides are extracted using a strong acid and oxidizing agent

Untargeted Metabolomics

oxidizing agent

– Derivatization of thiols

• The sample prep procedure has to be tailored to the metabolites of interest. The most comprehensive procedures allow for simultaneous isolation of ~300 compounds

• Important compounds may be missed using this approach.

• Data mining can be cumbersome and requires the use of software that facilitates metabolite identification and discovery

Fourier Transform Mass Spectrometry

• Pros

Ch t hi ti t i d

Technologies Used in Metabolomics

• Chromatographic separation not required

• Facilitates fast metabolic profiling

• Allow for ultra-high resolution

• Molecular formula can be assigned to ions

• Things to consider

• Isomers cannot be distinguished

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Gas Chromatography –Mass Spectrometry

• Pros


• Very high chromatographic resolving power

• Facile detection of non-polar metabolites


• The small molecules of interest must be sufficiently volatile and thermally stabley y

• Can be used for non-polar (and slightly polar) metabolites only

Liquid Chromatography –Mass Spectrometry

• Pros

• Broad detection capabilities


• Broad detection capabilities

• Can be used to measure metabolites that are highly polar and thermally labile


• HPLC interface can result in resolution that i li it d d t HPLCis limited as compared to uHPLC

• MS libraries may be less well developed

• Fragmentation patterns of many metabolites may not be well defined

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Capillary Electrophoresis –Mass Spectrometry

• Pros


• High separation capabilities and short analysis time

• Chiral separations are possible


• MS libraries are largely unavailableg y

• Method development can be complex

Untargeted Metabolomics Data Processing

• Due to data complexity software is required in order to identify metabolites

• XCMS (designed by Gary Siuzdak’s Laboratory at the Scripps Research Institute) is a LC-MS bioinformatics platform that incorporates novel feature detection and feature matching strategies to identify endogenous metabolites.

• XCMS software reads and processes LC-MS data that are stored as netcdf , mzXML, mzData and mzML files.

• Facilitates comparative analysis of multiple spectra

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• XCMS facilitates the detection of peaks, the detection of metabolites that are present at different concentrations in various samples, the identification of the identity of


various samples, the identification of the identity of metabolites and statistical analysis.

• This approach has successfully been used to identify metabolites involved in cancer, fruit quality/development and stem cell differentiation.

• This software removes the requirement for sophisticated knowledge of metabolitesknowledge of metabolites

• Any scientist conducting a metabolomic study using LC-MS can use this software

• Data analysis can be completed in minutes to hours depending on the size of the data files.

• METLIN (also developed by the Siuzdak Laboratory) is a freely accessible searchable web-based repository that contains accurate mass and tandem MS data for over


contains accurate mass and tandem MS data for over 64,000 metabolites.

• Queries are performed using metabolite mass-to-charge ratios as well as fragmentation data.

• Output includes the potential identities of metabolites: chemical formula and structure in addition, links to outside informationoutside information.

• MS/MS data used in the database were obtained using an Agilent 6510 Q-TOF operated in positive (ESI+) and negative (ESI-) electrospray ionization mode using four different collision energies (0, 10, 20 and 40V).

http://metlin.scripps.edu/

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Example: Subtraction of TIC

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Example: 16O-containing MS/MS spectra

Example: 18O-containing MS/MS spectra

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• The Human Metabolome Database (HMDB) is a freely available web-based metabolite database. The database contains over 40,000 metabolite entries including


contains over 40,000 metabolite entries including chemical data, clinical data and biochemistry data. http://www.hmdb.ca/

• Contains four additional databases – DrugBank, T3DB, SMPDB and FooDB.

– DrugBank contains on over 1500 drug and drug metabolitesmetabolites

– T3DB is a collection of over 3000 toxins/pollutants

– SMPDB is a collection of pathway diagrams for human metabolic disease pathways

– FooDB is a repository containing data for over 28,000 food components and additives.

• Extraction procedures can be optimized for molecules of interest

Targeted Metabolomics

• Selected reaction monitoring can be used for quantitation when synthetic standards are available

• Mass spec parameters can be optimized for detection of the compounds of interestdetection of the compounds of interest

• Fragmentation patterns can be more readily confirmed and analyzed

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• Common targeted metabolomics assays include detection of:

Targeted Metabolomics

– Glutathione pathway components

– The citric acid cycle components

– Pentose phosphate pathway components

– Amino acids

– Glycolysis metabolitesGlycolysis metabolites

– Purine and pyrimidines

– NAD‐related metabolites

Glutathione (GSH) Pathway

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GSH Targeted Metabolomics

bumpus metabolomics lecture

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