CN103936598B - The relative quantitation method of carboxylic acids signaling molecule in living things system based on novel mass discrepancy label - Google Patents
The relative quantitation method of carboxylic acids signaling molecule in living things system based on novel mass discrepancy label Download PDFInfo
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Abstract
The present invention provide a kind of based on novel mass discrepancy tag molecule to the method for the relative amount of carboxylic acids signaling molecule in quantitative biological sample system, also provide for a kind of by light/heavy isotope to the mass discrepancy tag molecule of labelling to and synthetic method.Described mass discrepancy tag molecule is to comprising light isotope tag molecule and heavy isotope tag molecule, two of which tag molecule is shown in formula (I), described gently/heavy isotope any portion to being present in described structure division A and B or two parts in, wherein: the light/heavy isotope in structure division A is to selecting free H/D12C/13C and14N/15One or more in the group of N composition, or the light/heavy isotope in structure division B is to selecting free H/D,12C/13C and16O/18One or more in the group of O composition.
Description
Technical field
The present invention provide a kind of based on novel mass discrepancy tag molecule to the method for the relative amount of carboxylic acids signaling molecule in quantitative biological sample system, described method is a kind of high sensitivity, high-throughout relative quantitation method.The present invention also provide for a kind of by light/heavy isotope to the mass discrepancy tag molecule of labelling to and synthetic method.
Background technology
Carboxylic acids signaling molecule is some carboxylic chemical molecular naturally occurring in biology, and its effect is in iuntercellular and intracellular delivery information, participates in a lot of processes of regulation and control biological growth and development, and its achievement in research has great using value.In order to further investigate the mechanism of action of carboxylic acids signaling molecule, it is necessary to their changes of contents is launched research.But, the quantitative analysis of carboxylic acids signaling molecule mainly faces following challenge: content is relatively low；Organism exists the interference of other high-load secondary metabolite；It is distributed and/or interaction network to study the time-space of signaling molecule, it is desirable to multi-signal molecule and precursor or metabolite are carried out content analysis simultaneously；Some biomaterials, such as mutant, the most rare preciousness；Sample throughput is big, and sample handling processes step is many, the longest.Therefore, the detection difficulty of carboxylic acids signaling molecule is the biggest, it is desirable to the sensitiveest, accurately, the method for high-flux analysis that simultaneously measures of multi-signal molecule.
Applying more quantitative approach at present is based on mass spectrometric stable isotope dilution, directly carboxylic acids signaling molecule is carried out absolute quantification analysis in the negative ion mode.But the method mainly has following two defect: cannot detect some signaling molecules or poor sensitivity；The signaling molecule standard substance of cold labeling need to be used as internal standard, and being difficult to of having obtains, and only standard substance kind is rare, expensive.Disadvantages described above all limits the quantitative analysis of carboxylic acids signaling molecule.
In the case of a lot, biological and botanist more pays close attention to carboxylic acids signaling molecule content difference in one group of sample of contrast, thus can the relative quantification thinking in proteomics be applied in the relative quantitative assay of this type of signaling molecule.The unique relevant technology having been reported that at present is the relative amount utilizing 3-bromo-2-oxopropyl pyridine and deuterium mark reagent thereof to have studied 7 Jasmonates signaling molecules for mass discrepancy label.But the target of the method is only a class signaling molecule, it is impossible to meet and relative quantitative assay while multiple types signaling molecule and interaction network are studied；In addition, the error of this labeled in vitro mode is bigger, because comparative sample have passed through individually extraction, derivatization labelling and Solid-Phase Extraction before combined analysis and processes, the error of these processes cannot compensate, and independent cross solid-phase extraction column add the cost of analysis, time and efforts, sample throughput relatively low [HuangYQ, etal. (2011) Useofisotopemassprobesformetabolicanalysisofthejasmonate biosyntheticpathway.Analyst136:1515-1522].
For making up the deficiency of said method, we have developed chromatograph-tandem mass spectrometry (LiquidChromatography-tandemMassSpectrometry, LC-MS/MS) based on novel mass discrepancy label and carboxylic acids signaling molecule is carried out relative quantitative assay.By chemical derivatization method, make two kinds of samples (such as matched group and process group) of contrast identical from a pair structure the most respectively but elementary composition different mass discrepancy label after a step solvent extraction, (2-bromoethyl) trimethylammonium bromide [BromocholineBromide (BETA), molecular formula C5H13NBr2, light isotope tag molecule] and its 9 deuterated isotope-labeled tag molecule D9-BETA [molecular formula C5H4D9NBr2, heavy isotope tag molecule] and reaction, after reaction, the product of two kinds of samples of contrast is merged and carries out follow-up sample pretreatment and LC-MS/MS analysis.The peak area ratio obtained i.e. this carboxylic acids signaling molecule relative amount in one group of comparative sample.While this method achieves multiple types carboxylic acids signaling molecule first, highly sensitive, high flux, accurately relative quantitative assay, contribute to promote carboxylic acids signaling molecule time-space distribution and the development of the correlational study such as interaction network, metabolism group.
Summary of the invention
In the first aspect, the present invention provides a kind of by light/heavy isotope mass discrepancy tag molecule pair to labelling, described mass discrepancy tag molecule is to comprising that structure is identical but elementary composition two kinds of different tag molecule, i.e. light isotope tag molecule and heavy isotope tag molecule, both tag molecule are shown in formula (I):
Wherein:
In structure division A, R1It is H or the low alkyl group containing 1-3 carbon atom,
R2It is H or the low alkyl group containing 1-3 carbon atom,
R3It is H or the low alkyl group containing 1-3 carbon atom,
Wherein R1、R2And R3Can be same or different；
In structure division B, Y is H or O,
[C]nIt is the straight chain containing 1-8 carbon atom, Branched fatty alkyl, or
It it is the cycloalkyl containing 5-7 carbon atom；
In structure division C, X is I, Br, Cl or F；
Described gently/heavy isotope any portion to being present in structure division A and B or two parts in, wherein:
Light/heavy isotope in structure division A to select free H/D,12C/13C and14N/15One or more in the group of N composition, or
Light/heavy isotope in structure division B to select free H/D,12C/13C and16O/18One or more in the group of O composition.
Specifically, table 1 is seen about the definition of each structure division in formula (I).
The definition of each structure division in table 1 formula (I):
In a preferred embodiment of the invention, it is provided that comprise (2-bromoethyl) trimethylammonium bromide (BETA) and its 9 deuterated isotope-labeled tag molecule D9The mass discrepancy tag molecule pair of-BETA, described tag molecule is corresponding to having formula (I) compound of following concrete substituent group: in wherein said structure division A, R1、R2And R3It is CH3, described structure division B is C2H4, the X in described structure division C is Br；Therein gently/heavy isotope to being H/D, be present in structure division A, i.e. in structure division A, 9 H in three methyl being connected with N are all replaced by its isotope D.
Generally, light isotope tag molecule can be purchased, or conventionally synthesizes.Heavy isotope tag molecule generally requires the new synthetic route of design and synthesizes.
Therefore, in second aspect, the present invention provides 9 deuterated isotope-labeled tag molecule D of a kind of synthesis (2-bromoethyl) trimethylammonium bromide (BETA)9The method of-BETA, described method includes D9-Trimethylamine is under dry methanol effect, with 1 in dry ice-propanone is bathed, 2-Bromofume reacts, question response mixture recovers to room temperature, it is stirred at room temperature 2 days, forming white solid, purification obtains 9 deuterated isotope-labeled tag molecule D of (2-bromoethyl) trimethylammonium bromide BETA9-BETA。
In a preferred embodiment of the invention, (2-bromoethyl) trimethylammonium bromide (BETA) is commercially available, such as, (Shanghai) chemical conversion industrial development company limited is liked purchased from ladder is uncommon, article No. is B0577,9 deuterated isotope-labeled tag molecule D of (2-bromoethyl) trimethylammonium bromide (BETA)9-BETA synthesizes in the steps below:
1.12gD9-Trimethylamine, under 6mL is dried methanol effect, reacts with 7.1mL1,2-Bromofume in dry ice-propanone is bathed；
Question response mixture recovers to room temperature, is stirred at room temperature 2 days；
The white solid formed with cold ether, finally gives 2.64g product D after filtering9-BETA, productivity 62%.
Utilize1H-nuclear magnetic resonance, NMR (1H-NMR), the product of high resolution mass spectrum (HR-MS) and high-resolution tandem mass spectrum (HR-MS/MS) furanone synthesis is D9-BETA。D9-BETA is synthesized first by the present inventor.
Those skilled in the art should understand that, read the present invention about design BETA in the mass discrepancy isotopic tag that replaced by 9 D of 9 H of quaternary ammonium group after the technology of relative quantitative assay, can predict: by designing a series of mass discrepancy label, method is expansible to be applied to organize in the relative quantitative assay of the carboxylic acids signaling molecule of sample (n ＞=2) more.The design (see Fig. 1) of a series of mass discrepancy label includes but not limited to:
A) the part A major function in formula (I) is that determinand is carried out structural modification, makes derivatization product can measure in the positive-ion mode thus the sensitivity of Enhancement Method, and its structure can be primary amine, secondary amine or tertiary amine, R therein1、R2And R3Can identical can also be different, but amine base is the strongest, and sensitivity is the highest, the heavy isotope of this part can be selected from D,13C、15One or more isotopes in N, it is also possible to be every kind of different number of various combinations of isotope, with D9As a example by-BETA, similar structure can also be D8-BETA, D7-BETA or D6-BETA etc.；
B) part B in formula (I) can be straight chain, Branched fatty alkyl or cycloalkyl, can also be Br atom (i.e., X in formula (I)) neighbouring containing carbonyl (i.e., Y in formula (I) is O atom) etc. the stronger group of electropositivity, according to SN2Nucleophilic substitution mechanism, this part electropositivity is the strongest, alkyl on α-C or β-C atom less or group more hour, the space bit of generation hinders the least, and reaction is the most easily carried out, the heavy isotope of this part can be selected from D,13C and18One or more isotopes in O, it is also possible to be every kind of different number of various combinations of isotope, as Y is18O, coupled C can be13C, it is also possible to be12C；And, it is also possible to it is heavy isotope and the combination of heavy isotope in part B in part A；
C) C portion in formula (I) is leaving group, and bromine atoms can be replaced by I, Cl and F, but partial alkaline of leaving away is the most weak, and reaction is the most easily carried out, such as I-＞ Br-＞ Cl-＞ F-。
Once synthesize the mass discrepancy label of seriation, a sample is merged into after the many groups sample Differential derivatization respectively contrasted, loading is purified and LC-MS/MS analysis to a solid-phase extraction column, improve sample throughput to a greater degree, save human and material resources, cost and the time required in sample handling processes.
In a third aspect, the present invention provide a kind of based on by light/heavy isotope to the mass discrepancy tag molecule of labelling to the method for the relative amount of carboxylic acids signaling molecule in quantitative biological sample system, described method is a kind of high sensitivity, high-throughout quantitative approach.
The method of the relative amount of carboxylic acids signaling molecule in quantitative biological sample system is comprised the steps: by the present invention by the mass discrepancy tag molecule of labelling based on by light/heavy isotope
(1) weigh etc. the biomaterial waiting to contrast wherein carboxylic acids signaling molecule content of quality respectively, be divided into control sample and process group sample；
(2) by the control sample of step (1) and process group sample respectively with suitable organic solvent extraction, centrifugal after take supernatant, ambient temperature under nitrogen dries up；
(3) product of step (2) is redissolved respectively in acetonitrile, it is sequentially added into triethylamine as acid binding agent, control sample performs the derivatization reaction with the light isotope tag molecule described in first aspect present invention, process group sample performs the derivatization reaction with the heavy isotope tag molecule described in first aspect present invention, product is dried up with nitrogen respectively；
(4) product of step (3) is redissolved respectively in water, merge the solution after redissolving, cation exchange column (is not particularly limited by loading to cation exchange column that is preactivated and that balanced, as long as can the product of purification step (3), such as, the CBA post of Agilent company of the U.S. can be available from), through drip washing, eluting, eluent nitrogen is dried up；
(5) eluted product of step (4) acetonitrile solution containing formic acid is redissolved, filter, carry out liquid chromatography-tandem mass spectrometry LC-MS/MS analysis in the positive-ion mode；
(6) by the relative amount of carboxylic acids signaling molecule in formula (II) calculating control sample and process group sample:
A1/A2=C1/C2 (II)
Wherein:
A1 represents the peak area of carboxylic acids signaling molecule marked product in the control sample by light isotope tag molecule labelling；
A2 represents by the peak area of carboxylic acids signaling molecule marked product in the process group sample of heavy isotope tag molecule labelling；
C1 represents the content of carboxylic acids signaling molecule in control sample；
C2 represents the content of carboxylic acids signaling molecule in process group sample.
In another preferred embodiment of the present invention, it is provided that a kind of based on comprising BETA and its 9 deuterated isotope-labeled tag molecule D9The mass discrepancy tag molecule of-BETA comprises the steps: biomaterial quality such as weighing respectively, to be contrasted to the method for the relative amount of carboxylic acids signaling molecule in quantitative biological sample system, described method, is divided into control sample and process group sample；
Control sample and process group sample are used organic solvent respectively, and such as methanol, 200 μ L extract, and take out supernatant after being centrifuged, and ambient temperature under nitrogen dries up；
Extraction product is redissolved respectively in 150 μ L acetonitriles, being sequentially added into 10 μ L2% triethylamine (TEA) is acid binding agent, the extraction product of control sample and 20 μ L200mMBETA perform the derivatization reaction, the extraction product of process group sample and 20 μ L200mMD9-BETA performs the derivatization reaction, reacts 3.0h after vortex at 95 DEG C；
The product of derivative reaction nitrogen respectively is dried up；
The product of the derivative reaction dried up is redissolved in water, merge the solution after redissolving, loading to activation, the cation exchange column balanced, through drip washing, eluting, eluent nitrogen is dried up；
The eluted product of cation exchange column 10% acetonitrile (containing 0.1% formic acid) 70 μ L is redissolved, filters, carry out liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the positive-ion mode and analyze, sample size 5 μ L；
The relative amount of carboxylic acids signaling molecule in control sample and process group sample is calculated by formula (II).
The strategy merged as early as possible in the present invention had both improve the accuracy of method, improve again sample throughput, had saved human and material resources, cost and time.
Principle and enforcement
Containing carboxyl in carboxylic acids signaling molecule structure, the conventional determining method of its LC-MS/MS is the most directly to measure.But the response of mass spectrum negative ion mode is generally low than positive ion mode.Carboxyl and halogenated alkane BETA or D9S is there is in-BETA under conditions of selected after series of optimumN2Nucleophilic substitution, generates esters and hydrobromic acid, sees Fig. 2.D9Fig. 3 is shown in the synthesis of-BETA.
The reaction condition optimized includes: solvent species, acid binding agent kind, BETA or D9The consumption of-BETA, response time and temperature.Condition after a kind of optimization is: use acetonitrile is solvent, and triethylamine (TEA) is acid binding agent, with a certain amount of BETA or D9-BETA performs the derivatization reaction, and the temperature of derivative reaction is 95 DEG C, and the response time is 3.0h.
The reaction of this chemical derivatization has two effects: on the one hand the carboxylic acids signaling molecule in one group of sample of contrast introduces mass discrepancy label respectively, makes product have mass discrepancy and be identified when Mass Spectrometer Method, as the basis of relative quantification；On the other hand the structure of carboxylic acids signaling molecule is modified, introduce the most positively charged quaternary ammonium group in the product, have when cation detects good mass spectrum to respond, thus greatly improve the sensitivity of method, reduce the consumption of test biomaterial, provide convenient for biological and botanist.Reacted by chemical derivatization, the one group of sample (such as matched group and process group) making contrast is identical with a pair structure the most respectively after a step solvent extraction, but elementary composition different mass discrepancy label, (2-bromoethyl) trimethylammonium bromide [BromocholineBromide (BETA), molecular formula C5H13NBr2, light isotope label] and its 9 deuterated isotopic tag D9-BETA [molecular formula C5H4D9NBr2, heavy isotope label] and reaction, two samples of contrast are merged into the follow-up sample pretreatment of row and LC-MS/MS analyzes.Differ the mass discrepancy label of 9 respectively to react due to the carboxylic acids signaling molecule in comparative sample with mass number, the molecular weight of product obtained also differs from 9, thus the quasi-molecular ion peak of product differs 9 and is distinguished by mass spectrum in first mass spectrometric, relative quantitative assay is carried out by many reaction detection (MultipleReactionMonitoring, MRM).The peak area ratio obtained i.e. this carboxylic acids signaling molecule relative amount in one group of comparative sample.
The explanation of computing formula
Control sample and the relative amount computing formula of carboxylic acids signaling molecule in process group sample:
A1/A2=C1/C2 (II)
Wherein:
A1 represents the peak area of carboxylic acids signaling molecule marked product in the control sample by light isotope tag molecule labelling；
A2 represents by the peak area of carboxylic acids signaling molecule marked product in the process group sample of heavy isotope tag molecule labelling；
C1 represents the content of carboxylic acids signaling molecule in control sample；
C2 represents the content of carboxylic acids signaling molecule in process group sample.
Such as, BETA and D is comprised when use9The mass discrepancy tag molecule of-BETA is in quantitative biological sample system during the relative amount of carboxylic acids signaling molecule, and in formula (II), the implication of A1, A2, C1 and C2 is as follows:
The peak area of carboxylic acids signaling molecule marked product in the control sample of A1:BETA labelling；
A2:D9The peak area of carboxylic acids signaling molecule marked product in the process group sample of-BETA labelling；
C1: the content of carboxylic acids signaling molecule in control sample；
C2: the content of carboxylic acids signaling molecule in process group sample.
Result
Utilize1H-nuclear magnetic resonance, NMR (1H-NMR), high resolution mass spectrum (HR-MS) and high-resolution tandem mass spectrum (HR-MS/MS) the furanone D synthesized first9The structure of-BETA.With TMS as internal standard, D2O is solvent,1H-NMR (300Hz, BrukerARX300NMRspectrometer) analysis result is as follows: 3.737 (s, 4H), Fig. 4.Mass spectrum is equipped with electric spray ion source (ESI) (WatersSynaptTMG2 high resolution mass spectrum), to monitor in the positive-ion mode, analysis result is as follows: HR-(ESI+) MS (mass-to-charge ratio, m/z): [M]+175.0797, (value of calculation C5H4D9NBr+, 175.0796, error 0.6ppm), see Fig. 5；HR-(ESI+) MS/MS (m/z): [M]+106.9492, (value of calculation C2H4Br+, 106.9491, error 0.9ppm), see Fig. 6.
The big class in 5 investigated, 8 kinds of carboxylic acids signaling molecules include salicylic acid (SA), auxin (indole-3-acetic acid (IAA), indole-3-monoprop (IPA), indole-3-butyric acid (IBA)), abscisic acid (ABA), Jasmonates (JA, (+)-7-iso-JA-L-Ile) and gibberellins (GA4).In order to investigate the temperature (70-95 DEG C) impact on derivatization efficiency, performing the derivatization reaction with the mixed mark of 8 kinds of signaling molecules respectively with BETA at 70,80 and 95 DEG C, the peak area obtaining 8 kinds of derivatization products the results are shown in Table 2.Result shows, at 95 DEG C, the area at all product peaks is the most maximum, therefore selectes 95 DEG C for preferably reaction temperature.Finally determine more excellent under the conditions of, the derivatization efficiency of above 8 kinds of signaling molecules is up to 78.9-99.9%, and relative error (SDs) is not more than 3.4% (n=3), and reaction efficiency is high, favorable reproducibility.
Due to the derivative reaction modification to structure, detection limit (LODs) of carboxylic acids signaling molecule reaches an angstrom mol level, and relative standard deviation (RSD) is not more than 9.5% (n=6)；nullThe detection sensitivity of new method more directly detects the method for original shape being obviously improved of 1-3 the order of magnitude (1.1-278 times)，Deliver relatively at present、Measure the sensitiveest method [ChenML of same carboxylic acids signaling molecule，Etal. (2012) Highlysensitiveandquantitativeprofilingofacidicphytohorm onesusingderivatizationapproachcoupledwithnano-LC-ESI-Q-TOF-MSanalysis.JournalofChromatographyB905:67-74] there is the lifting of 1-3 the order of magnitude (1.5-136 times)，The results are shown in Table 3.
By D-atom is designed near hydrophilic quaternary ammonium group, make to flow out altogether in a pair product chromatograph after difference labelling, eliminate potential isotope effect, it is ensured that the accuracy of method.
In the dynamic range of 2-3 the order of magnitude (0.1-10 and 0.003-20), the R of all carboxylic acids signaling molecule derivatization product standard curves2All reaching 0.99, RSD is not more than 10.1%, the results are shown in Table 3.
Establishing the response rate high (68.9-98.7%), the Solid-Phase Extraction processing method of favorable reproducibility (SDs is not more than 6.7%), purification effect is good；Method i.e. merges comparative sample after solvent extraction, derivatization, it with same Solid-Phase Extraction column purification and is carried out LC-MS/MS analysis, difference label internal standard each other to be measured after merging, not only substantially increase method accuracy, and improve sample throughput, save cost and time.
The peak area of carboxylic acids signaling molecule BETA marked product under table 2. different temperatures
LODs and two kinds of method sensitivity of the inventive method and other of the sensitiveest assay method that table 3. the inventive method measures carboxylic acids signaling molecule BETA marked product, conventional method directly measures original shape and has delivered at present increase multiple and compare
Note: sensitivity increases multipled=b/a；
Sensitivity increases multiplee=c/a；
N.a.: cannot provide
Table 4. present invention based on novel mass discrepancy label B ETA/D9The LC-MS/MS method of-BETA carries out the range of linearity and the regression equation parameter (n=3) of relative quantitative assay to carboxylic acids signaling molecule.
The Advantageous Effects of the present invention:
The present invention has synthesized D first9-BETA, and it is used as together with BETA the mass discrepancy label of novelty, the new method of foundation is successfully realized relative quantitative assay while multiple types carboxylic acids signaling molecule, has the advantage that or good effect:
1) being applicable to relative quantitative assay while multiple types carboxylic acids signaling molecule, the time-space at signaling molecule is distributed and significant in interaction network research；
2) synthesis and the supply of cold labeling standard substance it are not necessarily dependent on, to very difficult synthesis or there is no the signaling molecule of cold labeling standard substance and can provide content difference information, have broad application prospects in precursor, metabolite and the metabolism group research of signaling molecule；
3) sensitivity the relative amount information that on the one hand can provide Ionization Efficiency low, content relatively low carboxylic acids signaling molecule in comparative sample material is substantially improved, on the other hand specimen material consumption can be reduced, thus realize being difficult to the analysis of the carboxylic acids signaling molecule in acquisition, source the most precious abnormal material, facilitate the biological and preparation of samples of botanist and draw materials；
null4) sample contrasted extracts respectively、After derivatization，First merge again loading to be purified and LC-MS/MS analysis to same solid-phase extraction column，A pair product internal standard each other after labelling，Compensate for the error in purification and LC-MS/MS are analyzed，With [HuangYQ，Etal. (2011) Useofisotopemassprobesformetabolicanalysisofthejasmonate biosyntheticpathway.Analyst136:1515-1522] document report extract the most respectively、Derivatization、Cross different Solid-Phase Extraction column purifications，The method ratio of LC-MS/MS analysis is carried out after remerging，The strategy merged as early as possible in the present invention had both improve the accuracy of method，Improve again sample throughput，Save manpower、Material resources、Cost and time；
5) those skilled in the art have read the present invention about the mass discrepancy isotopic tag that replaced by 9 D of 9 H of quaternary ammonium group in design BETA after the technology of relative quantitative assay, can predict: by designing a series of mass discrepancy label, method is expansible to be applied to organize in the relative quantitative assay of the carboxylic acids signaling molecule of sample (n ＞ 2) more.After once having had the mass discrepancy label of seriation, a sample is merged into after many groups sample Differential derivatization respectively of contrast, loading is purified and LC-MS/MS analysis to a solid-phase extraction column, improve sample throughput to a greater degree, save human and material resources, cost and the time required in sample handling processes.
Accompanying drawing explanation
From detailed description below in conjunction with the accompanying drawings, features described above and the advantage of the present invention will be apparent from, wherein:
Fig. 1. a series of mass discrepancy label constructions and design.
Fig. 2. carboxylic acids signaling molecule and novel mass discrepancy label B ETA/D9The Differential derivatization reaction equation of-BETA；
Fig. 3 .D9The synthetic route of-BETA；
Fig. 4 .D9-BETA's1H-NMR spectrum (D2O is solvent)；
Fig. 5 .D9HR-(ESI+) the MS spectrogram of-BETA；
Fig. 6 .D9HR-(ESI+) the MS/MS spectrogram of-BETA；
Fig. 7 .BETA/D9-BETA mass discrepancy isotopic tag 8 kinds of carboxylic acids signaling molecules in 0.8mgDW12 days arabidopsiss process the relative quantitative assay result (n=8) responded to MeJA.
Detailed description of the invention
The present invention is further described, it will be appreciated by those skilled in the art that the present invention is not limited to these specific embodiments referring to specific embodiment.
Reagent used in following embodiment, except as otherwise noted, is the reagent of commercially available analytical pure rank.
Embodiment 1
9 deuterated isotope-labeled tag molecule D of synthesis (2-bromoethyl) trimethylammonium bromide (BETA)9-BETA
1.12gD9-Trimethylamine, under 6mL is dried methanol effect, reacts with 7.1mL1,2-Bromofume in dry ice-propanone is bathed；
Question response mixture recovers to room temperature, is stirred at room temperature 2 days；
The white solid formed with cold ether, finally gives 2.64g product D after filtering9-BETA, productivity 62%.
Utilize1H-nuclear magnetic resonance, NMR (1H-NMR), high resolution mass spectrum (HR-MS) and high-resolution tandem mass spectrum (HR-MS/MS) furanone have synthesized D9-BETA.With TMS as internal standard, D2O is solvent,1H-NMR (300Hz, BrukerARX300NMRspectrometer) analysis result is as follows: 3.737 (s, 4H), Fig. 4.Mass spectrum is equipped with electric spray ion source (ESI) (WatersSynaptTMG2 high resolution mass spectrum), to monitor in the positive-ion mode, analysis result is as follows: HR-(ESI+) MS (mass-to-charge ratio, m/z): [M]+175.0797, (value of calculation C5H4D9NBr+, 175.0796, error 0.6ppm), see Fig. 5；HR-(ESI+) MS/MS (m/z): [M]+106.9492, (value of calculation C2H4Br+, 106.9491, error 0.9ppm), see Fig. 6.
Embodiment 2
0.8mg dry weight (DW) model plant arabidopsis (Col-0) through (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate (MeJA) process and in untreated 12 days full stands more than the relative quantitative assay of 8 kinds of carboxylic acids signaling molecules
The process of process group sample: add 4mL100mMMeJA (solvent is 0.1% ethanol) in the grown under normal conditions arabidopsis full stand culture dish of 10 days, hot-house culture 2 days will be put into after 10min again, afterwards in pulverized under liquid nitrogen, lyophilizing after drawing materials
The process of control sample: add 4mL0.1% ethanol (blank solvent) in the grown under normal conditions arabidopsis full stand culture dish of 10 days, hot-house culture 2 days will be put into after 10min again, afterwards in pulverized under liquid nitrogen, lyophilizing after drawing materials
Weigh control sample and the process group sample of 0.8mg dry weight (DW) respectively, after methanol extraction, ambient temperature under nitrogen dries up respectively, redissolve in 150 μ L acetonitriles respectively, being sequentially added into 10 μ L2% triethylamine (TEA) is acid binding agent, control sample and 20 μ L200mMBETA perform the derivatization reaction, process group sample and 20 μ L200mMD9-BETA performs the derivatization reaction, and vortex reacts 3.0h at mixing latter 95 DEG C.Derivative reaction Nitrogen in Products air-blowing is done, redissolve in water, and the product of the derivative reaction of one group of sample of contrast is merged, loading is to the cation exchange column (purchased from the CBA post of Agilent company of the U.S.) activating, having balanced, after scrubbed, eluting, nitrogen dries up, filter after eluted product is redissolved with the acetonitrile solution containing formic acid, carry out LC-MS/MS analysis, sample size 5 μ L.
The peak area of carboxylic acids signaling molecule marked product during wherein A1 represents the control sample by BETA labelling；A2 represents by D9The peak area of carboxylic acids signaling molecule marked product in the process group sample of-BETA labelling；
According to public formula (II), with the A1/A2 that records as vertical coordinate, figure (see Fig. 7) is done for abscissa with each carboxylic acids signaling molecule, obtain in this embodiment 8 kinds of carboxylic acids signaling molecules content in control sample and the ratio of content, i.e. relative amount result in process group sample.As a example by Jasmonates JA, its A1/A2 is 0.00561 ± 0.00067, it is seen then that in process group sample, the content of JA has than the content in matched group and significantly promotes.
Should be appreciated that, although with reference to the embodiment that it is exemplary, the present invention carried out particularly shown and described, but it will be apparent to an ordinarily skilled person in the art that, under conditions of without departing substantially from by spirit and scope as defined by the claims of the present invention, the change of various forms and details can be carried out wherein, the combination in any of various embodiment can be carried out.
Claims (3)
1. one kind by light/heavy isotope mass discrepancy tag molecule pair to labelling, and described mass discrepancy tag molecule is to comprising light isotope tag molecule and heavy isotope tag molecule, and wherein both tag molecule are shown in formula (I):
Wherein:
In structure division A, R1、R2And R3It is CH3；
Structure division B is C2H4,
In structure division C, X is Br；
Described gently/heavy isotope to being H/D, be present in described structure division A.
2. 9 deuterated isotope-labeled tag molecule D of synthesis (2-bromoethyl) trimethylammonium bromide9The method of-(2-bromoethyl) trimethylammonium bromide, described method includes D9-Trimethylamine, under dry methanol effect, reacts with glycol dibromide in dry ice-propanone is bathed, question response mixture recovers to room temperature, being stirred at room temperature 2 days, form white solid, purification obtains 9 deuterated isotope-labeled tag molecule D of (2-bromoethyl) trimethylammonium bromide9-(2-bromoethyl) trimethylammonium bromide.
3. mass discrepancy tag molecule based on claim 1 is to a method for the relative amount of carboxylic acids signaling molecule in quantitative biological sample system, and described method comprises the steps:
(1) weigh etc. the biomaterial waiting to contrast wherein carboxylic acids signaling molecule content of quality respectively, be divided into control sample and process group sample；
(2) by the control sample of step (1) and process group sample respectively with suitable organic solvent extraction, centrifugal after take supernatant, ambient temperature under nitrogen dries up；
(3) product of step (2) is redissolved respectively in acetonitrile, it is sequentially added into triethylamine as acid binding agent, control sample performs the derivatization reaction with the light isotope tag molecule described in claim 1, process group sample performs the derivatization reaction with the heavy isotope tag molecule described in claim 1, product is dried up with nitrogen respectively；
(4) being redissolved respectively by the product of step (3) in water, merge the solution after redissolving, eluent nitrogen, to cation exchange column that is preactivated and that balanced, through drip washing, eluting, is dried up by loading；
(5) eluted product of step (4) acetonitrile solution containing formic acid is redissolved, filter, carry out liquid chromatography-tandem mass spectrometry LC-MS/MS analysis in the positive-ion mode；
(6) by the relative amount of carboxylic acids signaling molecule in formula (II) calculating control sample and process group sample:
A1/A2=C1/C2 (II)
Wherein:
A1 represents the peak area of carboxylic acids signaling molecule marked product in the control sample by light isotope tag molecule labelling；
A2 represents by the peak area of carboxylic acids signaling molecule marked product in the process group sample of heavy isotope tag molecule labelling；
C1 represents the content of carboxylic acids signaling molecule in control sample；
C2 represents the content of carboxylic acids signaling molecule in process group sample.
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