Lecture 9 - Metabolomics

Question 1

Traditional view of metabolism

Answer 1

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

Question 2

-omics view of metabolism

Answer 2

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

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

Question 3

What do changes in the metabolome reflect?

Answer 3

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

Question 4

Metabolomics definition

Answer 4

Quantitative analysis of all small molecules in a biological system

Question 5

Small molecules definition

Answer 5

Under 1500Da

Question 6

Metabolome

Answer 6

The full complement of small molecules in a sample

Question 7

Number of metabolites in a cell

Answer 7

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

Question 8

Why can studying metabolomics be very hard?
1)
2)
3)
4)
5)

Answer 8

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

Question 9

Possible analytical platforms for metabolomic analysis 
1)
2)
3)
4)

Answer 9

1) Nuclear magnetic resonance imagery (NMR)
2) Gas chromatography mass spectrometry
3) Liquid chromatography mass spectrometry
4) Imaging mass spectrometry

Question 10

Type of sample needed for mass spec analysis

Answer 10

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

Question 11

How can single-cells be analysed with mass spec?

Answer 11

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

Question 12

Nominal mass versus accurate mass spectrometry

Answer 12

Nominal mass has unit mass accuracy

Accurate mass has accuracy of fractions of a Dalton

Question 13

Metabolic analysis pipeline

Answer 13

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

Question 14

How does metabolomics differ from traditional biochemistry?

Answer 14

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

Trying to capture levels of metabolites in situ

Question 15

Human metabolome project

Answer 15

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

Question 16

Applications of metabolomics
1)
2)
3)
4)
5)
6)
7)

Answer 16

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

Question 17

Potential diagnostic use of metabolomics

Answer 17

Detect early biomarkers for disease.

Prognostic markers.

Question 18

Example of biomarkers for detection of Alzheimer’s disease
1)
2)
3)

Answer 18

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

Question 19

Tissue imaging mass spec process
1)
2)
3)

Answer 19

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

Question 20

MALDI-IMS
1)
2)
3)

Answer 20

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

Question 21

DESI-IMS
1)
2)
3)
4)

Answer 21

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

Question 22

Potential application of DESI-IMS

Answer 22

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

Question 23

Stable-isotope resolved metabolomics 
1)
2)
3)
4)
5)

Answer 23

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

Question 24

Organism that has been examined with stable-isotope resolved metabolomics

Answer 24

Toxoplasma gondii