Metabolomics Flashcards
Yang
Define metabolomics
Quantitative study of a whole set of small molecules (metabolome)
What is a primary metabolite?
Performs normal physiological functions such as cell growth
What is a intermediary metabolite?
Maintains homeostasis such as energy intermediates
What is the metabolome?
The complete set of small molecules, such as carbohydrates and
lipids, in a biological sample
The metabolome excludes: (3)
- Enzymes
- Genetic materials
- Structural molecules
The human metabolome is an analog of _____ ______
human genome
Metabolites are? (2)
- Final-result of cellular functions
- Quantifiable molecules with the closest link to phenotype
What are the techniques used in metabolomics? (4)
- LC-MS, especially HPLC-MS
- GC-MS
- NMR
- Capillary electrophoresis
- CD-MECC
What is liquid chromatography (LC)?
An analytical technique used to separate or purify molecules dissolved in a solvent (mobile phase = liquid)
What are the 4 types of LC used for separation or purification of small molecules?
- Partition chromatography: paper chromatography and thin-layer chromatography
- HPLC – High Performance Liquid Chromatography
- UPLC (UHPLC) – Ultra Performance Liquid Chromatography
- HPLC and UPLC are commonly used in metabolomics
What is the equation for resolution? (Rs)
Rs = (V2-V1)/((W1+W2)/2)
The cutoff for Rs value so that the peaks are completely separate is?
Rs ≥ 1.5
What are the 3 major players of liquid chromatography?
- Resolution
- Column
- Flow rate
In the LC column, what are the effects of resin particle size? (2)
- Smaller particle size –> higher pressure (related to flow rate)
- Smaller particle size –> better resolution
Why is flowrate important in LC? (2)
- Loss of resolution and capacity at high flow rate
- Backpressure on column resin
How does HPLC work? (4)
- Separation of small molecule compounds dissolved in a solution
- Mixture of compounds is injected into the column
- Compounds are separated based on difference in partition
coefficients between the mobile phase and stationary phase
- Mobile phase: solvent
- Stationary phase: column - Mobile phase should be degassed to avoid air bubbles
In HPLC, what are the normal column sizes? (4)
- 5 cm
- 10 cm
- 15 cm
- 25 cm
In HPLC, what is the normal particle size?
Normally 5 micron
In HPLC, what is the usual pressure range?
200-400 bar
In HPLC, what is the typical flow rate?
~1mL/min
What is normal-phase HPLC? (2)
- Retention is decided by the interaction of the polar parts of the stationary phase and solute
- Packing material (stationary phase) must be more polar than mobile phase with respect to the sample
Common column ligands in normal-phase HPLC are? (3)
- Silica
- CN
- NH2
Common mobile phase solvents in normal-phase HPLC are?
Nonpolar solvents or solvent mixture such as hexane
What is reverse-phase HPLC? (2)
- Retention is decided by the interaction of the nonpolar parts between the stationary phase and the solute
- Packing material (stationary phase) must be relatively nonpolar than mobile phase with respect to the sample
Common column ligands in reverse-phase HPLC are?
Bonded hydrocarbons such as C18 and C8
Common mobile phase solvents in reverse-phase HPLC are?
Polar solvents or solvent mixtures such as methanol and acetonitrile
UPLC (aka UHPLC) can detect particle sizes of?
What is the column pressure?
1.7-1.8 micrometers
Column pressure up to 1030 bar
True or False? Compared to UPLC, HPLC has increased resolution, sensitivity, and speed
False - UPLC > HPLC in these things
What is the mobile phase of gas chromatography?
What is the stationary phase?
- Mobile phase = gas (helium most common, also hydrogen and nitrogen)
- Stationary phase = liquid (GLC, liquid coated on a solid) and solid (GSC, not widley used)
When is gas chromatography used?
What does it require?
What does it do?
- Widely used in analytical chemistry
- Requires vaporization of compounds without decomposition
- Separation of gaseous and volatile substances
GLC principle is?
separation by partition coefficient
The stationary phase of gas chromatography capillary columns can be what substances? (5)
- Polyethylene glycol
- Polysiloxanes
- Hydrocarbons
- Esters
- Amides
What is the diameter and length of gas chromatography capillary columns?
Diameter = 0.025-0.075cm
Length = 30-90m
Compare helium, hydrogen, and nitrogen as gasses used for GC. Two each.
- Helium
- Excellent thermal conductivity
- Expensive - Hydrogen
- Better thermal conductivity
- Reacts with unsaturated compounds and inflammable - Nitrogen
- Reduced sensitivity
- Inexpensive
What is the Human Metabolome Database (HMDB) of Canada?
- Detailed information of small molecule metabolites found in human body
- Chemical data
- Clinical data
- Molecular biology/biochemistry data - Contain 220,945 metabolite entries
What 4 things does metabolomics study?
- Metabolite target analysis
- Metabolite profiling
- Metabolic fingerprinting
- Metabolic profiling
What is metabolite target analysis?
Quantitative or semi-quantitative clinical and pharmaceutical analysis of a specific group of metabolites (for example, lipids)
What is metabolite profiling?
Analysis of a large group of metabolites that is either related to a
specific metabolic pathway or a class of compounds
- More targeted than metabolite fingerprinting
What is metabolic fingerprinting? (4)
- Providing information from spectra of total composition of
metabolites - 1H NMR metabolic fingerprinting
- Powerful method for discriminating between biological samples on the basis of differences in metabolism
- Disease diagnosis
What is metabolic profiling? (2 - different from metabolite profiling I guess)
- How metabolites are organized into pathways
- Understand mechanism of diseases or drugs
