Lecture 9 - Metabolomics Flashcards Preview

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Flashcards in Lecture 9 - Metabolomics Deck (24):
1

Traditional view of metabolism

Gene->mRNA->enzyme (enzyme affects substrate->product reaction)

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-omics view of metabolism

Genome->transcriptome->proteome->metabolome->phenotype.

Genome, trascriptome, proteome and metabolome all affect each other and themselves

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What do changes in the metabolome reflect?

All up-stream regulatory events (genetic, transcription, translation, post-translational) and extracellular factors.

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Metabolomics definition

Quantitative analysis of all small molecules in a biological system

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Small molecules definition

Under 1500Da

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Metabolome

The full complement of small molecules in a sample

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Number of metabolites in a cell

Varies from a few hundred in mycoplasma to 4-5 thousand in yeast or mammalian cells

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Why can studying metabolomics be very hard?
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1) 2,000-200,000 metabolites
2) Extreme range of concentrations (from nMol to mMol)
3) Diverse chemistries, with no amplification (like with DNA/RNA)
4) Changes very quickly
5) Integration of non-linear networks

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Possible analytical platforms for metabolomic analysis
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1) Nuclear magnetic resonance imagery (NMR)
2) Gas chromatography mass spectrometry
3) Liquid chromatography mass spectrometry
4) Imaging mass spectrometry

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Type of sample needed for mass spec analysis

A crude sample, prepared with organic solvents (EG: chloroform)

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How can single-cells be analysed with mass spec?

Nanoelectrospray tip is inserted into cell cytoplasm, sucks up cytoplasm into a mass spec

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Nominal mass versus accurate mass spectrometry

Nominal mass has unit mass accuracy
Accurate mass has accuracy of fractions of a Dalton

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Metabolic analysis pipeline

1) Sample preparation
2) Sample extraction
3) Sample analysis
4) Data analysis

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How does metabolomics differ from traditional biochemistry?

Breadth of coverage, not necessarily resolving or quantifying individually the large numbers of analysed metabolites

Trying to capture levels of metabolites in situ

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Human metabolome project

1) NIG-funded
2) Identified 2,900 metabolites in human body
3) Developed comprehensive databases (chemical, pathways, analytical approaches, disease associations)

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Applications of metabolomics
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1) Systems biology
2) Bioengineering
3) Diagnostics
4) Toxicology
5) Therapeutics
6) Microbiology
7) Functional genomics

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Potential diagnostic use of metabolomics

Detect early biomarkers for disease.
Prognostic markers.

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Example of biomarkers for detection of Alzheimer's disease
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1) Detected using liquid chromatography mass spec
2) Three groups - normal, converters, symptomatic
3) 10 plasma phospholipid metabolites identified that distinguished controls from those who converted to memory impairment

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Tissue imaging mass spec process
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1) Frozen or chemically-fixed samples coated with a chemical matrix
2) Matrix assisted laser desorption ionisation (MALDI) or desorption electrospray ionisation mass spec performed on samples
3) Spatial mapping of thousands of individual metabolites on sample

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MALDI-IMS
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1) Matrix assisted laser desorption ionisation imaging mass spec
2) Sample placed in a vacuum, where a laser excites ions in tissue.
3) Ions escape sample, are detected by mass spec

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DESI-IMS
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1) Desorption electrospray ionisation imaging mass spec
2) Spray of nebulised solvent onto sample
3) Nebulises back out of sample, detected by mass spec.
4) Can be performed at room temperature, which is an advantage over MALDI

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Potential application of DESI-IMS

Could be used in surgery, to detect biomarkers when resecting a tumour.

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Stable-isotope resolved metabolomics
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1) Stable isotope labelling of sample to be analysed (EG: glucose with C13)
2) Sample is allowed to undergo reaction, whether in a cell culture, live animal bacterium, etc
3) Sample is extracted at set timepoints, analysed with mass spec
4) Data analysis
5) Flux/network map of what happens to the labelled metabolite

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Organism that has been examined with stable-isotope resolved metabolomics

Toxoplasma gondii