METABOLOMIKA

METABOLOMICS

The study of global metabolite profiles in a system (cell, tissue, or organism) under a given set of conditions.

25,000-30,000 several 100,000 up to 7,000

METABOLOME: the complete set of small-molecule metabolites

- metabolic intermediates- hormones - other signalling molecules- secondary metabolites

Primary metabolites are organic compounds that are directly involved in normal growth, development, and reproduction.

Secondary metabolites are organic compounds that are not directly involved in the normal growth, development, or reproduction of an organism. Usually has an important ecological function (e.g. antibiotics, pigments).

Primary metabolites are organic compounds that are directly involved in normal growth, development, and reproduction, e.g.:

- organic acids (e.g. acetic acid, lactic acid ...) - alcohols (e.g. ethanol, glycerol, polyols...) - amino acids- nucleotides, coenzymes - vitamins - carbohydrates - peptides - other

Secondary metabolites are organic compounds that are not directly involved in the normal growth, development, or reproduction of an organism:

A/ Small "small molecules“: - alkaloids (e.g. atropine, cocaine, codeine, morphine ...) - terpenoids (e.g. carotenes, some steroids (e.g. sapinons), geraniol, limonene ...) - glycosides (e.g. salicin, flavonoic and phenolic glycosides ...) - flavonoids (e.g. catechins, quercetin, luteolin, rutin ...) - natural phenols (e.g. resveratrol, eugenol ...) - phenazines (e.g. pyocyanin, phenazine-1-carboxylic acid ...)

B/ Big "small molecules“: - polyketides (e.g. erythromycin ...) - fatty acid synthase products (e.g. phloroglucinols ...) - nonribosomal peptides (e.g. vancomycin, gramicidin, bacitracin ...) - polyphenols (e.g. tannins, flavonoids

Structural analysisHomogenization

Extraction:- using a solvent (water, hot water; polar solvent (e.g. methanol, methanol/formic acid); nonpolar solvent (e.g. chloroform, chlorophorm/phenol) - liquid liquid extraction - solid phase extraction (e.g. on C18 sorbent cartridges) - ultrasonic extraction- heat reflux extraction- microwave-assisted extraction- critical carbon dioxide- pressurized liquid extraction

Concentration: by evaporization, ultrafiltration ....

Purification: by crystalization, preparative chromatography ...

Analysis (fractionation & spectrometry)- paper chromatography - thin layer chromatography (TLC) - gass chromatography (GC)- liquid chromatography (FPLC, HPLC, RPLC) - electrophoretic techniques (capillary electrophoresis) - spectroscopy (IR, UV, NMR, MS) - chemical characterisation

Liquid chromatography (LC)The mobile phase is a liquid. LC can be carried out either in a column or a plane. HPLC generally utilizes very small packing particles and a relatively high pressure.

HPLC: the sample is forced by a liquid (mobile phase) at high pressure, through a column that is packed with a stationary phase composed of irregularly or spherically shaped particles, a porous monolithic layer, or a porous membrane.

NPLC (normal phase LC): stationary phase is more polar than the mobile phase (e.g. toluene as the mobile phase, silica as the stationary phase). RPLC (reverse phase LC): stationary phase is less polar than the mobile phase (e.g. water-methanol mixture as the mobile phase and C18 = octadecylsilyl as the stationary phase).

Preparative HPLC apparatus

UV-VIS spectroscopy:

Organic compounds, especially those with a high degree of conjugation, absorb light in the UV or visible regions of the electromagnetic spectrum. Ultraviolet-visible spectroscopy (UV-vis) can distinguish between enantiomers by showing a distinct Cotton effect (change in optical rotation) for each isomer.

Spectrophotometry is the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength. Throughcalculations of observed wavelengths, a spectrophotometer is able to determine, depending on the control, what substances are present in a sampleand exactly how much.

IR spectroscopy:

The infrared spectrum of a sample is recorded by passing a beam of infrared light through the sample. Examination of the transmitted light reveals how much energy was absorbed at each wavelength. From this, a transmittance or absorbance spectrum can be produced, showing at which IR wavelengths the sample absorbs. Analysis of these absorption characteristics reveals details about the molecular structure of the sample.

Nuclear magnetic resonance (NMR): All stable isotopes that contain an odd number of protons and/or of neutronshave an intrinsic magnetic moment and angular momentum - a nonzero spin.All nuclides with even numbers of both have spin 0. The most commonly studied nuclei are 1H (the most NMR-sensitive isotope after the radioactive 3H) and 13C.

Magnetic nuclei in a magnetic field absorb and re-emit electromagnetic (EM) energy. This energy is at a specific resonance frequency which depends on the strength of the magnetic field and other factors. This allows the observation of specific quantum mechanical magnetic properties of an atomic nucleus.

NMR usually involves two sequential steps:

A/ The alignment (polarization) of the magnetic nuclear spins in an applied, constant magnetic field H0.

B/ The perturbation of this alignment of the nuclear spins by employing an electro-magnetic (radio frequency) pulse.

When a sample is placed in a non-uniform magnetic field then the resonance frequencies of the sample's nuclei depend on where in the field they are located.

The electron distribution of the same type of nucleus (e.g. 1H or 13C) usually varies according to the local geometry (binding partners, bond lengths, angles between bonds, ...), and with it the local magnetic field at each nucleus. This is reflected in the spin energy levels (and resonance frequencies). The variations of nuclear magnetic resonance frequencies of the same kind of nucleus, due to variations in the electron distribution, is called the chemical shift. The size of the chemical shift is given with respect to a reference frequency of a reference sample.

Mass spectrometry (MS) is an analytical technique that measures the mass-to-charge ratio of charged particles. It is used for determining masses of particles, for determining the elemental composition of a sample or molecule, and for elucidating the chemical structures of molecules, such as peptides and other chemical compounds. The MS principle consists of ionizing chemical compounds to generate charged molecules or molecule fragments and measuring their mass-to-charge ratios.

MS procedure:1. A sample is loaded onto the MS instrument, and undergoes vaporization.

2. The components of the sample are ionized by one of a variety of methods (e.g., by impacting them with an electron beam), which results in the formation of charged particles (ions).

3. The ions are separated according to their mass-to-charge ratio in an analyzer by electromagnetic fields.

4. The ions are detected, usually by a quantitative method.

5. The ion signal is processed into mass spectra.

parent peak (M = 92)

Human Metabolome Project is a $7.5 million Genome Canada funded project launched in January 2005. They estimated approximately 2900 endogenous or common metabolites that are detectable in the human body. Not all of these metabolites can be found in any given tissue or biofluid. This is because different tissues/biofluids serve different functions or have different metabolic roles. Until 2006, the HMP has identified and quantified (i.e. determined the normal concentration ranges for) 309 metabolites in CSF, 1122 metabolites in serum, 458 metabolites in urine and approximately 300 metabolites in other tissues and biofluids.

In January 2007 scientists at the University of Alberta and the University of Calgary finished a draft of the human metabolome. They have catalogued and characterized 2,500 metabolites, 1,200 drugs and 3,500 food components that can be found in the human body.

The data are freely accessible in an electronic format to all researchers through the Metabolome Database (www.hmdb.ca). In addition, all compounds will be publicly available through our Human Metabolome Library (www.metabolibrary.ca).