Metabolomics 1 Flashcards
What is metabolomics?
quantitative study of a whole set of small molecules (metabolome)
What is a metabolome?
complete set of small molecules, such as carbohydrates and lipids, in a biological sample
What is a primary metabolite?
performs normal physiological functions such as cell growth
What is an intermediary metabolite?
maintains homeostasis such as energy intermediates
What is the human metabolome?
an analog of human genome
What does a metabolome provide information on?
important information on biological, physiological and pathophysiological processes
What is excluded from a metabolome?
enzymes
genetic materials
structural molecules
True or false: all changes in genome or transcriptome cause abnormality or disease
false
True or false: not all enzymes and protein products detected by proteomics are functional
true
What are metabolites?
final result of cellular functions
quantifiable molecules with the closest link to phenotype
What are the techniques used in metabolomics?
LC-MS, especially HPLC-MS
GC-MS
NMR
capillary electrophoresis
-CD-MECC
What is liquid chromatography?
an analytical technique used to separate or purify molecules dissolved in a solvent
for separation or purification of small molecules
What is the mobile phase in LC?
liquid
What are examples of liquid chromatography?
partition chromatography
HPLC
UPLC
HPLC and UPLC are commonly used in metabolomics
What is the equation for resolution?
Rs = V2-V1/(W1+W2)/2
What does Rs of 1 mean?
the molecules are not yet separated
-not pure
What does Rs of 1.5 mean?
peaks are separated
- > 1.5 is the cut off for good separation
Describe the effect of resin particle size on resolution.
smaller particle size –> higher pressure
smaller particle size –> better resolution
small size + low flow rate = good resolution
Describe HPLC.
separation of small molecule compounds dissolved in a solution
mixture of compounds is injected onto 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
How is retention decided for normal-phase HPLC?
retention is decided by the interaction of the polar parts of the stationary phase and solute
What is the difference between the stationary phase and mobile phase in normal-phase HPLC?
packing material (stationary phase) must be more polar than mobile phase with respect to the sample
What are the common column ligands in normal-phase HPLC?
silica
CN
NH2
What are the mobile phase solvents for normal-phase HPLC?
nonpolar solvents or solvent mixtures such as hexane
How is retention decided for reverse-phase HPLC?
retention is decided by the interaction of the nonpolar parts between the stationary phase and the solute
What is the difference between the stationary phase and mobile phase in reverse-phase HPLC?
packing material (stationary phase) must be relatively nonpolar than the mobile phase with respect to the sample
What are the common column ligands used in reverse-phase HPLC?
bonded hydrocarbons such as C18 and C8
What are the common mobile phase solvents used in reverse phase HPLC?
polar solvents or solvent mixtures such as methanol and acetonitrile
What is the particle size for UPLC?
1.7-1.8 um
What is the column pressure for UPLC?
up to 1030 bar
What are the advantages of UPLC?
increased resolution, sensitivity, and speed
What is the mobile phase in gas chromatography?
gas
What is the stationary phase in gas chromatography?
liquid (GLC, liquid coated on a solid) and solid (GSC, not widely used due to limited number of stationary phase)
Which gases are used in gas chromatography?
helium (most common)
hydrogen
nitrogen
Which field is gas chromatography widely used in?
analytical chemistry
What is the purpose of gas chromatography?
separation of gaseous and volatile substances
GLC principle - separation by partition coefficient
What can be used as a stationary phase for GC capillary columns?
polyethylene glycol
polysiloxanes
hydrocarbons
esters
amides
What are the carrier gases?
helium
hydrogen
nitrogen
Differentiate the carrier gases.
helium:
-excellent thermal conductivity
-expensive
hydrogen:
-better thermal conductivity
-reacts with unsaturated compounds and inflammable
nitrogen:
-reduced sensitivity
-inexpensive
How are molecules characterized in metabolomics?
mass spectrometry
NMR
What is the HMDB?
detailed information of small molecule metabolites found in human body
-chemical data
-clinical data
-molecular biology/biochemistry data
How many metabolite entries are in the HMDB?
220,945
-water soluble and lipid soluble metabolites
-metabolites that would be regarded as either abundant or relatively rare
What are the different metabolomics studies?
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
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?
providing information from spectra of total composition of metabolites
H NMR metabolic fingerprinting
powerful method for discriminating between biological samples on the basis of differences in metabolism
disease diagnosis
What is metabolic profiling?
how metabolites are organized into pathways
understand mechanism of diseases or drugs
