Metabolomics Flashcards
What is the definition of metabolomics?
It is a quantitive study of a whole set of small molecules (metabolome)
What is a metabolome?
A metabolome can contain the following:
- The molecules are contained in a human cell, tissue, or organ
- The metabolism are involved in primary and intermediary metabolism
- Primary metabolite: performs normal physiological functions such as cell growth
- Intermediary metabolite: maintains homeostasis such as energy intermediates
What is excluded from the metabolome?
- Enzymes
- Genetic materials
- Structutal molecules
No large + non-functional proteins
What are some techniques used in metabolomics?
- LC-MS (especially HPLC-MS)
- GC(gas chromotography)-MS
- NMR
- Capillary electrophoresis
What are some characteristics of LC (liquid chromotography)?
It is an analytical technique used to separate or purify molecules dissolved in a solvent
It is a technique used to separate or purify small molecules
ex. HPLC and UPLC
What are the three variables that can be adjusted in LC?
- Resolution
- Column
- Flow rate
How is resolution for liquid chromatography calculated?
Review slide 9 and 11
What is the optimal resolution volume for adjacent peaks?
At least 1.5 to prevent peak overlap (results in pure samples)
What is the impact of resin particle size on the liquid chromatography variables?
Smaller particle size = higher pressure & better resolution
What is the impact of flow rate on chromatography variables?
Loss of resolution and capacity at high flow rate
Due to backpressure on column, the flow rate tends to slow without continuous pumping
Slow flow rate = increased resolution
What are some characteristics of HPLC?
Mixture of compunds is injected into the column
Results in the separation of small molecule compounds dissolved in a solution
Separation is based on differences in partition coefficients between moble and stattionary phase
What are some standard HPLC variables?
Column size: normally 5cm, 10cm, 15cm, 25cm
Particle size: normally 5 micron (some at 3 micron for increased resolutioon and pressure)
Pressure: 200 to 400 bar
Flow rate: 1mL/min (start at a low flow rate and increase accordingly)
What are some examples of columns used in HPLCs?
Non-polar:
- C18 (alkyl)
- C18 (polar embedded)
Polar:
- Pentafluorophenyl
- Aminopropyl
What are some characteristics of normal-phase HPLC?
Stationary phase: polar
Mobile phase: non-polar (ex. hexanes)
slide 17
What are some characteristics associated with reverse-phase HPLC?
Stationary phase: non-polar
Mobile phase: Polar solvents (methanol and acetonitrile)
slide 18
What is UPLC?
It is similar to HPLC, but flow rates and pressures are higher (more expensive to buy and operate)
Particle size (1.7-1.8 microns)
UPLC also yield increased resolution (slide 20), sensitivity, and speed
What is gas chromatography?
Stationary phase: liquid (GLC, liquid coated on a solid)
Mobile phase: gas (not charged, ex. He, H2, N2)
Requires fine control to prevent decomposition while maintaining vapourization
Review slide 22
What are some characteristics of gas chromatography capillary columns?
Stationary phase: PEG, polysiloxanes, hydrocarbonsm esters (can be polar or non-polar)
Diameter: 0.025cm to 0.075cm
Length: 30-90cm
What are some characteristics of helium in gas chromatography?
- Excellent thermal conductivity (transfers heat effectively, which is good fro preventing decomposition/condensation of sample)
- Expensive
What are some characteristics of hydrogen gas in gas chromatography?
- Better thermal conductivity (not as good as He)
- Reacts with unsaturated compounds and inflammable (conversion of double/triple bonds into single bonds)
What are some characteristics of nitrogen gas in gas chromatography?
- Reduced sensitivity (broader liquid chromatography)
- Inexpensive
How are the results of liquid chromatography characterized?
MS and NMR are used to characterize the results from LC
What is the HMDB (Human Metabolome Database)?
It is a detailed catalogue of small molecules found in the human body (more than 200,000 entries)
Contains chemical, clinical, and molecular biology/biochemistry data
At what concentrations are metabolites considered to be significant?
When concentrations of metabolites exceed 1micromolar, they are considered to be abundant
Lower than 1 nanomolar, these concentrations are considered to be relatively rare
Review slide 30
What are the components of a metabolomics study?
- Metabolite target analysis
- Metabolite profiling
- Metabolite fingerprinting
- Metabolic profiling
What is metaboliute target analysis?
It is a quantitive or semi-quantitive clinical and pharmaceutical analysis of a specific group of metabolites (ex. all lipids)
What is metabolite profiling?
It is the analysis of a large group of metabolites that is either related to a specific metabolic pathway or a class of compounds (ex. only fatty acid metabolites are studied)
slide 35
What is metabolic fingerprinting?
It provides information from spectra of total composition of metabolites
NMR metabolic fingerprinting
Powerful method for discriminating between biological samples on the basis of differences in metabolism
slide 37
What is metaboilc profiling?
- Organization of metabolites into pathways
- Understand mechanism of diseases or drug
ex. intra/interindividual metabolic profiling for T2DM
