lecture 9: metabolomics Flashcards
- what is metabolomics - what are the technologies that underpin metabolomics - what questions can metabolite profiling be used to address - metabolomics at the tissue, single cell level
1
Q
What is metabolism in the 21st century?
A
- metabolic defects underlie nearly all diseases, including many not traditionally associated with altered metabolism (i.e. cancers, Alzheimer’s disease)
- most drugs and diagnostic biomarkers are small molecule metabolites (or derived from)
- ‘personalised medicine’ requires accurate and rapid methods for measuring global metabolic responses and drug metabolism
- attempts to bioengineer new plant crops/microbes hampered by lack of understanding of metabolism in vivo
2
Q
What are current challenges in metabolomics?
A
- we know a lot about individual metabolic pathways, but relatively little about regulation in vivo
- measurements of gene transcription, protein translation/post-translational modifications are often poor indicators of metabolic fluxes
3
Q
what is the traditional view of metabolism?
A
- gene → mRNA → enzyme → acts on substrate → product
- gene → mRNA → enzyme = central dogma of molecular biology
- substrate → product = metabolism
- if this was true you would be able to look at levels of mRNA and predict the rate at which the reaction was going to occur
- this is not the case and every attempt to manipulate cells in this manner has resulted in failure
4
Q
what is the OMICs view of metabolism?
A
- genome ↔ transcriptome ↔ proteome (can also act on genome) → metabolome → phenotype
- metabolome → proteome
- metabolome → transcriptome
- metabolome → genome
- metabolome → metabolome
- also inputs from the outside that are not part of the genome → further complexity
- genome = ultimate potential of a cell → what is possible
- transcriptome = the current direction of the cell → what appears to be happening
- proteome = functional capabilities of a cell → what makes it happen
- metabolome = the actual capabilities of the cell in context of cellular environment → what is happening
- changes in the metabolome reflect all up-stream regulatory events (genetic, transcription, translation, post-translational) as well as extracellular factors (nutrient availability etc)
- potentially highly sensitive read-out of physiological state of cell
5
Q
What is metabolomics?
A
- the quantitative analysis of all small molecules in a biological system
- synonyms: metabolomics, metabonomics, metabolite profiling, fluxomics
6
Q
What is the metabolome?
A
- the full complement of small molecules (less than 1500 dalton) in a sample
7
Q
What is the scope of metabolomics?
A
- 2000 - 200,000 metabolites
- primary and secondary metabolites
- analysis of metabolic flux
- identification of metabolic networks
8
Q
What are metabolites?
A
- all low molecular weight metabolites (less than 1500 daltons)
- includes, but not limited to:
- sugars, sugar phosphates
- organic acids
- amino acids
- fatty acids
- nucleosides
- lipids (i.e. sterols, phospholipids, lipidomics)
- small peptides
- other secondary metabolites
- drugs, insecticides, other xenobiotics
- number of metabolites per cell varies (few hundred in Mycoplasma to 4-5 thousand in yeast, mammalian cells)
- total number of metabolites is greater than 500,000
9
Q
What are challenges to doing metabolomics?
A
- there are a lot of metabolites (varies from cell to cell, 2,000 - 200,000)
- extreme range of concentrations (nM to mM)
- need very senstive instruments
- diverse chemistries, no amplification
- changes very quickly (even more so than other parameters in a cell)
- integration of non-linear networks
10
Q
How do you go about doing metabolomics?
A
- analytical platform:
- NMR - nuclear magnetic spectroscopy
- GC-MS - Gas chromatography-mass spectrometry
- LC-MS: liquid chromatrography-mass spectrometry
- imaging MS
- image frozen/fixed sections of tissue instead of extract
- analytical data
- informatic analysis
- at the moment there is no one technology that will be able to detect all the metabolites in a single cell extract
- need a complementary set of different analytical platforms
11
Q
What is the metabolomic analysis pipeline?
A
- sample preparation e.g. body fluids (serum, urine, saliva, CSF, faeces, breath), cultured cells, microbes, tissue biopsy/section
- sample extraction
- sample analysis
- data analysis
12
Q
What is metabolomic analysis of single cells?
A
- metabolite ‘features’ identified by accurate mass and MS/MS
- electrospray MS analysis of single plant cell (leaf) sampled with a nanoelectrospray tip
13
Q
What is nominal mass?
A
- unit mass accuracy
- 180 daltons = 141 predicted elemental compositions with C, N, O, S, Cl, Br, and I
14
Q
What is accurate mass?
A
- 180.06339 = 2 possible elemental compositions
- Only one sensible
- however, 32 steroisomers
- need FTICR or Orbitrap MS instruments with mass accurace of 0.5 ppm and mass resolution of 100,000
15
Q
What is the difference between a biochemistry lab back in the 50s or 60s and a metabolomics lab today?
A
- traditionally you would be interested in characterising an enzyme, putting in one reactant and looking at the products that came out of it
- might use a range of similar analytical techniques but you are just looking for a very small number of metabolites
- can do targeted analysis of multiple compounds of a single class etc but still looking at a limited number of metabolites
- now looking at either metabolite fingerprinting (metabolites not necessarily resolves or quantitated individually) or Metabolite profiling of high complexity
- metabolomics differs fundamentally from traditional biochemistry in both breadth of coverage and attemp to capture levels in situ
18
Q
What are biomarkers for health and disease?
A
- a need to be able to detect diseass as early as possible in order to be able to in order to be able to take immediate action and potentially develop new therapeutics
- in some cases you don’t need a sophisticated biomarker for some conditions
- but in many cases it would be very useful to know long before they have obvious physical manifestations
- commonly sampled ‘tissues’ include blood, urine, saliva
- e.g.
- alzheimer’s disease currently affects 35 million people worldwide, and predicted to affect 115 million by 2050
- no treatment and existing biomarkers are limited (i.e. tau, amyloid-b), time consuming and expensive (i.e. MRI)
- need new biomarkers for preclinical disease diagnosis in order to develop disease
- modifying or preventative therapies
- untargeted LC/MS analysis of serum from 525 individuals, >70 years age
21
Q
What is tissue imaging mass spectrometry?
A
- frozen or chemically fixed tissue sections
- +/– coated with chemical matrix
- matrix assisted laser desorption ionisation (MALDI)-MS or desorption electrospray ionisation (DESI)-MS
- spatial mapping of 1000s of individual metabolites, lipids
Mass spectrometers
- low resolution; 1 dalton/ quadrupole-MS
- high mass resolution; 1:100 (time of flight; TOF)
- ultra high mass resolution; 1:10,000 (FTICR = fourier transform ion cyclotron resonance) can be used to determine elemental composition
MALDI-IMS performed in tight vacuum, requires a bit of prep
laser rasters across the tissue - ions are excited etc
DESI: can be done at ambient temp, spray of nebulised solvent, solubilises, re-nebulises → analyte
- portable DESI-MS instruments are being made that can be used in rapid microbial diagnosis, testing for pesticides on surface of food etc
- nanoDESI imaging or metabolites in colon cancer biopsies at 12µm resolution
- DESI-IMS suitable for intra-operative diagnosis
22
Q
How do we go about measurng metabolic dynamics in vivo?
A
- doesn’t tell you why
- stable-isotope resolved metabolomics
- take tissue sample etc
- stable isotope labelling (i.e. with 12C/15N-labelled glucose or amino acids)
- sample extraction at defined time points (mins to hours)
- sample analysis (mass spec/NMR)
- data analysis (labelling of metabolites)
- flux/network (model data)
stable isotope labelling of carbon skeletons
mass of derivative blah blah
measure of turnover , kinetics of production
23
Q
Why explore the metabolism of Toxoplasma gondii? What has been discovered?
A
- infects ~1/3 of the world’s population
- most infections are asymptomatic
- associated with serious disease in newborn babies and mothers, and in immunocompromised individuals
- live in a specialised vacuole in just about any nucleated cell in the body
- genome-based annotation of T. gondii central carbon metabolism → T. gondi lacks a mitochondrial pyruvate dehydrogenase (PDH) and was predicted to exclusively generate ATP by glycolysis
- however in this organism the PDH is located in another organelle called the apicoplast → fatty acid biosynthesis
- labelling experiment with C13 glucose found that all the TCA cycle intermediates were strongly labelled suggesting not just glycolysis
- tells us there must be an enzyme that wasn’t able to be recognised by the genome annotations that converts pyruvate to acetyl-CoA
- has been identified → completely new pathways and drug targets
