CN102830193A - Serum metabonomic study method based on gas chromatography-mass spectrometry technology - Google Patents
Serum metabonomic study method based on gas chromatography-mass spectrometry technology Download PDFInfo
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Abstract
The invention relates to a serum metabonomic study method based on a gas chromatography-mass spectrometry technology. According to the invention, non-target metabonomic analysis can be carried out upon an experiment sample by using a gas chromatography-mass spectrometry metabolomic platform, and data analysis can be carried out upon produced metabonomic data. Data analysis mainly comprises the contents of: (1) providing metabonomic original data; (2) visually displaying metabolic spectrum differences among different components through multidimensional statistical analysis; (3) metabolic spectrum differences among different components and characteristic metabolites causing the differences; and (4) metabolic spectrum differences among different components and characteristic metabolites causing the differences.
Description
Technical field
This patent relates to a kind of being applicable to that the gas chromatography-mass spectrum technology carries out the method for blood serum metabolic group research, can carry out the metabolism group instrumental analysis of non-targeted; Experiment sample is carried out the metabolism group data analysis.
Background technology
Metabolism group is the metabolism group (metabolomes, all metabolin components of a certain biology) through comparative control group and experimental group, to seek the different research method of other metabolism spectral difference.Some disease association of research during these differences possibly found with clinical biomarker also possibly change relevant with the metabolism after drug candidate in the medicament research and development toxicological study is taken in.The environment metabolism group comes into one's own aspect the influence of wildlife and environment in the contact of research chemicals day by day.At agricultural/chemical field, metabolism group can be used for the exploitation of herbicide and pesticide.
The analysis of metabolism group spectral pattern is different with gene expression research or these researchs that disclose a part of behavior that takes place in the cell of proteome analysis, and it can describe the complete physiological status of a certain moment cell.The more important thing is, if can be in the same place the data integration of proteomics, transcription group and metabolism group (a kind of conformability research ideas and methods that call systems biology), with obtaining the more complete image of living organism biology.
Mass spectrum (MS) is because of having widely dynamic range, can carry out reproducible quantitative test, and can analyze very complex physical body fluid, has been used in the research of metabolism group.Because the complicacy of this type sample in order to detect metabolin as much as possible, usually also will be separated before mass spectrophotometry (gas chromatography, liquid chromatography or Capillary Electrophoresis).
Gas chromatography and mass spectrum (GC/MS) are effective combinations of analyzing volatile chemical.Gas chromatography uses delivery gas to promote analyte through being coated with the fused quartz kapillary of stain.Distribute realization to separate based on the difference of analyte between gas phase and kapillary internal coating.GC/MS requires analyte to volatilize, so that in kapillary, move.Therefore, analyte must have volatility, maybe can pass through the chemically derived volatility that has.For example plant terpenes and essential oil.Free fatty acid also can be analyzed with GC/MS through after deriving.Gas chromatography has high chromatogram degree of separation, and for the analysis of free fatty acid, GC/MS is higher than method for distinguishing sensitivity.
Summary of the invention
The objective of the invention is to be applicable to that the gas chromatography-mass spectrum technology carries out the method for blood serum metabolic group research, can carry out the metabolism group instrumental analysis of non-targeted; Experiment sample is carried out the metabolism group data analysis, and, reduced the error that the sample process process is brought because step is merged.
The technical scheme that the present invention adopts is:
Sample preparation: accurately pipette serum sample that 40 μ l frozen water thaw and manage to EP, add 120 μ l chromatogram methyl alcohol, leave standstill 30min (4 ℃) behind the vortex 30s; The centrifugal 15min of 20000g (4 ℃) gets 120 μ l supernatants to the high-recovery sample bottle, and gentle nitrogen dries up; The 15mg/ml methoxamine pyridine solution that adds 80 μ l, vortex concussion 30s is in 37 ℃ of reaction 90min; Add 80 μ l BSTFA reagent (containing 1%TMCS) then, 70 ℃ of following reactions are carried out GC/MS and are analyzed after 60 minutes.
GC/MS analyzes: the instrumental analysis platform of this experiment is an Agilent 7890A GC/5975C MS system, and capillary chromatographic column is the HP-5MS (30m * 250 μ m i.d.) of Agilent J& W Scientific company.Instrument parameter is set at: 270 ℃ of injector temperatures, and 230 ℃ of EI ion source temperatures, 150 ℃ of quadrupole rod temperature, carrier gas is a helium, split sampling not, sample size 1.0 μ l.Heating schedule is: 70 ℃ of initial temperatures, keep 2min, and the speed of 10 ℃/min rises to 140 ℃, and the speed of 4 ℃/min rises to 240 ℃, rises to 300 ℃ and keep 8min with the speed of 10 ℃/min at last.Adopt the full scan form to carry out Mass Spectrometer Method, the Mass Spectrometer Method scope is 50-600 (m/z).
The data analysis content: the metabolism group raw data is extracted in (1); (2) the metabolism spectral difference of analyzing between showing not on the same group visually through multidimensional statistics is different; (3) the different and characteristic metabolin that makes a difference of the metabolism spectral difference between the different component; (4) the different and characteristic metabolin that makes a difference of the metabolism spectral difference between the different component.
The present invention's advantage compared with prior art is:
1. non-targeted ground is studied all metabolins, has avoided the one-sidedness of traditional analysis research, has embodied systematicness and globality characteristic;
2. created trace detection technology, only need current metabolism group sample demand 20% in addition lower, filled up domestic blank, occupy the leading position in the world;
3. develop processing large sample metabolism group complex data treatment technology, removed noise jamming;
Embodiment
Concrete grammar is described below:
1. accurately pipette serum sample that 40 μ l frozen water thaw and manage to EP, add 120 μ l chromatogram methyl alcohol
2. leave standstill 30min (4 ℃) behind the vortex 30s, the centrifugal 15min of 20000g (4 ℃)
3. get 120 μ l supernatants to the high-recovery sample bottle, gentle nitrogen dries up, and adds the 15mg/ml methoxamine pyridine solution of 80 μ l, and vortex concussion 30s is in 37 ℃ of reaction 90min
4. add 80 μ l BSTFA reagent (containing 1%TMCS), 70 ℃ of following reactions are carried out GC/MS and are analyzed after 60 minutes.
5.GC/MS instrument parameter is: 270 ℃ of injector temperatures, 230 ℃ of EI ion source temperatures, 150 ℃ of quadrupole rod temperature, carrier gas is a helium, split sampling not, sample size 1.0 μ l.Heating schedule is: 70 ℃ of initial temperatures, keep 2min, and the speed of 10 ℃/min rises to 140 ℃, and the speed of 4 ℃/min rises to 240 ℃, rises to 300 ℃ and keep 8min with the speed of 10 ℃/min at last.Adopt the full scan form to carry out Mass Spectrometer Method, the Mass Spectrometer Method scope is 50-600 (m/z).
6. the data that instrument produced are carried out data analysis; Under R software (www.r-project.org/) platform, adopt and extract original data signal from the program code of writing; Import TagFinder software then and carry out retention time correction, peak alignment and the deconvolution analysis of ms fragment, all parameters are default setting, in EXCEL software, carry out later stage compilation at last; The result is organized as two-dimensional data matrix, comprises variable (rt_mz), observed quantity (sample) and integral area.Before carrying out formal statistical study, the mass signal total mark area of sample is carried out normalization, promptly the total mark area of each sample all normalizes to 1000.At last, normalized data importing Simca-P software (version 11.5) is carried out principal component analysis (PCA) (PCA), offset minimum binary side's discriminatory analysis (PLS-DA) and quadrature offset minimum binary side discriminatory analysis (OPLS-DA) respectively.
More than be the description of this invention and non-limiting, based on other embodiment of inventive concept, all among protection scope of the present invention.
Claims (2)
1. method that the blood serum metabolic group that is applicable to phase chromatography-mass spectroscopy technology is studied is characterized in that:
Step 1: sample preparation: accurately pipette serum sample that 40 μ l frozen water thaw and manage to EP, add 120 μ l chromatogram methyl alcohol, leave standstill 30min (4 ℃) behind the vortex 30s; The centrifugal 15min of 20000g (4 ℃) gets 120 μ l supernatants to the high-recovery sample bottle, and gentle nitrogen dries up; The 15mg/ml methoxamine pyridine solution that adds 80 μ l, vortex concussion 30s is in 37 ℃ of reaction 90min; Add 80 μ l BSTFA reagent (containing 1%TMCS) then, 70 ℃ of following reactions are carried out GC/MS and are analyzed after 60 minutes.
Step 2: the instrumental analysis platform of this experiment is an Agilent 7890A GC/5975C MS system, and capillary chromatographic column is the HP-5MS (30m * 250 μ m i.d.) of Agilent J& W Scientific company.Instrument parameter is set at: 270 ℃ of injector temperatures, and 230 ℃ of EI ion source temperatures, 150 ℃ of quadrupole rod temperature, carrier gas is a helium, split sampling not, sample size 1.0 μ l.Heating schedule is: 70 ℃ of initial temperatures, keep 2min, and the speed of 10 ℃/min rises to 140 ℃, and the speed of 4 ℃/min rises to 240 ℃, rises to 300 ℃ and keep 8min with the speed of 10 ℃/min at last.Adopt the full scan form to carry out Mass Spectrometer Method, the Mass Spectrometer Method scope is 50-600 (m/z).
Step 3: advance the data analysis content: the metabolism group raw data is extracted in (1); (2) the metabolism spectral difference of analyzing between showing not on the same group visually through multidimensional statistics is different; (3) the different and characteristic metabolin that makes a difference of the metabolism spectral difference between the different component; (4) the different and characteristic metabolin that makes a difference of the metabolism spectral difference between the different component.
2. data analysing method flow process
Under the R software platform, adopt and extract original data signal from the program code of writing; Import TagFinder software then and carry out retention time correction, peak alignment and the deconvolution analysis of ms fragment; All parameters are default setting; In EXCEL software, carry out later stage compilation at last, the result is organized as two-dimensional data matrix, comprise variable (rt_mz), observed quantity (sample) and integral area.Before carrying out formal statistical study, the mass signal total mark area of sample is carried out normalization, promptly the total mark area of each sample all normalizes to 1000.At last, normalized data importing Simca-P software (version 11.5) is carried out principal component analysis (PCA) (PCA), offset minimum binary side's discriminatory analysis (PLS-DA) and quadrature offset minimum binary side discriminatory analysis (OPLS-DA) respectively.
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| CN103592389A (en) * | 2013-11-15 | 2014-02-19 | 湖州市中心医院 | LC/MS (liquid chromatography-mass spectrometer) metabonomics analysis method based on serum of GDM (gestational diabetes mellitus) patient |
| CN103616450A (en) * | 2013-11-29 | 2014-03-05 | 湖州市中心医院 | Serum specificity metabolite spectrum for patient with lung cancer, and building method thereof |
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| CN104713971A (en) * | 2015-04-01 | 2015-06-17 | 山东省肿瘤医院 | Method for analyzing serum metabolomics on basis of LC-MS (liquid chromatogram-mass spectrograph) serum metabolomics technology |
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| CN110967436A (en) * | 2018-09-28 | 2020-04-07 | 中国人民解放军军事科学院军事医学研究院 | Method for typing different serotypes of clostridium botulinum based on mass spectrometry detection |
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| CN114487188A (en) * | 2022-01-25 | 2022-05-13 | 上海交通大学 | Metabonomics method for preparing animal model for treating primary dysmenorrhea based on radix aconiti kusnezoffii slices |
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