WO2006112841A1 - Procedes de profilage metabolique - Google Patents

Procedes de profilage metabolique Download PDF

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WO2006112841A1
WO2006112841A1 PCT/US2005/013242 US2005013242W WO2006112841A1 WO 2006112841 A1 WO2006112841 A1 WO 2006112841A1 US 2005013242 W US2005013242 W US 2005013242W WO 2006112841 A1 WO2006112841 A1 WO 2006112841A1
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faah
sample
metabolites
enzyme
metabolite
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Benjamin F. Cravatt
Alan Saghatelian
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The Scripps Research Institute
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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
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
    • 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
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
    • G01N30/724—Nebulising, aerosol formation or ionisation
    • G01N30/7266—Nebulising, aerosol formation or ionisation by electric field, e.g. electrospray

Definitions

  • This invention relates to methods for metabolite profiling, and, more specifically, to using liquid chromatography in conjunction with mass spectroscopy to determine the assignment of endogenous substrates to enzymes.
  • Enzymes are central components of nearly all signal transduction cascades and metabolic pathways that regulate biological processes through the conversion of specific substrates to products. Therefore, it is desirable to be able to determine for every enzyme the assignment of endogenous (natural) substrates to that enzyme.
  • in vitro enzyme analysis does not always provide the results that can be readily translated into a comprehensive understanding of the scope of substrates utilized by the enzymes in vivo. Since in the living cell and organism, enzymes do not function in isolation, but rather as parts of large and complex biochemical networks. Accordingly, in vitro assays may fail to account for many potentially competitive metabolic pathways that alter or restrict the substrates utilized by a particular enzyme in vivo. Second, efforts to assign natural substrates to enzymes in vitro are not well suited to discover novel metabolites that are regulated by enzymes in vivo. Finally, many enzymes can be subject to post-translational regulation in vivo, including covalent modification (e.g., phosphorylation) and protein-protein interactions, which may alter substrate recognition and catalysis.
  • covalent modification e.g., phosphorylation
  • metabolites are typically measured in biological samples by "targeted" gas or liquid chromatography (LC)-MS techniques, in which the levels of specific compounds are determined using isotopic variants as internal standards coupled with MS analysis by selected ion monitoring.
  • LC liquid chromatography
  • FIG. IA metabolites can be detected by selected ion monitoring (shown for a metabolite of the mass of 347.5) and their levels can be quantified by comparing their mass signals to those of isotopically distinct internal standards.
  • a method for metabolite profiling including analyzing metabolites using untargeted liquid chromatography-mass spectroscopy.
  • the method includes extracting metabolites from a biological object, separating the metabolites using liquid chromatography, and subjecting the metabolites to mass ionization.
  • the method can allow identification endogenous substrates for enzymes, for example, N-acyl taurines can be identified as endogenous substrates for the fatty acid amide hydrolase enzyme.
  • FIG. IA schematically illustrates untargeted LC-MS methods for comparative metabolite analysis.
  • FIG. IB schematically illustrates a method for discovery metabolite profiling, according to embodiments of the present invention.
  • FIG. 2 A illustrates a global view of the relative levels of metabolites obtained by a method for discovery metabolite profiling, according to one embodiment of the present invention.
  • FIG. 2B illustrates a close-up view of the relative levels of metabolites obtained by a method for discovery metabolite profiling, according to one embodiment of the present invention.
  • FIG. 3 A illustrates results of analysis of a metabolite obtained by a method for discovery metabolite profiling, according to one embodiment of the present invention.
  • FIG. 3B illustrates results of analysis of a metabolite obtained by a method for discovery metabolite profiling, according to one embodiment of the present invention.
  • FIG. 3C illustrates results of analysis of a metabolite obtained by a method for discovery metabolite profiling, according to one embodiment of the present invention.
  • FIG. 4A illustrates schematically the enzyme-catalyzed hydrolysis of two substrates.
  • FIG. 4B shows LC-MS traces illustrating the enzyme-catalyzed hydrolysis of two substrates
  • enzyme is defined as a protein produced by living organisms to catalyze a specific chemical or biochemical reaction involving other substances, without altering the direction or nature of the reaction.
  • an enzyme in an active, unaltered form is referred to as a "wild type enzyme.”
  • Activity of a wild type enzyme can modified, for example, by administration of pharmaceutical compound.
  • hydrolase is refers as an enzyme that catalyzes bond cleavage by hydrolysis.
  • FAH refers to a particular hydrolase, the fatty acid amide hydrolase.
  • FAH(+/+) refers to a tissue that has FAAH enzyme.
  • FAAH(-/-) refers to a tissue that lacks FAAH enzyme.
  • substrate is defined as a compound upon which the enzyme will act to catalyze transformation of the compound.
  • the term "metabolite” is defined as a compound resulting from enzymatic reactions, i.e., the compound synthesized by a process in which an enzyme takes part.
  • the term “metabolomics” refers to the study of the repertoire of non-proteinaceous, endogenously-synthesized small molecules present in an organism. Representative small molecules include well-known compounds like glucose, cholesterol, ATP and lipid signaling molecules. These molecules are the ultimate product of cellular metabolism.
  • the "metabolome” refers to the catalogue of those molecules in a specific organism, e.g. the human metabolome. In the present invention, the metabolomics profile refers to a pattern of the metabolome in an untreated and a treated sample and the resulting differences provides a metabolomic profile of the sample.
  • endogenous is defined as a substance occurring naturally in a living organism.
  • the abbreviation "CNS” refers to central nervous system; the abbreviation “DMP” refers to discovery metabolite profiling; the abbreviation “LC” refers to liquid chromatography; the abbreviation “MS” refers to mass spectrometry; the abbreviation “FTMS” refers to Fourier transform mass spectrometry.
  • the abbreviation “NAE” refers to N-acyl ethanolamine, which is a fatty acid having the general structure (I):
  • NAT refers to N-acyl taurine, which is a fatty acid having the general structure (II):
  • Fatty acids are designated by using the symbol for carbon, followed by a first numeral and a second numeral, where the numerals are separated by a colon sign.
  • the first numeral refers to the number of carbon atoms in the fatty acid
  • the second numeral refers to the number of unsaturated areas in the acyl chain of the fatty acid.
  • designations "C22:0” and “C24:0” describe a fatty acid molecules having 22 and 24 carbon atoms, with no unsaturation in the acyl chain.
  • designation "Cl 8:1 NAT” describes a molecule of N-acyl taurine having 18 carbon atoms and numeral 1 refers to the number of degrees of unsaturation in the NAT acyl chain.
  • the present invention provides for identifying those of the many substrates accepted by enzymes in vitro that are actually utilized in vivo.
  • the present invention also provides for identifying previously uncharacterized metabolites regulated by these enzymes.
  • the present invention additionally allows to obtain and compare metabolomic profiles of the treated (e.g., with a pharmaceutical agent) and untreated biological samples. The differences between the profile can serve as a biomarker for the pharmaceutical agent's activity in vivo.
  • the method of the present invention can be illustrated as shown schematically on FIG. IB. As shown by FIG.
  • metabolites can be detected in the broad mass scanning mode (e.g., between about 200 and 1200 Daltons) and their levels can be quantified by measuring direct mass ion intensities, without the inclusion of internal standards. Enzyme-regulated metabolites can be identified by comparison of mass ion intensities between wild type and knockout samples. From the schematic illustration shown by FIG. IB, metabolite 3 can be identified as an enzyme-regulated metabolite.
  • a discovery metabolite profiling (DMP) method for identification endogenous substrates for enzymes can include analyzing metabolites using untargeted (i.e., without using an internal standard) liquid chromatography in conjunction with mass spectroscopy.
  • the method can include extracting the metabolites, separating the metabolites into fractions using LC, and mass ionization of tissue lipid metabolites using a diverse set of purified standards.
  • Example of the standards that can be used include NAEs, ceramides, phospholipids, fatty acids, and glycerol esters, in both the positive and negative ionization modes, as shown in Table 1.
  • Negative oleoyl lysophosphatidic acid 33 200 dioleoyl phosphatidyl glycerol 40 80 dioleoyl phosphatidyl ethanolamine 60 5 dioleoyl phosphatidyl choline 50 100 dioleoyl phosphatidyl serine 52 2
  • tissue included an active enzyme being studied and the other tissue lacked such an enzyme (i.e., had the enzyme in an unactivated form). Any tissue and enzyme can be used, to be selected by those having ordinary skill in the art.
  • CNS tissue was used, which is one non-limiting example of the tissue that can be used.
  • the enzyme that was studied was FAAH, which is one non-limiting example of the enzyme that can be studied.
  • tissue was extracted from CNS of two animals (e.g., mice).
  • One sample included a tissue extracted from a FAAH(+/+) mouse, and another sample included a tissue extracted from a FAAH(-/-) mouse.
  • Both samples were then subjected to LC-MS analysis, without using an internal standard.
  • LC-MS analysis without using an internal standard.
  • LC/electrospray ionization (ESI)-MS was used, in both the positive and negative ion modes, scanning across a mass range of between about 200 and 1200 Dalton.
  • chromatograms received as a result of the analysis were then be compared to determine upon which endogenous substrate(s) a particular enzyme acts. Those having skill in the art can make the comparison. As one illustrative, non-limiting example, a comparative analysis of the resulting total ion chromatograms was performed manually by generating extracted ion chromatograms in 5 Dalton increments (e.g., 200-205, 205-210, etc., up to 1195-1200).
  • FIG. 2 A illustrates a global view of the relative levels of metabolites in FAAH (+/+)and FAAH(-/-) brains, plotted over a mass range of 200-1200 Daltons and liquid chromatography retention times of 0-105 minutes (the plot is shown for negative ionization mode).
  • FAAH(-/-) brains possessed highly elevated levels of NAEs (lipid group 4) and an unknown class of metabolites (group 5), while other lipids, e.g., free fatty acids (group 1), phospholipids (group 2), ceramides (group 3), were unaltered in these samples.
  • FIG. 2B illustrates a close-up view of the LC-MS region containing elevated metabolites in FAAH(V-) brains, showing representative known (e.g., C18:l) and novel (e.g., C:24:l) NAE substrates of FAAH, as well as an unknown family of metabolites also upregulated in FAAH(-/-) brains. Data represent the ratios of the averages standard errors of six independent experiments per group.
  • NAE substrates e.g., C 18:1, C 18:0
  • Fig. 2B, group 4 known endogenous substrates of FAAH
  • Table 2 the data represent the mass ion intensity ratios of the averages of six independent experiments per group.
  • the data in parentheses were determined by targeted LC-MS methods, which are provided for comparison.
  • the average ion intensities and relative levels of NAEs measured by a targeted LC/MjS method are also provided in Table 3 for comparison.
  • deuterated (d4 - Cl 8:2 to C20:0) standards in spinal cord and brain from FAAH (+/+) and FAAH (-/-) mice were used.
  • NAEs such as anandamide (C20:4)
  • DMP was also detected by DMP in FAAH(-/-) tissues, establishing a sensitivity limit for this method that was within 5-10 fold of the sensitivity of targeted LC-MS analysis.
  • NAEs several heretofore unrecognized members of this lipid family were also found by DMP to be significantly elevated ( ⁇ 4-10 fold) in CNS tissues from FAAH(V-) mice, including C 16:1, C22:0, and C24:l.
  • enzyme-regulated metabolites can be identified and structurally characterized. This allows to verify which of the substrates accepted by an enzyme in vitro are actually utilized in vivo. For example, as demonstrated by the data provided above, for FAAH-regulated metabolites, lipid species examined, other than NAEs, remained unchanged in FAAH(+/+) and (-/-) tissues, including free fatty acids, phospholipids, and ceramides (see, Table 1 and FIGs. 2A and 2B, groups 1, 2 and 3, respectively). In addition to NAEs, using DMP allows detection of an unknown a class metabolites the level of which was elevated in the brains and spinal cords of FAAH(-/-) mice (FIG. 2B, group 5).
  • FIG. 3 A shows that an unknown class of FAAH-regulated brain metabolite is chemically N-acyl taurine (NAT).
  • NAT N-acyl taurine
  • FIG. 3B shows the results of the MS/MS analysis of the natural m/z 474 metabolite (upper trace) leading to its structural assignment as C24:0 NAT.
  • MS/MS data were obtained on a Micromass Q-TOF instrument. Highlighted are prominent fragments corresponding to taurine (124), vinyl sulfonic acid (107), and sulfur trioxide (80), as well as a pattern of progressive loss of 14 mass units from m/z 150-430 indicative of a fatty acyl chain (inset). This fragmentation spectrum matched closely the MS/MS data of a synthetic C24:0 NAT standard (lower trace).
  • FIG. 1B shows the results of the MS/MS analysis of the natural m/z 474 metabolite (upper trace) leading to its structural assignment as C24:0 NAT.
  • MS/MS data were obtained on a Micromass Q-TOF instrument. Highlighted are prominent fragments corresponding to taurine (124), vinyl sulfonic acid (107), and sulfur trioxide
  • 3C shows that LC-MS spectra of natural and synthetic samples of C18:l, C22:0, and C24:0 NATs are very close.
  • m/z 446, 472, and 474 metabolites have a consensus molecular formula (i.e., a conserved heteroatomic component, NO 4 S, plus a variable hydrocarbon portion) that they may belong to the same structural class.
  • a tandem MS analysis generated highly related fragmentation patterns for the 446, 472, and 474 metabolites, which a series of shared lower molecular weight ions (m/z 80, 107, 124) suggesting the presence of taurine (FIG. 3B), a bioactive amino acid known to those having ordinary skill in the art to be highly enriched in the brain.
  • NATs are substrates for FAAH.
  • a general LC-MS assay was used. FAAH was indeed found to hydrolyze representative long and very long chain members of both the NAE and NAT classes of lipids, as well as members of the monoacyl glycerol family of fatty acid esters.
  • the general scheme illustrating the FAAH-catalyzed hydrolysis of NAEs and NATs is shown on FIG. 4A.
  • the lower trace on FIG. 4B demonstrates the results for reaction between NAT and FAAH.
  • the upper trace demonstrates the spectrum when NAT alone is present.
  • deuterated standards were included in the mixture as known in the art.
  • FAAH(+/+) and (-/-) mice were sacrificed at the same time of day and tissues were immediately isolated, weighed, placed into the CHCl 3 /MeOH/l%NaCl solution and homogenized using dounce tissue grinders. Each sample was then centrifuged at 2500 rpm for 10 min at 4 °C in a glass vial.
  • LC-MS analysis was performed using the instrument Agilent 1 100 LC-MSD SL.
  • a HAISIL 300 Cl 8 column (5 ⁇ m, 4.6 x 100 mm) from Higgins Analytical was used together with a precolumn (Cl 8, 3.5 ⁇ m, 2 x 20 mm).
  • Mobile phase A consisted of 95/5 water/methanol and mobile phase B was made up of 50/45/5 isopropanol/methanol/water.
  • Solvent modifiers such as 0.1 % formic acid, for positive ionization mode, and 0.1 % ammonium hydroxide, for negative ionization mode, were used to assist ion formation as well as improve the LC resolution.
  • the flow rate for each run started at 0.1 mL/min for 5 minutes, to alleviate the back pressure associated with injecting CHCl 3 , followed by a flow rate of 0.4 mL/min for the duration of the gradient.
  • the gradient started at 0% B and then linearly increased to 100% B over 60 minutes followed by an isocratic gradient of 100% B for 30 minutes before equilibrating for 10 minutes at 0% B.
  • the total analysis time including 5 minutes at 0.1 mL/min, was 105 minutes.
  • MS analysis was performed with an electrospray source ionization (ESI) interface.
  • the capillary voltage was set to 3.0 kV and the fragmentor voltage to 100 V.
  • the drying gas temperature was 350°C, the drying gas flow was 10 L/min, and the nebulizer pressure was 35 psi. Data was collected using a mass range of 200-1200 Da and each run was performed using 40 ⁇ L injections of tissue metabolite extract.
  • the peak ratios between FAAH(+/+) and (-/-) samples provided a quantitative measure of the relative metabolite levels.
  • a lower cutoff ion intensity of 32,500 was used.
  • the average ion intensity values are reported as equal to, or less than, the calculated ion intensity and the resulting FAAH (-/-)/FAAH(+/+) ratios are reported as equal to, or greater than, the calculated ratio.
  • N-acyl taurines were purified as follows.
  • the metabolite extracts from five FAAH (-/-) spinal cords were combined for a single LC purification using a Hitachi 7000 series HPLC.
  • a Clipeus Cl 8 column (5 ⁇ m, 10 x 150 mm) from Higgins Analytical was used.
  • the mobile phase A consisted of 95/5 water/methanol/0.1 % ammonium hydroxide and mobile phase B was made up of 50/45/5 isopropanol/methanol/water/0.1% ammonium hydroxide.
  • the gradient started at 0% B and then linearly increased to 100% B over 60 minutes followed by an isocratic gradient of 100% B for 20 minutes at a flow rate of 2.5 mL/min.
  • Fractions (1 per minute) were collected using a Gilson FC 203B fraction collector. Fractions containing the 446, 460, 472, and 474 metabolites were identified by MS analysis. These fractions were then collected and the solvent removed using a rotary evaporator. The samples were then dissolved in a minimal amount of solvent B, such as 200-300 ⁇ L for exact mass and MS/MS analysis.
  • FTMS FTMS
  • the title experiments were conducted as follows. The high accuracy measurements were performed in negative ion mode using a Bruker APEX III (7.0 T) FTMS (Billerica, MA) equipped with an ApolloTM electrospray source. The collected LC fractions were mixed with a collection of small molecule standards and directly infused at 3 ⁇ L/min using a Harvard Apparatus (Holliston, MA) syringe pump. Pneumatic assist at a backing pressure of 60 psi was used along with an optimized flow rate of heated counter- current drying gas (300 0 C). Ion accumulation was performed using SideKickTM without pulsed gas trapping.
  • MS/MS experiments were performed in the negative ion mode using a Micromass QTof-MicroTM (Manchester, UK) equipped with a Z-sprayTM electrospray source and a lockmass sprayer.
  • the source temperature was set to 110°C with a cone gas flow of 150 L/hr, a desolvation gas temperature of 365°C, and a nebulization gas flow of 350 L/hr.
  • the capillary voltage was set at 3.2 kV and the cone voltage at 30 V. Collision energy was set at 40-45 V. Samples were directly infused at 4 ⁇ l/min using a Harvard apparatus syringe pump (Holliston, MA). MS/MS data were collected in centroid mode over a scan range of 50-500 m/z for acquisition times of 2 minutes.
  • NATs were quantified by using a C17:0 NAT standard (500 pmol), which was synthesized according to the technique described in Example 7, and added to the extraction solution.
  • mice were sacrificed and tissues immediately isolated, weighed, placed into the CHCl 3 /MeOH/l%NaCl solution and homogenized using dounce tissue grinders. Each sample was then processed as described above and analyzed by targeted LC-MS using selected ion monitoring. Concentrations of NATs were estimated with respect to the C 17:0 NAT standard.
  • Assays of the enzyme fatty acid amide hydrolase (FAAH) were performed by following the conversion of substrates to their corresponding fatty acids by LC-MS.
  • FAAH was recombinantly expressed and purified from E. coli as known to those having ordinary skill in the art. Reactions were conducted with 1.25-125 nM FAAH and 12.5-150 ⁇ M N-acyl ethanolamine (NAE) or NAT substrate in a reaction buffer of 100 mM Tris- HCl, 1 mM EDTA, 0.1% Triton X-100, pH 8 (adjusted using HCl or NaOH). Reactions were quenched with 0.5 N HCl.
  • LC-MS analysis was performed using an Agilent 1100 LC-MSD SL.
  • LC analysis a HAISIL 100 C8 column (5 ⁇ m, 4.6 x 50 mm) from Higgins Analytical was used.
  • the mobile phase A consisted of 95/5 water/methanol/0.1 % ammonium hydroxide and mobile phase B was made up of 50/45/5 isopropanol/methanol/water/ 0.1 % ammonium hydroxide.
  • the gradient started at 10% B and then linearly increased to 100% B over 10 minutes followed by an isocratic gradient of 100% B for 5 minutes at a flow rate of 0.5 mL/min. Aliquots of the quenched solutions were directly injected to the LC-MS for analysis.
  • Selected ion monitoring was used to measure both the starting NAE or NAT and the corresponding fatty acid hydrolysis product.
  • Standard curves of fatty acids (Cl 8: 1 , C22:0 and C24:0) allowed the conversion of the ion intensities into a molar quantities.
  • Each substrate was tested at four independent concentrations and, at each concentration, four separate time points were measured (4-90 min) such that no greater than 20% formation of product was observed at the final time point. Linear kinetics was observed for each substrate at each concentration tested and, from these data, initial velocities were calculated and used to determine k cat , K m , and k cat /K m .
  • K m and k cat values not be separately determined due to substrate solubility limits ( ⁇ 200 ⁇ M in reaction buffer: 100 mM Tris- HCl, pH 8.0, 1 mM EDTA, 0.1% Triton X-100, 2.5% DMSO).
  • substrate solubility limits ⁇ 200 ⁇ M in reaction buffer: 100 mM Tris- HCl, pH 8.0, 1 mM EDTA, 0.1% Triton X-100, 2.5% DMSO.
  • the rates of hydrolysis of these substrates by FAAH increased linearly over a concentration range of 25-150 and, from these data, specificity constants (k cat /K m ) were determined, as shown in Table 6.

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Abstract

La présente invention concerne un procédé de profilage métabolique, comprenant une analyse des métabolites par chromatographie liquide non ciblée couplée à la spectroscopie de masse.
PCT/US2005/013242 2005-04-19 2005-04-19 Procedes de profilage metabolique WO2006112841A1 (fr)

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WO2008097491A2 (fr) * 2007-02-05 2008-08-14 Wisconsin Alumni Research Foundation Biomarqueurs de réponse au rayonnement ionisant
CN103529139A (zh) * 2013-09-18 2014-01-22 上海交通大学 用自然变异分析转基因水稻代谢差异的方法
CN103604875A (zh) * 2013-06-08 2014-02-26 江苏警官学院 甲基苯丙胺滥用者血清代谢标志物的测定方法
US8703424B2 (en) 2010-03-22 2014-04-22 Stemina Biomarker Discovery, Inc. Predicting human developmental toxicity of pharmaceuticals using human stem-like cells and metabolomics
CN105548404A (zh) * 2016-01-15 2016-05-04 安徽农业大学 一种基于代谢组学鉴别霍山米斛与霍山铁皮石斛品种的方法
US10168342B2 (en) 2015-03-27 2019-01-01 The Scripps Research Institute Lipid probes and uses thereof
US10782295B2 (en) 2013-08-13 2020-09-22 The Scripps Research Institute Cysteine-reactive ligand discovery in proteomes
US11535597B2 (en) 2017-01-18 2022-12-27 The Scripps Research Institute Photoreactive ligands and uses thereof

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008097491A2 (fr) * 2007-02-05 2008-08-14 Wisconsin Alumni Research Foundation Biomarqueurs de réponse au rayonnement ionisant
WO2008097491A3 (fr) * 2007-02-05 2008-12-24 Wisconsin Alumni Res Found Biomarqueurs de réponse au rayonnement ionisant
US8703424B2 (en) 2010-03-22 2014-04-22 Stemina Biomarker Discovery, Inc. Predicting human developmental toxicity of pharmaceuticals using human stem-like cells and metabolomics
US10473641B2 (en) 2010-03-22 2019-11-12 Stemina Biomarker Discovery, Inc. Predicting human developmental toxicity of pharmaceuticals using human stem-like cells and metabolomics
CN103604875A (zh) * 2013-06-08 2014-02-26 江苏警官学院 甲基苯丙胺滥用者血清代谢标志物的测定方法
US10782295B2 (en) 2013-08-13 2020-09-22 The Scripps Research Institute Cysteine-reactive ligand discovery in proteomes
CN103529139A (zh) * 2013-09-18 2014-01-22 上海交通大学 用自然变异分析转基因水稻代谢差异的方法
US10168342B2 (en) 2015-03-27 2019-01-01 The Scripps Research Institute Lipid probes and uses thereof
CN105548404A (zh) * 2016-01-15 2016-05-04 安徽农业大学 一种基于代谢组学鉴别霍山米斛与霍山铁皮石斛品种的方法
US11535597B2 (en) 2017-01-18 2022-12-27 The Scripps Research Institute Photoreactive ligands and uses thereof

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