As a powerful tool for the qualitative and quantitative study of small molecules (metabolites) in biological samples, metabolomics has become a major tool for annotating gene functions and revealing various endogenous physiological and biochemical reactions produced by cells after exogenous stimuli in various research fields, including life sciences, disease diagnosis, drug development, nutrition, toxicology, environmental science, and botany.
Glycolysis pathway analysis
Glycolysis is an energy-generating process that produces adenosine triphosphate (ATP), which catabolize glucose into phosphorylated sugar metabolites and finally pyruvate and lactate, as well as ATP. The physicochemical properties of pyruvate and lactate are different from those of the upstream metabolites and usually require Tricarboxylic acid cycle (TCA) analysis.
TCA is a core catabolic pathway that provides energy for cellular life activities and is present in all aerobic organisms. Its intermediate products are also precursors for the synthesis of other biomolecules. The detailed metabolomic analysis of this pathway usually involved components of interest including citric acid, isocitric acid, ketoglutaric acid, succinic acid, jasmonic acid, malic acid, and oxaloacetic acid. These substances were extracted in the same way as glycolytic metabolites. The LC-MS analysis method is identical to lactic acid, which requires reversed-phase chromatography conditions for separation and negative ion mode with mass spectrometry. In contrast, oxaloacetate is as unstable as pyruvic acid and can be treated with the same derivatization reagents to obtain reliable quantitative results.
Acyl coenzyme A and acylcarnitine analysis
Acyl-CoA is a high-energy compound that plays a key role in major metabolic pathways as an intermediate metabolite. Usually, the fatty acylcarnitine content can reflect the efficiency of its corresponding fatty acyl-CoA entering mitochondria and being utilized. The number of carbon chains of fatty acyl-CoA is widely distributed from short-chain with C2-C6, medium-chain with C7-C14, to long-chain with ≥ C16 and have a broad polarity span. The pretreatment method cannot take into account the short-chain polar components and the extraction efficiency of long-chain weakly polar components. It is also difficult to separate these full-chain metabolites effectively under a single chromatographic condition.
Nucleotide analysis
The basic unit of nucleic acid is a nucleotide. Nucleic acid metabolism is closely related to nucleotide metabolism. Nucleic acid metabolism carries out replication, recombination, and transcription of genetic information. Nucleotides are involved in almost all metabolic processes of cells and are highly hydrophilic, therefore revealing well retention and separation with normal-phase chromatography. NAD+, FAD, NADP, and other electron carriers are involved in important redox reactions and continuously provide the body with ATP, so the quantitative analysis of these components can provide important biological information.
Amino acid analysis
Amino acid metabolism is closely linked to the glycolytic pathway, pentose phosphate pathway, and the TCA cycle and, therefore, is a component of great interest in metabolomic analysis. These metabolites are highly water-soluble and are usually extracted with a certain percentage of organic solvent in the aqueous solution system, such as 80% methanol or 80% acetonitrile. Some amino acids with important biological functions are chemically unstable and require special pretreatment methods. For example, homocysteine plays a key role in maintaining redox homeostasis and is closely related to the mechanism of many human diseases. Since the hydrophobic group in its structure is highly reactive and forms disulfides bonds easily, derivatization reactions are required to stabilize such metabolites with hydrophobic groups to ensure accurate quantitative results.
Steroid hormone analysis
Steroid hormones play an important role in regulating many biological responses in the body, including androgens, estrogens, progesterone, salt corticosteroids, and glucocorticoids. These components have important biological functions and can be used as stimulants by athletes. Therefore, the quantitative analysis of steroid hormones is particularly important. Since the content of steroid hormones in biological fluids is very low, thus requirements for these pretreatment extractions are relatively strict. For extraction of components in urine or blood, the most commonly used solvent involves methyl tert-butyl ether, along with a solid-phase extraction strategy. Components exist in the urine mainly in the form of glucosinolates and sulfates combined, so they need to be hydrolyzed before extraction, and glucuronide glucosidase can be used to obtain free steroid hormones. Reversed-phase chromatography is generally used to separate hormonal components, and mass spectrometry is performed using an ESI or APCI ion source.
Bile acid analysis
Bile acids are end products of cholesterol metabolism and are thought to facilitate the emulsification and absorption of fats as well as fat-soluble vitamins. Bile acids are involved in various physiological processes, including energy utilization, bile transport, small intestinal motility, bacterial growth, and inflammatory responses. Bile acids are also closely associated with the development of degenerative liver and kidney diseases, chronic inflammation, intestinal mucosal dysfunction, biliary stasis, and cancer. For the extraction of such components, polar organic solvents, such as methanol and acetonitrile are usually used. For the extraction of tissue samples, pre-grinding with liquid nitrogen or breaking up with magnetic beads is required. Chromatographic separation is performed by conventional reversed-phase chromatography.
Lipidomic analysis
As an important component of cell membranes, lipids are characterized by structural and functional diversity, and their content in tissues or body fluids of living organisms is much higher than that of other metabolites. Lipids mainly include fatty acids, glycerol esters, glycerophospholipids, sterol esters, sphingolipids, glycolipids, and polyketides. Fatty acids are the core building blocks of most lipid molecules and have important biological functions, which can be used as energy sources and precursors of cell membrane lipids. Fatty acid derivatives function as hormones and intracellular messengers and are involved in a variety of disease processes. Conventional extraction solvents for lipidomes include chloroform, methanol, methyl tert-butyl ether, isopropanol/methanol, etc. However, using these organic reagents in the pretreatment of samples allows the extraction of most lipids at high and representative levels, while an optimized extraction solvent system is required to extract specific lipids of interest.
In order to obtain maximized information regarding the whole metabolome in a biological sample, a combination of different pretreatment methods, multiple chromatographic conditions (normal-phase, reversed-phase), untargeted scanning by high-resolution mass spectrometry with different ionization methods (ESI, APCI), and a combination of highly sensitive targeted detection methods are required. Creative Proteomics can provide customers with multi-omics solutions with extensive experience in metabolite analysis. We are equipped with various pretreatments optimized for a wide range of sample types and have introduced a variety of mass spectrometers to meet different detection needs.
More can be found at https://metabolomics.creative-proteomics.com/fatty-acids-analysis-service.htm
