Metabolomics

Question 1

What is the definition of metabolomics?

Answer 1

It is a quantitive study of a whole set of small molecules (metabolome)

Question 2

What is a metabolome?

Answer 2

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

Question 3

What is excluded from the metabolome?

Answer 3

• Enzymes
• Genetic materials
• Structutal molecules

No large + non-functional proteins

Question 4

What are some techniques used in metabolomics?

Answer 4

• LC-MS (especially HPLC-MS)
• GC(gas chromotography)-MS
• NMR
• Capillary electrophoresis

Question 5

What are some characteristics of LC (liquid chromotography)?

Answer 5

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

Question 6

What are the three variables that can be adjusted in LC?

Answer 6

• Resolution
• Column
• Flow rate

Question 7

How is resolution for liquid chromatography calculated?

Answer 7

Review slide 9 and 11

Question 8

What is the optimal resolution volume for adjacent peaks?

Answer 8

At least 1.5 to prevent peak overlap (results in pure samples)

Question 9

What is the impact of resin particle size on the liquid chromatography variables?

Answer 9

Smaller particle size = higher pressure & better resolution

Question 10

What is the impact of flow rate on chromatography variables?

Answer 10

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

Question 11

What are some characteristics of HPLC?

Answer 11

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

Question 12

What are some standard HPLC variables?

Answer 12

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)

Question 13

What are some examples of columns used in HPLCs?

Answer 13

Non-polar:
- C18 (alkyl)
- C18 (polar embedded)

Polar:
- Pentafluorophenyl
- Aminopropyl

Question 14

What are some characteristics of normal-phase HPLC?

Answer 14

Stationary phase: polar

Mobile phase: non-polar (ex. hexanes)

slide 17

Question 15

What are some characteristics associated with reverse-phase HPLC?

Answer 15

Stationary phase: non-polar

Mobile phase: Polar solvents (methanol and acetonitrile)

slide 18

Question 16

What is UPLC?

Answer 16

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

Question 17

What is gas chromatography?

Answer 17

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

Question 18

Review slide 22

Question 19

What are some characteristics of gas chromatography capillary columns?

Answer 19

Stationary phase: PEG, polysiloxanes, hydrocarbonsm esters (can be polar or non-polar)

Diameter: 0.025cm to 0.075cm

Length: 30-90cm

Question 20

What are some characteristics of helium in gas chromatography?

Answer 20

• Excellent thermal conductivity (transfers heat effectively, which is good fro preventing decomposition/condensation of sample)
• Expensive

Question 21

What are some characteristics of hydrogen gas in gas chromatography?

Answer 21

• Better thermal conductivity (not as good as He)
• Reacts with unsaturated compounds and inflammable (conversion of double/triple bonds into single bonds)

Question 22

What are some characteristics of nitrogen gas in gas chromatography?

Answer 22

• Reduced sensitivity (broader liquid chromatography)
• Inexpensive

Question 23

How are the results of liquid chromatography characterized?

Answer 23

MS and NMR are used to characterize the results from LC

Question 24

What is the HMDB (Human Metabolome Database)?

Answer 24

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

Question 25

At what concentrations are metabolites considered to be significant?

Answer 25

When concentrations of metabolites exceed 1micromolar, they are considered to be abundant

Lower than 1 nanomolar, these concentrations are considered to be relatively rare

Question 26

Review slide 30

Question 27

What are the components of a metabolomics study?

Answer 27

• Metabolite target analysis
• Metabolite profiling
• Metabolite fingerprinting
• Metabolic profiling

Question 28

What is metaboliute target analysis?

Answer 28

It is a quantitive or semi-quantitive clinical and pharmaceutical analysis of a specific group of metabolites (ex. all lipids)

Question 29

What is metabolite profiling?

Answer 29

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

Question 30

What is metabolic fingerprinting?

Answer 30

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

Question 31

What is metaboilc profiling?

Answer 31

• Organization of metabolites into pathways
• Understand mechanism of diseases or drug

ex. intra/interindividual metabolic profiling for T2DM