Flashcards in Lecture 9 - Metabolomics Deck (24):
Traditional view of metabolism
Gene->mRNA->enzyme (enzyme affects substrate->product reaction)
-omics view of metabolism
Genome, trascriptome, proteome and metabolome all affect each other and themselves
What do changes in the metabolome reflect?
All up-stream regulatory events (genetic, transcription, translation, post-translational) and extracellular factors.
Quantitative analysis of all small molecules in a biological system
Small molecules definition
The full complement of small molecules in a sample
Number of metabolites in a cell
Varies from a few hundred in mycoplasma to 4-5 thousand in yeast or mammalian cells
Why can studying metabolomics be very hard?
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
Possible analytical platforms for metabolomic analysis
1) Nuclear magnetic resonance imagery (NMR)
2) Gas chromatography mass spectrometry
3) Liquid chromatography mass spectrometry
4) Imaging mass spectrometry
Type of sample needed for mass spec analysis
A crude sample, prepared with organic solvents (EG: chloroform)
How can single-cells be analysed with mass spec?
Nanoelectrospray tip is inserted into cell cytoplasm, sucks up cytoplasm into a mass spec
Nominal mass versus accurate mass spectrometry
Nominal mass has unit mass accuracy
Accurate mass has accuracy of fractions of a Dalton
Metabolic analysis pipeline
1) Sample preparation
2) Sample extraction
3) Sample analysis
4) Data analysis
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
Human metabolome project
2) Identified 2,900 metabolites in human body
3) Developed comprehensive databases (chemical, pathways, analytical approaches, disease associations)
Applications of metabolomics
1) Systems biology
7) Functional genomics
Potential diagnostic use of metabolomics
Detect early biomarkers for disease.
Example of biomarkers for detection of Alzheimer's disease
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
Tissue imaging mass spec process
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
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
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
Potential application of DESI-IMS
Could be used in surgery, to detect biomarkers when resecting a tumour.
Stable-isotope resolved metabolomics
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