Metabolomics is a field of life science about chemical processes involving metabolites. The combination of these metabolites is called the metabolome
This is one of the most perspective direction among molecular –omics. Metobolomic today is not only postgenomic analysis, but full value direction. Metabolic profile can be an alternative view to biological systems, different from proteomic genomic and transcriptomic points of view. Metabolome is one of the most changeable systems in biology systems. That’s why metabolomics research is a brilliant way for ecological, functional genomics, toxicology, medicine and many other areas.
But metabolomiс approach is accompanied by many difficulties: the approximate average number of metabolites in metabolite network is 5000. Therefore metabolomic approach requires high-precision analytic technics with modern equipment installed in our resource center
Our equipment for gas chromato-mass-spectroscopy allows us conduct analyzes of different compounds such as:
-sugars and them derivatives
- aminoacids
- other organic acids
- plant hormones (for example: auxin, salicylic acid, ethylene, abscisic acid)
- waxes (for example: esters of fatty acids and fatty alcohol)
Our equipment for liquid chromato-mass-spectroscopy allows us conduct analyzes of:
- - Proteins
- - Peptides
- - Glucosinolates
- - Phenol derivatives
Analysis of component composition of essential oils with two-dimensional gas chromatography.
GC-MS – metabolic analysis of peritoneal fluid
Analysis of fatty acid methyl esters
Analysis of component composition of essential oils with two-dimensional gas chromatography.
Resource center equipment:
Comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometer (GC x GC-TOF) LECO Pegasus 4D;
Autosampler vials, septs.
Method description:
This method allows us to characterize difficult mixes of flying phytogenic compounds. Using this method you can solve non-standard problems, for example: research and control aromatherapy drugs quality and research allelopathic connections implemented by flying compounds.
Solvents: hexane or chloroform.
Sample preparation: dissolve analyte in hexane or chloroform to concentration 1 mg/ml. Then filtrate it using paper filters.
NOTE: If you used hydrodistillation for essential oils extraction there can be some water in sample. To avoid contamination by water you should dry sample using sodium sulfate with subsequent filtration before dissolving in hexane or chloroform.
Analyzis conditions:
Carrier gas: helium
Flow: 1 ml/min., constant.
Evaporator temperature: 250 °С
First column: BPX-5 (Supelco) 30m. х 0.25mm. x 0.25 um.
First thermostat temperature: from 70 to 270 °С, 4 °С/min.
Second column: BPX-50 (SGE) 1.5m. х 0.1 mm. x 0.1 um.
Second thermostat temperature: from 75 to 275 °С, 4 °С/min.
Thermal modulator overheating: 15 °С.
Modulation time: 4 sec.
Time of hot impuls: 1.3 sec.
Transfer Line: 250 °С.
MS parameters: LECO Pegasus 4D GCxGC-TOFMS
Ionization type: EI
Ionization energy: 70 eV
Source temperature: 220 °С.
m/z range: 50 – 500.
Speed of data collectionСкорость сбора данных: 100 spec/sec.
Data analysis: data collection and procession occur in an automatic deconvolution mode with identification of compounds using mass spectrometric NIST10 library.
Quantitative evaluation of data using normalization method with results visualization are obtained in ChromTOF®.
Source:
Буданцев, А. Л., & Шаварда, А. Л. (2013). Анализ эфирного масла Dracocephalum oblongifolium (Lamiaceae) с использованием полной двумерной хроматографии. Растительные ресурсы, 49(1), 107–117.
GC-MS – metabolic analysis of peritoneal fluid.
Resource center equipment:
Comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometer (GC x GC-TOF) LECO Pegasus 4D;
Vacuum concentrator CentriVac Labconco;
Autosampler vials, septs.
Method description:
This method is a good example of untarget metabolomic analysis using GC-MS of metabolic pool.
It can be used for metabolomics research of flaying small molecules or small molecules with fluing derivates (organic acids, alcohols, amines, mono- or disaccharides and other) from any biological objects.
Reagents: N,O-Bistrifluoroacetamide (BSTFA) (Fluka), pyridine.
Standard tricosane (Sigma) solution in pyridine with 1000 ppm (1 mg/ml) concentration.
Sample preparation:
First step is sample homogenization with methanol or acetonitrile. Optionally there possibly ultrafiltration to remove all macromolecules. Then were leofilisation in vacuum concentrator. After leofelisation sample mix with 20-100 ul of internal standard (1000 ppm tricosane in pyridine). Then we need to get the trimethylsilyl derivatives of analytes. To do this, there are two ways. First option. Mix sample with 20 ul of BSTFA and incubate it in 100 °С by 15 min. Second option. Automatic silicilation in Gerstel autosampler which installed in to Pegasus 4D.
Analysis conditions:
Carrier gas: helium
Flow: 1 ml/min., constant.
Evaporator temperature: 300 °С
First column: BPX-5 (Supelco) 30m. х 0.25mm. x 0.25 um.
First thermostat temperature: from 70 to 320 °С, 6 °С/min., 15 min isotherm.
Second column: BPX-50 (SGE) 1.5m. х 0.1 mm. x 0.1 um.
Second thermostat temperature: from 75 to 325 °С, 6 °С/min.
Thermal modulator overheating: not used.
Transfer Line: 250 °С.
MS parameters: LECO Pegasus 4D GCxGC-TOFMS
Ionization type: EI
Ionization energy: 70 eV
Source temperature: 220 °С.
m/z range: 50 – 1000.
Speed of data collectionСкорость сбора данных: 2 spec/sec.
Data analysis: Metabolomic matrix were created using free soft AMDIS and UniChrom.
NOTE: If you want to use AMDIS or UniChrom you should convert your chromatogram to AIA (netCDF) format.
Quantitative evaluation of metabolomics profile occur by full ion current using internal standart method without sensitivity factor (semi-quant).
Source:
Shender, V. O., Pavlyukov, M. S., Ziganshin, R. H., Arapidi, G. P., Kovalchuk, S. I., Anikanov, N. A., … Govorun, V. M. (2014). Proteome-metabolome profiling of ovarian cancer ascites reveals novel components involved in intercellular communication. Molecular & Cellular Proteomics : MCP, 13(12), 3558–3571. doi:10.1074/mcp.M114.041194
Analysis of fatty acid methyl esters
Resource center equipment:
Comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometer (GC x GC-TOF) LECO Pegasus 4D;
Autosampler vials, septs.
Method description:
Two-dimension realization of FAME- analysis. This method allows identification of fatty acids trans-isomers and distinguish between the position of the double bounds. Two dimensional chromatographic method allow us to divide analytes by two characteristic. On the first column where it is effective to divide saturated fats and on the second column where it is effective to divide the unsaturated fats. Together it gives essential chromatographic resolution that allows you to distinguish a large number of isomers.
Sample preparation:
Our Resource Center does not provide sample hydrolysis for fatty acid methyl esters extraction.
Analyzis conditions:
GC parameters: LECO Pegasus 4D с термомодулятором и вторичным термостатом.
Injection: 1 ul, 250 °С.
First column: DB-5MS 30 м. х 0.25 мм. х 0,25 мкм. (Agilent).
Second column: BPX-50 2 м. х 0.1 мм. х 0.1 мкм. (SGE)
Carrier gas: helium
Flow: 1 ml/min., constant.
First thermostat temperature: 40°С, 2 min., 30°С/min. till 160°С, 2°С till 300°С, 5 min. Isoterm.
Second thermostat temperature: 45°С, 2 min., 30°С/min. till 165°С, 2°С till 305°С, 5 min. Isoterm.
Thermal modulator overheating: 30 °С.
Modulation time: 5 sec.
Time of hot impuls: 1 sec.
Transfer Line: 250 °С.
Analyzis time: 83.5 min.
MS parameters: LECO Pegasus 4D GCxGC-TOFMS
Ionisation type: EI
Ionisation energy: 70 eV
Source temperature: 220 °С.
m/z range: 45 – 650.
Speed of data collection: 100 spec/sec.
Quantitative evaluation of data using normalization method with results visualization are obtained in ChromTOF®.
Source:
LECO Application Notes: http://www.leco.com/support/application-support-separation-sciences/app-notes-separation-science?view=category&id=144
