Metabolomics

Metabolomics is an emerging field of “omics” research specializing in the near global analysis of small molecule metabolites (< 1500 Daltons) found in living organisms. Though the field of metabolomics is relatively new, its applications are already being seen in many disciplines including disease diagnostics, agriculture food and safety, and pharmaceutical research and development. These applications are leading to the discovery of many biomarkers and the development of improved screening methods. Metabolomics utilizes a number of different assays for quantitative analysis and identification of metabolites in a variety of sample types.

Specific information relating to the assays is provided below. Each description includes a sample list of possible metabolites identifiable by each method as well as a sample spectra.

Nuclear Magnetic Resonance (NMR)

Nuclear Magnetic Resonance (NMR) is a powerful spectroscopic technique that measures the absorption of radiofrequency radiation by different nuclei (within a chemical compound) when that compound is placed under a strong magnetic field. It gives information about both the structural and chemical properties of certain molecules. NMR is unique as it is one of the few non-destructive methods for analyzing 3D structure and molecular dynamics.

Basically, when a sample is placed in the magnet the nuclei of the proton atoms align with the magnetic field in a way analogous to when the needle of a compass aligns itself in the earth's magnetic field. The magnets used in NMR spectroscopy are ten thousand times stronger than the earth’s magnetic field. The NMR experiment consists of the application of radio frequency pulses of energy to the sample. These pulses stimulate the nuclei to rotate away from their equilibrium position and they start to rotate around the axis of the magnetic field. The exact frequency at which the nuclei rotate is related to both the chemical and physical environment of the atom in the molecule. So therefore, by using different combinations of pulses and delays it is possible to determine how each atom in the molecule interacts with other atoms in the molecule. Finally, using a large set of these interactions it is possible to calculate the three-dimensional structures of molecules. Nuclear Magnetic Resonance can also be used to look at dynamic processes. Some of which are internal motions within regions of larger molecules such as loops in a protein or the base pairs in DNA or RNA. NMR can also be used to monitor chemical reactions.

The Chenomx NMR Suite Professional v. 6.0 is used for identifying and quantifying individual compounds based on the respective signature spectra compiled in libraries. Employing the unique technology, called “targeted” metabolite profiling”, ChenoMX suite currently offers identification and quantification of more than 300 compounds from 1H NMR spectra of complex analyte mixtures. For the conducted experiments, samples are analyzed in D2O by 1H NMR. One significant downside of NMR-based metabolomics is that this technique is not as sensitive as GC- or LC-MS-based techniques and often requires sample amount on milli- or submilli- gram level. Recent advances in modern NMR instrumentation, such as introduction of microcoil NMR probes, clustering first stage preamplifiers and application of cryogenic probes, bring sensitivity of NMR analyses to nano-gram level.

Lipidomics

Lipids have been the Cinderellas of the biological world for far too long, and it is indeed inspiring to see the sudden burst of interest in these fascinating molecules and the techniques for their analysis (Christie 2010). The recognition of lipidomics as a discipline received an impetus with the emergence of new and advanced high-throughput technologies.

The full spectrum of lipids in a biological system is called the lipidome. Mapping of the lipidome is referred to as lipidomics.

Lipidomics is the analysis of lipids on the systems-level scale together with their interacting factors (Feng and Prestwich 2005). Lipidomics involves the quantification and characterization of all lipids in cells/organisms to determine the molecular mechanisms by which they operate. The aim of lipidomics is more than simply to analyze lipids in biological systems; it is to relate lipid compositions of tissues or membranes of animals, plants or microorganisms to their physical properties, enzymes and biology.

The biochemical experiments involved in lipidomics begin with extraction of lipids from various samples such as tissues, cells, different organisms, biofluids etc. The complex lipid mixture - either in its unprocessed form or after sample modification - is analyzed by one or more analytical techniques (GC-MS, HPLC, LC-MS, etc.) to obtain a lipid profile that contains information on the lipid composition of different classes of lipids such as monoacylglycerols (MAG), diacylglycerols (DAG), triacylglycerols (TAG), free fatty acids (FFA), cholesterol, glycolipids and phospholipids present in the starting material. These individual lipid classes are further analyzed in detail to quantify the abundance of saturated, unsaturated, polyunsaturated fatty acids associated with it. Fatty acids analyzed and quantified from stereospecific positions of TAG are subjected to combinatorial lipid reconstruction (CLR) to acquire comprehensive information about different molecular species of TAG.

Analogous to other large-scale approaches, the experimental results are referenced to a control condition to infer distinct metabolites whose levels change upon perturbation of the biological system under investigation. A number of other experimental techniques can be applied to obtain information of lipid inventory from tissues, cells or organism. However, stereospecific fatty acid analysis of TAG, identification and characterization of candidate pathways and effectors of lipids involves additional efforts (affinity probes, optical cell-based assays, enzymatic assays, bioinformatics and so on).

Direct Flow Injection Mass Spectrometry (DI-MS)

We have adapted a targeted quantitative metabolomics approach to analyze biofluids using direct flow injection mass spectrometry (AbsoluteIDQ™ Kit). The Kit is a commercially available assay from BIOCRATES Life Sciences AG (Austria), and it was originally validated for plasma samples. Recently, the kit has been optimized for the analysis of human CSF and urine samples. This kit assay in combination with a 4000 QTrap (Applied Biosystems/MDS Sciex) mass spectrometer is used for targeted identification and quantification of a large number (160) of endogenous metabolites including amino acids, acylcarnitines, glycerophospholipids, sphingolipids and sugars. The method used combines the derivatization and extraction of analytes, and the selective mass-spectrometric detection using multiple reation monitoring (MRM) pairs. Isotope-labeled internal standards are integrated in Kit plate filter for metabolite quantification.

The AbsoluteIDQ kit contains a 96 deep-well plate with a filter plate attached with sealing tape, and reagents and solvents used to prepare the plate assay. The first eight wells in the Kit are used for one blank, three zero samples (urea solution), one standard and three quality control samples that are provided with each kit. A straightforward sample preparation step is used for the assay. Biofluid samples are left to thaw on ice, and are vortexed and centrifuged at 13,000x g. 10 µL of supernatants for each sample is loaded on a filter paper of the kit plate and dried in a stream of nitrogen. Extraction of the metabolites is then achieved using methanol containing 5 mM ammonium acetate. The extracts are analyzed using a 4000 QTrap (Applied Biosystems/MDS Sciex) mass spectrometer. A standard flow injection protocol consisting of two 20 μL injections (one for the positive and one for the negative ion detection mode) is applied for all measurements. MRM detection is used for quantification. MetIQ software, which is a proprietary of Biocrates and is included in the Kit, controls the entire assay workflow, from sample registration to automated calculation of metabolite concentrations to the export of data into other data analysis programs.

Gas Chromatography—Mass Spectrometry (GC-MS)

Gas Chromatography – Mass Spectrometry (GC-MS) is a very powerful technique and has been regarded as the gold standard for analysing lipids, drug metabolites and environmental analysis for many years. One of the advantages of GC-MS is that the identification is based on both a retention time and a mass spectrum (a compounds’ specific fragmentation pattern). Compounds produce reproducible fragmentation patterns when ionized by a fixed electron voltage (usually 70eV) making the technique particularly valuable for identification and quantification for small molecules as it means it is instrument independent and allows databases to be created and shared between users. The NIST08 Mass Spectral Library is one of the most widely used GC-MS reference libraries. It contains more that 220,000 electron ionization mass spectra for a wide variety of metabolites. Reproducibility and robustness of GC-MS methodology allows quantitative detection of analytes unlike proteomic mass spectrometry which tends to be more qualitative.

GC-MS is ideally suited and has traditionally been used for analysis of nonpolar analytes like synthetic organic compounds and hydrophobic natural products. Polar compounds have to be derivatized to make them amenable to analysis by GC-MS. This means that care has to be taken to ensure that the variability inevitably introduced by this preprocessing step is kept to an absolute minimum. Over the past few years, robust sample preparation and analysis methods have been developed. GC-MS has very high sensitivity and can therefore be used for the analysis of less commonly encountered types of samples that might only be available in minute amounts.

Gas chromatography–mass spectrometry (GC-MS)-based metabolomics profiling methods have been developed and used for plant metabolite profiling since the 1980s. Recent introduction of modern GC-TOF and GC-TOF/TOF instruments brings gas chromatography to a new level of sensitivity and allows high throughput analysis of biological samples. As a result, during the past few years GC technology has been more widely used for metabolomics studies in animals and humans with the aim of toxicology and biomarker discovery, disease diagnosis and classification.

High Performance Liquid Chromatography (HPLC)

HPLC, High Performance Liquid Chromatography is one of the most powerful and versatile analytical techniques for separation, identification, and quantification of metabolites present in different biofluids and plant materials.

HPLC is a highly improved form of column chromatography. Instead of a solvent being allowed to drip through a column under gravity, it is forced through under pressures of up to 400 atmospheres. This increase in pressure accelerates the speed of the solvent moving through the column. It also allows use of smaller particle size for the column packing material. This, in turn, provides greater surface area for interactions between the stationary phase and the molecules flowing past it. This translates to better separation of the components of the biological samples. The other major improvement over column chromatography concerns the detection methods that provide a characteristic retention time for the analyte. These methods are highly automated and extremely sensitive.

Though the HPLC is effective in separation, it needs to be coupled with a detector in order to read the results. The detectors coupled to HPLC used in our laboratory are UV, Fluorescence, and Evaporative Light Scattering Detector (ELSD).

The HPLC/Fluorescence Detector is a fast, reliable, and reproducible method for separation and quantification of metabolites. Because the major endogenous metabolites are not naturally fluorescent, we developed the derivatization of metabolites by using dansyl chloride as fluorescent reagent. The dansylation reaction is fast and produces little or no side products. The coupling of HPLC with the fluorescence detector is useful for identifying amines and phenols. We are also using the HPLC/fluorescence detector for the identification and quantification of melatonin in milk.

HPLC/ELSD is used for separation and quantification of lipid classes (neutral lipids, phospholipids). Further separation of neutral lipids (CEs, FFAs, MAGs, DAGs, TAGs) and phopspholipids (PC, LysoPC, PE, LysoPE, PI, LysoPI, PS, LysoPS, SM) is also done using HPLC/ELSD.

Liquid chromatography–mass spectrometry (LC-MS)

Examples of Metabolomics Applications

• Characterize the precise chemical composition of biofuels and biofuel feedstock, including metabolites, lipids and carbohydrates
• Map genotypic changes to quantifiable metabolic outputs
• Assist in the metabolic engineering of both microbes and plants
• Facilitate the design, testing, monitoring and optimization of microbial fermentations
• Comprehensively characterize the phytochemical and nutrient content in plants
• Identify and quantify new chemical biomarkers associated with diseases and disease symptoms
• Assess the physiological effects of drugs or foods on human health