CN106018626A - Tobacco stigma metabonomic analysis method based on gas chromatography-mass spectrometry - Google Patents

Tobacco stigma metabonomic analysis method based on gas chromatography-mass spectrometry Download PDF

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CN106018626A
CN106018626A CN201610608785.XA CN201610608785A CN106018626A CN 106018626 A CN106018626 A CN 106018626A CN 201610608785 A CN201610608785 A CN 201610608785A CN 106018626 A CN106018626 A CN 106018626A
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stigma
metabolite
sample
nicotiana tabacum
mass spectrometry
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李勇
逄涛
师君丽
马文广
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Yunnan Academy of Tobacco Agricultural Sciences
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Yunnan Academy of Tobacco Agricultural Sciences
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N2030/022—Column chromatography characterised by the kind of separation mechanism
    • G01N2030/025—Gas chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N2030/065—Preparation using different phases to separate parts of sample

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Abstract

The invention discloses a tobacco stigma metabonomic analysis method based on a gas chromatography-mass spectrometry. The tobacco stigma metabonomic analysis method comprises the steps of pre-treating a sample, carrying out chromatographic-mass spectrum separation, identifying a metabolite structure, quantitatively analyzing metabolites and analyzing data. The tobacco stigma metabonomic analysis method takes a polar solvent and a non-polar solvent to extract polar metabolites and non-polar metabolites of tobacco stigmas respectively, and all the collected metabolites collected by the gas chromatography-mass spectrometry are qualitatively identified through adopting retention indexes and a mass spectrogram library; and all the collected metabolites are quantitatively analyzed by adopting a non-target quantitative analysis method. The establishment of the method provides important references to development of relative researches of tobacco stigma metabonomics.

Description

A kind of Nicotiana tabacum L. stigma metabonomic analysis methods based on gaschromatographic mass spectrometry
Technical field
The invention belongs to technical field of analytical chemistry, be specifically related to a kind of Nicotiana tabacum L. stigma metabolism based on gaschromatographic mass spectrometry Group credit analysis method.
Background technology
Metabolism group is because of pathology physiological stimulation and the change such as gene, environment caused dynamically multiparameter generation to biosystem Thank to the qualitative, quantitative research of response.Metabolism group is the research field developed rapidly in recent years, proposes metabolism from 1997 Since the concept that group is learned, achieve fruitful achievement, especially at plant metabolism in plant, animal and microbe research field Group learns the many domestic and international well-known universities of research aspect and research unit has all carried out correlational study work.
The analysis method that metabolism group institute uses mainly has nuclear magnetic resonance spectrometry, liquid chromatography-mass spectrometry, gas phase Chromatographic mass spectrometry method etc..Up to the present, gaschromatographic mass spectrometry owing to its running cost is low, qualitative, quantitative ability strong and to metabolism The features such as the broad covered area of thing, have become as Plant Metabolome and study one of most important analysis method.
Nicotiana tabacum L. is the very important a kind of model plant of Plant Physiology research, is also that very important a kind of economy is planted Thing.At present, people have carried out substantial amounts of research at cigarette quality, the aspect such as disease-resistant, pest-resistant, and these researchs are mainly from base The aspect such as cause, environment is launched, and the relevant report conducted a research from the angle of metabolism group is less.This is primarily due to metabolism Group is learned and is still a kind of constantly improving and the research method of development at present.Owing to Nicotiana tabacum L. is the Main Economic organ of Nicotiana tabacum L., people couple Research in Nicotiana tabacum L. is concentrated mainly on Nicotiana tabacum L..Nicotiana tabacum L. stigma is the organ accepting pollen during Nicotiana tabacum L. breeding, educates with tobacco hybridization Plant directly related, be the direct object of different cultivars Nicotiana tabacum L. intermolecular hybrid affinity research.Hence set up the metabolism group of Nicotiana tabacum L. stigma Science study method is significant.
Summary of the invention
It is an object of the invention to provide a kind of Nicotiana tabacum L. stigma metabonomic analysis methods based on gaschromatographic mass spectrometry.
The object of the present invention is achieved like this, including sample pre-treatments, chromatographic mass spectrometry separate, metabolite structures identify, The quantitative analysis of metabolite, data analysis step, specifically include:
A, sample pre-treatments: fresh Nicotiana tabacum L. stigma sample is put into mortar, adds liquid nitrogen freezing, grind, obtain after lyophilization Lyophilizing Nicotiana tabacum L. stigma sample, preserves in 4 DEG C；
B, chromatographic mass spectrometry separate:
1) extraction of polar metabolite and derivatization: the lyophilizing Nicotiana tabacum L. stigma sample accurately weighing 20.0 mg is centrifuged in 1.5 mL Pipe, adds 370 μ L methanol, 480 μ L deionized waters, 450 μ L methyl tertiary butyl ether(MTBE)s and 200 μ L inner mark solutions, ultrasonic, from The heart, takes off clear liquid 600 μ L, puts into freeze dryer centrifugal concentrating 2 hours to remove solvent, takes out sample and add 100 μ L methoxamine Solution, 37 DEG C of constant temperature oximate 90 min, add 80 μ L MSTFA, 37 DEG C of constant temperature silanization 30 min, the sample derived turns To gas chromatography mass spectrometer analysis, sample size 1 μ L；
2) extraction of nonpolar metabolite: accurately weigh 100.0 mg lyophilizing Nicotiana tabacum L. stigma samples, add 4.9 mL dichloromethane With 100 μ L inner mark solutions, ultrasonic 30 min, it is centrifuged, takes supernatant 2 mL nitrogen and dry up, add 200 μ L dichloromethane and redissolve, After redissolution, extracting solution goes to gas chromatography mass spectrometer analysis, sample size 1 μ L；
C, metabolite structures are identified: use mass spectrum to deconvolute and spectrogram library searching, retention index, n-compound checking combine Method to carry out compound qualitative；
D, the quantitative analysis of metabolite: the peak area of the peak area of metabolite with internal standard substance is divided by, it is thus achieved that relative quantitation information, Specific formula for calculation is as follows:
Wherein, CxAnd C1Represent metabolite x and interior target content, μ g/g respectively；
AxAnd A1Represent the chromatographic peak area of metabolite x and content respectively；
E, data analysis: the quantitative analysis results of polar metabolite and nonpolar metabolite in each analysis sample is united Meter, uses principal component analysis and cluster analysis as multivariable technique, carries out the relativeness research between sample, use T inspection analyzes method as univariate statistics, finds key metabolites.
The present invention uses polar solvent and non-polar solven extracted tobacco stigma polar metabolite and nonpolar metabolism respectively Thing, all metabolite using retention index and mass spectrogram storehouse Qualitative Identification gaschromatographic mass spectrometry to be gathered, use non-target All metabolite that quantitative analysis method quantitative analysis is gathered.Being established as of the method is carried out Nicotiana tabacum L. stigma metabolism group and is correlated with Research provides important reference.
Accompanying drawing explanation
Fig. 1 is principal component analysis shot chart (left) and the load diagram (right) of K326 and alata stigma；
Fig. 2 is K326 and alata stigma metabolite Unsupervised clustering analysis chart；
Fig. 3 is the T assay volcano figure of K326 and alata stigma metabolite；
Fig. 4 is the gaschromatographic mass spectrometric analysis total ion current figure of Nicotiana tabacum L. stigma polar metabolite；
Fig. 5 is the gaschromatographic mass spectrometric analysis total ion current figure of the nonpolar metabolite of Nicotiana tabacum L. stigma.
Detailed description of the invention
Below in conjunction with embodiment and accompanying drawing, the present invention is further illustrated, but never in any form to the present invention in addition Limiting, based on present invention teach that any conversion or replacement made, belonging to protection scope of the present invention.
Nicotiana tabacum L. stigma metabonomic analysis methods based on gaschromatographic mass spectrometry of the present invention, locates including before sample Reason, chromatographic mass spectrometry separation, metabolite structures qualification, the quantitative analysis of metabolite, data analysis step, specifically include:
A, sample pre-treatments: fresh Nicotiana tabacum L. stigma sample is put into mortar, adds liquid nitrogen freezing, grind, obtain after lyophilization Lyophilizing Nicotiana tabacum L. stigma sample, preserves in 4 DEG C；
B, chromatographic mass spectrometry separate:
1) extraction of polar metabolite and derivatization: the lyophilizing Nicotiana tabacum L. stigma sample accurately weighing 20.0 mg is centrifuged in 1.5 mL Pipe, adds 370 μ L methanol, 480 μ L deionized waters, 450 μ L methyl tertiary butyl ether(MTBE)s and 200 μ L inner mark solutions, ultrasonic, from The heart, takes off clear liquid 600 μ L, puts into freeze dryer centrifugal concentrating 2 hours to remove solvent, takes out sample and add 100 μ L methoxamine Solution, 37 DEG C of constant temperature oximate 90 min, add 80 μ L MSTFA, 37 DEG C of constant temperature silanization 30 min, the sample derived turns To gas chromatography mass spectrometer analysis, sample size 1 μ L；
2) extraction of nonpolar metabolite: accurately weigh 100.0 mg lyophilizing Nicotiana tabacum L. stigma samples, add 4.9 mL dichloromethane With 100 μ L inner mark solutions, ultrasonic 30 min, it is centrifuged, takes supernatant 2 mL nitrogen and dry up, add 200 μ L dichloromethane and redissolve, After redissolution, extracting solution goes to gas chromatography mass spectrometer analysis, sample size 1 μ L；
C, metabolite structures are identified: use mass spectrum to deconvolute and spectrogram library searching, retention index, n-compound checking combine Method to carry out compound qualitative；
D, the quantitative analysis of metabolite: the peak area of the peak area of metabolite with internal standard substance is divided by, it is thus achieved that relative quantitation information, Specific formula for calculation is as follows:
Wherein, CxAnd C1Represent metabolite x and interior target content, μ g/g respectively；
AxAnd A1Represent the chromatographic peak area of metabolite x and content respectively；
E, data analysis: the quantitative analysis results of polar metabolite and nonpolar metabolite in each analysis sample is united Meter, uses principal component analysis and cluster analysis as multivariable technique, carries out the relativeness research between sample, use T inspection analyzes method as univariate statistics, finds key metabolites.
Sample particle diameter after grinding in step A is less than 40 mesh.
In step A, cryodesiccated vacuum is 0.18 mbar, and temperature is-6 DEG C.
Step B 1) described in inner mark solution be 100 μ g/mL to hydroxyl coumaric acid aqueous solution.
Step B 1) in ultrasonic frequency be 25 ~ 45 kHz, the time is 20 ~ 30 min.
Step B 1) in centrifugal centrifugal force >=5000g.
Step B 1) in the vacuum of freeze dryer centrifugal concentrating be 0.18 mbar, temperature is-6 DEG C.
Step B 1) described in the concentration of methoxamine solution be 20 mg/ml.
Step B 2) described in the dichloromethane solution of the peach aldehyde that inner mark solution is 100 μ g/mL；Described centrifugal from Mental and physical efforts >=10000g.
In step B, chromatographiccondition is: chromatographic column is DB-5MS (30 mm × 0.25, m × 0.25 μm), journey Sequence Elevated Temperature Conditions is 60 DEG C and keeps 1 min, 5 DEG C/min to rise to 280 DEG C, keeps 15 min；Injector temperature is 250 DEG C； Sampling volume is 1 μ L；Carrier gas is helium；Post flow is 1.1 mL/min；Split ratio is 20:1；Mass spectral analysis condition is: transmission Line temperature is 230 DEG C；Ion source temperature 210 DEG C；The solvent delay time of nonpolar metabolite is 2 min, polarity derivatization The solvent delay time of sample is 7 min；Mass scan range is m/z 35-400；Scanning of the mass spectrum speed is 5 Hz.
It is analyzed as a example by phase Nicotiana tabacum L. stigma in full bloom below, particularly as follows:
1, sample pre-treatments
1.1 experiment reagents and device
Methanol (chromatographic grade) is bought in Merck company of Germany；Ultra-pure water is prepared by Millipore purification system.To hydroxyl tonkabean Acid and peach aldehyde (internal standard compound) are bought in lark prestige company；0#Light diesel (calculating for retention index) refuels purchased from locality Stand.PE Clarus 600 GC-MS gas chromatography mass spectrometer (PE company of the U.S.)；SB-50D ultrasonic extraction instrument (the new sesame in Ningbo Biotech inc)；DB-5 MS capillary chromatographic column (30 mm × 0.25, m × 0.25 the μm) (U.S. Agilent company)；MILLI-Q water purification machine (MILLIPORE company)；(system in Beijing Jing founds the limited public affairs of centrifuge to LD5-2A centrifuge Department)；CP2245 analytical balance (sensibility reciprocal 0.0001g, Sartorious company of Germany).
1.2 sample pre-treatments
Prepared by sample: fresh Nicotiana tabacum L. stigma sample is put into mortar, adds liquid nitrogen freezing, and grind rapidly (less than 40 Mesh).Milled sample proceeds to rapidly freeze dryer and carries out lyophilization (vacuum 0.18 mbar, temperature-6 DEG C, freeze-drying time 72 H), remove moisture, be placed in 4 DEG C of Refrigerator stores.
Sample extraction and derivatization: metabolite in Nicotiana tabacum L. stigma is divided into polar metabolite and nonpolar metabolite.Wherein The extraction of polar metabolite and derivatization method be: the lyophilizing Nicotiana tabacum L. stigma sample accurately weighing 20.0 mg is centrifuged in 1.5 mL Pipe, adds 370 μ L methanol, 480 μ L deionized waters, 450 μ L methyl tertiary butyl ether(MTBE)s and 200 μ L inner mark solutions (fragrant to hydroxyl Bean aqueous acid 100 μ g/mL), ultrasonic 30 min, centrifugal (centrifugal force >=5000g), take off clear liquid 600 μ L, put into freeze dryer Within 2 hours, to remove solvent, it is molten that taking-up sample adds 100 μ L methoxamine to (vacuum 0.18 mbar, temperature-6 DEG C) centrifugal concentrating Liquid (20 mg/mL, pyridine solution), 37 DEG C of constant temperature oximate 90 min, add 80 μ L MSTFA, 37 DEG C of constant temperature silanizations 30 Min, the sample derived goes to gas chromatography mass spectrometer analysis, sample size 1 μ L.
The extracting method of nonpolar metabolite: accurately weigh 100.0 mg lyophilizing Nicotiana tabacum L. stigma samples, add 4.9 mL bis- Chloromethanes and 100 μ L inner mark solutions (dichloromethane solution of peach aldehyde, 100 μ g/mL), ultrasonic 30 min, centrifugal (centrifugal force >= 10000g), taking supernatant 2 mL nitrogen and dry up, add 200 μ L dichloromethane and redissolve, after redissolution, extracting solution goes to gas chromatogram Spectrometer analysis, sample size 1 μ L.
1.3 chromatograph mass spectrum analysis conditions
Contrast for convenience of polar metabolite and nonpolar metabolite, the chromatographic separation condition of two kinds of metabolite and mass spectrum in this method Analysis condition essentially identical (except solvent clipping time), concrete chromatographic mass spectrometry condition is as follows:
Chromatographiccondition: chromatographic column is DB-5MS (30 mm × 0.25, m × 0.25 μm), and temperature programming condition is 60 DEG C keep 1 min, 5 DEG C/min rise to 280 DEG C, keep 15 min；Injector temperature is 250 DEG C；Sampling volume is 1 μ L； Carrier gas is helium；Post flow is 1.1 mL/min；Split ratio is 20:1.
Mass spectral analysis condition: transmission line temperature is 230 DEG C；Ion source temperature 210 DEG C；The solvent of nonpolar metabolite prolongs Time is 2 min late, and the solvent delay time of polarity derivatization sample is 7 min；Mass scan range is m/z 35-400；Matter Spectrum scanning speed is 5 Hz.
The Structural Identification of 1.4 metabolite
Owing to metabolism group is concerned with the situation of change of all metabolite that can detect someway, and wherein most Metabolite is likely to not pay close attention in traditional metabolite analysis, and therefore the Structural Identification to unknown metabolite is metabolism group Highly important research contents.This experiment uses mass spectrum to deconvolute and spectrogram library searching, retention index, n-compound checking phase In conjunction with method to carry out compound qualitative.
Mass spectrum deconvolutes and spectrogram library searching: owing to all metabolite cannot be completely separated by gas chromatogram, therefore gas phase The mass spectrum put sometime that chromatograph mass spectrum analysis instrument obtains is in practice likely to be the mixing of the mass spectrum of several compound.This Experiment uses AMDIS(V 2.7) software carries out mass spectrum and deconvolutes and peak identification, it is thus achieved that eliminate substrate background and fused peaks Pure mass spectrum.NIST MS Search 2.0 is used to join NIST mainlib data base, wileyregistry8e data base The preliminary structure carrying out metabolite with fiehn metabolism group data base is qualitative.
Retention index is qualitative: owing to metabolite retention index on same type gas chromatographic column is the most stable, this reality Testing employing retention index, to carry out supplementary structure qualitative.Method particularly includes: by carbon number continuous print linear paraffin (0# diesel oil) according to this The chromatographic mass spectrometry method of experiment is analyzed, it is thus achieved that the retention time of these alkane, then carries out metabolite according to below equation Retention index calculate:
Wherein:RI x Refer to that retention time is in the retention index that carbon number is certain metabolite between n and n+1；N represents linear paraffin Carbon number；tx、tn、tn+1Refer to respectively when metabolite x, carbon number are the reservation of linear paraffin etc. that the linear paraffin of n, carbon number are n+1 Between.
N-compound is verified: be standardized compound checking to through mass spectral database and retention index compound qualitatively.I.e. N-compound is analyzed according to this chromatographic mass spectrometry method tested, by the retention time obtained and mass spectrum and tobacco column In head sample, compound undetermined is compared, if both of which can be coincide, the most finally determines the qualitative results of this compound.
1.5 the quantitative analysis method of metabolite
Owing to or cannot be difficult to obtain the n-compound of all metabolite, and metabolism group is typically without absolute quantitation, The quantitative analysis of metabolite uses the method for relative quantification.I.e. assume that metabolite response on the detector is consistent with internal standard substance, The peak area of the peak area of metabolite with internal standard substance is divided by, it is thus achieved that relative quantitation information.Specific formula for calculation is as follows:
Wherein, CxAnd CIRepresent metabolite x and interior target content, A respectivelyxAnd AIRepresent metabolite x and interior target chromatographic peak respectively Area.
1.6 data analysis
The quantitative analysis results of polar metabolite and nonpolar metabolite in each analysis sample is integrated into Excel data Table (peak table), in peak table, the quantitative information of some metabolite is included into specific a (row), in order to metabolism between multisample The statistical analysis of thing content.Use principal component analysis and cluster analysis as multivariable technique, the phase between research sample To relation research, use T inspection to analyze method as univariate statistics, find key metabolites.
To be embodied as case, the present invention will be further described below:
Embodiment 1
The stigma metabolism group research of different cultivars (K326 and alata) Nicotiana tabacum L.
Experiment material: each 6 of the stigma sample of K326 and the alata kind Nicotiana tabacum L. of phase in full bloom, sample is prepared according to " before sample Processing " the described method of part carries out.
Experimental technique:
The extraction of polar metabolite and derivatization method be: accurately weighs the lyophilizing Nicotiana tabacum L. stigma sample of 20.0 mg in 1.5 mL Centrifuge tube, adds 370 μ L methanol, 480 μ L deionized waters, 450 μ L methyl tertiary butyl ether(MTBE)s and 200 μ L inner mark solutions (to hydroxyl Basic note bean aqueous acid 100 μ g/mL), ultrasonic 30 min, centrifugal (centrifugal force >=5000g), take off clear liquid 600 μ L, put into and freeze Dry machine (vacuum 0.18 mbar, temperature-6 DEG C) centrifugal concentrating 2 hours, removes solvent, takes out sample and adds 100 μ L methoxamine Solution (20 mg/mL, pyridine solution), 37 DEG C of heating in water bath 90 min, add 80 μ L MSTFA, 37 DEG C of water-bath 30 min, spread out The sample given birth to goes to gaschromatographic mass spectrometric analysis instrument analysis, sample size 1 μ L.
The extracting method of nonpolar metabolite: accurately weigh the lyophilizing Nicotiana tabacum L. stigma sample of 100.0 mg, add 4.9 mL Dichloromethane and 100 μ L inner mark solutions (dichloromethane solution of peach aldehyde, 100 μ g/mL), ultrasonic 30 min, centrifugal (centrifugal force >=10000g), take supernatant 2 mL nitrogen and dry up, add 200 μ L dichloromethane and redissolve, after redissolution, extracting solution goes to gas phase color Spectrum mass spectrometer analysis, sample size 1 μ L.
Chromatographiccondition: chromatographic column is DB-5MS (30 mm × 0.25, m × 0.25 μm) temperature programming condition Being 60 DEG C keeps 1 min, 5 DEG C/min to rise to 280 DEG C, keeps 15 min；Injector temperature is 250 DEG C；Sampling volume is 1 μL；Carrier gas is helium；Post flow is 1.1 mL/min；Split ratio is 20:1.
Mass spectral analysis condition: transmission line temperature is 230 DEG C；Ion source temperature 210 DEG C；The solvent of nonpolar metabolite prolongs Time is 2 min late, and the solvent delay time of polarity derivatization sample is 7 min；Mass scan range is m/z 35-400；Matter Spectrum scanning speed is 5 Hz.Use mass spectrum to deconvolute and spectrogram library searching, retention index, n-compound verify the side combined It is qualitative that method carries out compound.The method using internal standard relative quantification is carried out quantitatively.
The Structural Identification of metabolite: use mass spectrum to deconvolute and spectrogram library searching, retention index, n-compound checking phase In conjunction with method to carry out compound qualitative.Using AMDIS(V 2.7) software carries out mass spectrum and deconvolutes and peak identification, it is thus achieved that go Except substrate background and the pure mass spectrum of fused peaks.Use NIST MS Search 2.0 join NIST mainlib data base, The preliminary structure that wileyregistry8e data base and fiehn metabolism group data base carry out metabolite is qualitative.Use and retain It is qualitative that index carries out supplementary structure.Method particularly includes: the chromatograph that carbon number continuous print linear paraffin (0# diesel oil) is tested according to this Mass spectrometry method is analyzed, it is thus achieved that the retention time of these alkane, then carries out the retention index of metabolite according to below equation Calculate:
Wherein: RIxRefer to that retention time is in the retention index that carbon number is certain metabolite between n and n+1；N represents linear paraffin Carbon number；tx、tn、tn+1Refer to respectively when metabolite x, carbon number are the reservation of linear paraffin etc. that the linear paraffin of n, carbon number are n+1 Between.
The quantitative analysis of metabolite: owing to or cannot be difficult to obtain the n-compound of all metabolite, and metabolism Group is learned typically without absolute quantitation, and the quantitative analysis of metabolite uses the method for relative quantification.I.e. assume that metabolite is at detector On response consistent with internal standard substance, the peak area of the peak area of metabolite with internal standard substance is divided by, it is thus achieved that relative quantitation information.Tool Body computing formula is as follows:
Wherein, Cx and CI represents metabolite x and interior target content respectively, Ax and AI represents metabolite x and interior target chromatograph respectively Peak area.
Data analysis: use principal component analysis and cluster analysis as multivariable technique, the phase between research sample To relation research, use T inspection to analyze method as univariate statistics, find key metabolites.
Experimental result: experimental result is listed in table 1, table 2, Fig. 1, Fig. 2, Fig. 3.K326 Yu alata exists as can be seen from Figure 1 There is significantly separation on the two dimensional surface of principal component analysis shot chart (Fig. 1 is left), illustrate that the stigma metabolite of both Nicotiana tabacum L.s is deposited In significantly difference, and concrete difference metabolite is embodied on principal component analysis shot chart (Fig. 1 is right).
Metabolite data carrying out cluster analysis (Fig. 2) find, two breed formations of alata and K326 two are the most poly- Class, and the hierarchical relationship between two indivedual samples of big apoplexy due to endogenous wind finds full expression the most in fig. 2.
In order to find further and verify diversity metabolite, all metabolite are carried out T check analysis, by arriving of analyzing P value and class between average ratio (alata is divided by K326) make volcano figure (Fig. 3), take p value less than 0.05, and average ratio more than 2 or Metabolite less than 0.5 marks by yellow in the drawings.K326 Yu alata two can be can be visually seen from the figure of volcano grow tobacco stigma Significance difference opposite sex metabolite.
The structure-characterized result of table 1 part stigma metabolite
The relative quantitative assay peak table of table 2 part stigma metabolite

Claims (10)

1. a Nicotiana tabacum L. stigma metabonomic analysis methods based on gaschromatographic mass spectrometry, it is characterised in that locate before including sample Reason, chromatographic mass spectrometry separation, metabolite structures qualification, the quantitative analysis of metabolite, data analysis step, specifically include:
A, sample pre-treatments: fresh Nicotiana tabacum L. stigma sample is put into mortar, adds liquid nitrogen freezing, grind, obtain after lyophilization Lyophilizing Nicotiana tabacum L. stigma sample, preserves in 4 DEG C；
B, chromatographic mass spectrometry separate:
1) extraction of polar metabolite and derivatization: the lyophilizing Nicotiana tabacum L. stigma sample accurately weighing 20.0 mg is centrifuged in 1.5 mL Pipe, adds 370 μ L methanol, 480 μ L deionized waters, 450 μ L methyl tertiary butyl ether(MTBE)s and 200 μ L inner mark solutions, ultrasonic, from The heart, takes off clear liquid 600 μ L, puts into freeze dryer centrifugal concentrating 2 hours to remove solvent, takes out sample and add 100 μ L methoxamine Solution, 37 DEG C of constant temperature oximate 90 min, add 80 μ L MSTFA, 37 DEG C of constant temperature silanization 30 min, the sample derived turns To gas chromatography mass spectrometer analysis, sample size 1 μ L；
2) extraction of nonpolar metabolite: accurately weigh 100.0 mg lyophilizing Nicotiana tabacum L. stigma samples, add 4.9 mL dichloromethane With 100 μ L inner mark solutions, ultrasonic 30 min, it is centrifuged, takes supernatant 2 mL nitrogen and dry up, add 200 μ L dichloromethane and redissolve, After redissolution, extracting solution goes to gas chromatography mass spectrometer analysis, sample size 1 μ L；
C, metabolite structures are identified: use mass spectrum to deconvolute and spectrogram library searching, retention index, n-compound checking combine Method to carry out compound qualitative；
D, the quantitative analysis of metabolite: the peak area of the peak area of metabolite with internal standard substance is divided by, it is thus achieved that relative quantitation information, Specific formula for calculation is as follows:
Wherein, CxAnd C1Represent metabolite x and interior target content, μ g/g respectively；
AxAnd A1Represent the chromatographic peak area of metabolite x and content respectively；
E, data analysis: the quantitative analysis results of polar metabolite and nonpolar metabolite in each analysis sample is united Meter, uses principal component analysis and cluster analysis as multivariable technique, carries out the relativeness research between sample, use T inspection analyzes method as univariate statistics, finds key metabolites.
Nicotiana tabacum L. stigma metabonomic analysis methods based on gaschromatographic mass spectrometry the most according to claim 1, its feature exists Sample particle diameter after grinding in step A is less than 40 mesh.
Nicotiana tabacum L. stigma metabonomic analysis methods based on gaschromatographic mass spectrometry the most according to claim 1, its feature exists In step A, cryodesiccated vacuum is 0.18mbar, and temperature is-6 DEG C.
Nicotiana tabacum L. stigma metabonomic analysis methods based on gaschromatographic mass spectrometry the most according to claim 1, its feature exists In step B 1) described in inner mark solution be 100 μ g/mL to hydroxyl coumaric acid aqueous solution.
Nicotiana tabacum L. stigma metabonomic analysis methods based on gaschromatographic mass spectrometry the most according to claim 1, its feature exists In step B 1) in ultrasonic frequency be 25 ~ 45 kHz, the time is 20 ~ 30min.
Nicotiana tabacum L. stigma metabonomic analysis methods based on gaschromatographic mass spectrometry the most according to claim 1, its feature exists In step B 1) in centrifugal centrifugal force >=5000g.
Nicotiana tabacum L. stigma metabonomic analysis methods based on gaschromatographic mass spectrometry the most according to claim 1, its feature exists In step B 1) in the vacuum of freeze dryer centrifugal concentrating be 0.18 mbar, temperature is-6 DEG C.
Nicotiana tabacum L. stigma metabonomic analysis methods based on gaschromatographic mass spectrometry the most according to claim 1, its feature exists In step B 1) described in the concentration of methoxamine solution be 20 mg/ml.
Nicotiana tabacum L. stigma metabonomic analysis methods based on gaschromatographic mass spectrometry the most according to claim 1, its feature exists In step B 2) described in the dichloromethane solution of the peach aldehyde that inner mark solution is 100 μ g/mL；Described centrifugal centrifugal force >= 10000g。
Nicotiana tabacum L. stigma metabonomic analysis methods based on gaschromatographic mass spectrometry the most according to claim 1, its feature exists In step B, chromatographiccondition is: chromatographic column is DB-5MS (30 mm × 0.25, m × 0.25 μm), temperature programming Condition is 60 DEG C and keeps 1 min, 5 DEG C/min to rise to 280 DEG C, keeps 15 min；Injector temperature is 250 DEG C；Sample introduction body Amassing is 1 μ L；Carrier gas is helium；Post flow is 1.1 mL/min；Split ratio is 20:1；Mass spectral analysis condition is: transmission line temperature It it is 230 DEG C；Ion source temperature 210 DEG C；The solvent delay time of nonpolar metabolite is 2 min, polarity derivatization sample The solvent delay time is 7 min；Mass scan range is m/z 35-400；Scanning of the mass spectrum speed is 5 Hz.
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