CN110551143A - Novel derivatization method for measuring aldehyde ketone compound in biological sample - Google Patents
Novel derivatization method for measuring aldehyde ketone compound in biological sample Download PDFInfo
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- C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
- C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
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- G—PHYSICS
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- 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
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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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
- G01N30/04—Preparation or injection of sample to be analysed
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- G01N2030/067—Preparation by reaction, e.g. derivatising the sample
Abstract
The invention discloses a novel derivatization reagent and a derivatization method for determining aldehyde ketone compounds in a biological sample by using the reagent. The derivatization reagent is named CILAT (clean Isobic Labeled affinity tag), and can selectively mark aldehyde ketone compounds from metabolite environments. This derivatizing agent consists of three parts: 1) an aminooxy functional group capable of reacting specifically with a carbonyl group to form a covalent bond; 2) a photocleavable biotin group for affinity enrichment of the labeled aldehyde ketone compound; 3) an Isobaric Tag (Isobaric Tag) containing a stable isotope label was used for quantitative analysis of the aldone compounds. The detection method comprises the following steps: and (2) respectively reacting the derivatization reagent with aldehyde ketone compounds in different samples to be detected, mixing the reaction products together, then enriching the molecules marked by the CILAT by using the strong acting force between biotin and streptavidin protein, finally cutting off the biotin part by using ultraviolet light, and analyzing the generated residual molecules by using high-resolution mass spectrometry LC-MS/MS.
Description
Technical Field
The invention relates to the technical field of chemical small molecule detection reagents, in particular to a novel derivatization method for measuring aldehyde ketone compounds in biological samples.
Background
Quantitative studies of small biological molecule metabolites have great potential to discover enzyme function, reveal previously unknown pathways, and discover new disease markers. Of hundreds of metabolites having a wide range of structures and functions, aldehydes and ketones (aldehyde ketone compounds) containing carbonyl groups are generally associated with oxidative stress of biological individuals. Since oxidative stress plays an important role in numerous human diseases (e.g., diabetes, epilepsy, autism, cancer), quantitative measurement of aldoketones can facilitate the study of disease mechanisms and the development of novel therapeutic approaches.
The determination of aldo-keto compounds in biological samples is usually carried out by gas or liquid chromatography-mass spectrometry (GC-or LC-MS). Because these molecules have poor ionization capacity in mass spectrometry, they often require derivatization to improve the sensitivity of detection. For example, tertiary or quaternary amines can be introduced by specific reaction of a carbonyl group with an aminooxy group (aminooxy group) or by reductive amination between a carbonyl group and an amino group (reductive amination). Therefore, the ionization of the positive mode of the mass spectrum can be obviously improved, and the sensitivity of mass spectrum detection is greatly improved. Meanwhile, the derivation method also allows the introduction of stable isotope labels, so that quantitative comparison can be carried out on aldehyde ketone compounds in different biological samples. However, since the concentration of aldoketonic compounds in biological samples is much lower than that of other classes of metabolites (e.g., amino acids and thiols), the signal of aldoketonic compounds in complex biological mixtures is easily suppressed, thereby increasing the difficulty of detecting such compounds.
Disclosure of Invention
In order to solve the problems, the invention discloses a novel derivatization method for measuring aldehyde ketone compounds in biological samples.
In order to solve the problem of accurately and quantitatively determining the type and the content of an aldehyde ketone compound in a complex biological sample, the invention discloses a novel derivatization reagent, which is named CILAT (clean Isobasic laboratory Affinity tag) and can selectively mark the aldehyde ketone compound from a metabolite environment. This derivatizing agent consists of three parts: 1) an aminooxy functional group capable of reacting specifically with a carbonyl group to form a covalent bond; 2) a photocleavable biotin group for affinity enrichment of the labeled aldehyde ketone compound; 3) an Isobaric Tag (Isobaric Tag) containing a stable isotope label was used for quantitative analysis of the aldone compounds. This derivatizing agent has several advantages simultaneously. For example, the derivatized aldehyde ketone compound also contains tertiary amine, which can enhance ionization and sensitivity of the compound to be detected in mass spectrum; the existence of the biotin group allows the enrichment and purification of the derivatized aldehyde ketone compound, further improving the sensitivity by eliminating other interferences; the isobaric label allows the aldehyde ketone compounds in a plurality of biological samples to be quantified simultaneously in one experiment, and greatly improves the flux of sample analysis. This isobaric tag is part of a CILAT reagent whose structure comprises a reporter group (reporter) and an equilibrating group (balancer), wherein the reporter group and the equilibrating group are connected by a MS/MS cleavable bond. The complete set of CILAT derivatization reagents can comprise 2-10 molecules with identical chemical structures, and 13C or 15N isotopes are contained at different positions of isobaric structures in each molecule. The mass of the reporter group is different, but the different mass of the reporter group is compensated by the balancing group. So that the mass of reporter + counter group in each reagent is the same. With such isobaric tags, aldehyde ketone compounds in up to 10 biological samples can be quantified in one assay. Meanwhile, aldehyde ketone compounds in different biological samples are marked by the isobaric labels with the same mass, so that extra complexity is not introduced into the primary mass spectrum, and data analysis can be greatly simplified.
10 molecules of derivatizing agent (named CILAT-10Plex derivatizing agent) were assayed simultaneously for 10 biological samples. The derivatization reagent is a derivatization reagent comprising 10 molecules, and the structure comprises CILAT-10Plex114, CILAT-10Plex115C, CILAT-10Plex116C, CILAT-10Plex117C, CILAT-10Plex118C, CILAT-10Plex119, CILAT-10Plex115N, CILAT-10Plex116N, CILAT-10Plex117N and CILAT-10Plex 118N; the structure of 10 molecular derivatization reagents is as follows:
Two molecule derivatizing reagents (named CILAT-2Plex derivatizing reagents) for simultaneous measurement of 2 biological samples the structure of CILAT-2Plex derivatizing reagents, comprising two molecules CILAT-2Plex114 and CILAT-2Plex115 CILAT-2Plex114 has position 1 of 13 C, position 2 of 14 N, CILAT-2Plex115 has position 1 of 12 C, position 2 of 15 N, the arrow points are the breaks in the mass spectrum of the tag, and the mass to charge ratios of the formed reporter ions are 114.12 and 115.12, respectively, the following structure:
The invention also discloses an application of the derivatization reagent, which is used for qualitative and quantitative analysis of aldehyde ketone compounds in biological samples.
The invention also discloses a derivatization method for determining the aldehyde ketone compound in the biological sample by using the derivatization reagent, which comprises the following steps: and (3) respectively reacting the derivatization reagents with aldehyde ketone compounds in a sample to be detected, mixing the reaction products together, enriching molecules containing the same amount of ectopic labels by using strong acting force between biotin and streptavidin, and finally cutting off the biotin part by using ultraviolet light to generate molecules which can be analyzed by using high-resolution mass spectrometry LC-MS/MS. The reporter group and the counter group are connected by a mass MS/MS cleavable bond and the intensity of the reporter ion formed by cleavage in tandem mass spectrometry is used for quantitative analysis.
The invention also discloses another application of the derivatization reagent, which is used for determining the absolute content of the aldehyde ketone compound in a biological sample.
Since the stable isotope raw materials required to prepare the CILAT reagent (CILAT-10Plex) that can quantify 10 samples simultaneously are expensive and in many applications it is only necessary to compare the concentrations of the aldehyde ketone compounds in two biological samples. The invention synthesizes CILAT-2Plex reagent and is applied to relative quantification of aldehyde ketone compounds in two biological samples. Wherein CILAT-2Plex114 and CILAT-2Plex115 are reacted with aldehyde ketone compounds in two samples respectively, mixed together, then enriched with CILAT-tagged molecules by the strong interaction between biotin and streptavidin, and finally cleaved with UV light, so that the resulting molecules (aldehyde ketone compound + CILAT structure of the moiety) can be analyzed by high resolution mass spectrometry LC-MS/MS. Since the reporter and counter-group are connected by a mass MS/MS cleavable linkage, the intensity of the reporter ion formed by cleavage in tandem mass spectrometry (mass to charge ratios of 114.12 and 115.12, respectively) can be quantified. CILAT-2Plex can also be used to determine the absolute content of aldehyde ketone compounds in a biological sample. For example, CILAT-2Plex114 can be used to label an aldehyde ketone compound in a biological sample, while CILAT-2Plex115 can be used to label a corresponding standard of precisely measured concentrations of an aldehyde ketone compound to determine the absolute amount of the aldehyde ketone compound in the biological sample.
Compared with the prior art, the invention has the following beneficial effects:
The problem of accurately and quantitatively determining the type and the content of the aldehyde ketone compound in a complex biological sample is solved;
The ionization and sensitivity of the compound to be detected in the mass spectrum can be enhanced; the existence of the biotin group allows the enrichment and purification of the derivatized aldehyde ketone compound, further improving the sensitivity by eliminating other interferences;
The isobaric label allows the aldehyde ketone compounds in a plurality of biological samples to be quantified simultaneously in one experiment, so that the flux of sample analysis is greatly improved;
Because the aldehyde ketone compounds in different biological samples show the same mass after being marked by the isobaric labels, no additional complexity is introduced into the primary mass spectrum, and the data analysis can be greatly simplified.
drawings
FIG. 1 is a diagram of the structure of the derivatizing reagent for two molecules;
FIG. 2 is a flow chart of the application of CILAT-2 Plex;
FIG. 3 is a synthetic scheme of example 1;
FIG. 4 is an HPLC chromatogram of CILAT-2Plex 114;
FIG. 5 is a MALDI-MS spectrum of CILAT-2Plex 114. CILAT-2Plex115 maps were similar;
FIG. 6 shows the general chemical structure of aldehyde ketone compound after CILAT-2Plex reagent treatment, where R 1 and R 2 are the substituent groups on both sides of carbonyl group in aldehyde ketone compound;
FIG. 7 shows the structure of CILAT-10 Plex.
Detailed Description
the present invention will be further illustrated with reference to the accompanying drawings and specific embodiments, which are to be understood as merely illustrative of the invention and not as limiting the scope of the invention.
Example one: preparation of CILAT-2Plex
2 3 2 3 +after adding Biotin resin (0.51mmol/g,200mg) prepared by solid phase synthesis to a reaction vessel, adding 5mL of 20% solution of Piperidine/DMF and shaking for 30 minutes to remove Fmoc group, washing with DMF 3 times, adding 1.5mL of DMF solution containing Fmoc-aminoethylether bondker 110mg, HBTU 76mg, HoBt 27mg, 250. mu. LDIPEA/DMF (2M), after shaking for 2 hours, filtering off the resin, washing with DMF 3 times, adding 20% solution of Piperidine/DMF and shaking for 30 minutes to remove Fmoc group, washing with DMF 3 times, adding 3. mu. of DMF solution containing Fmoc-Dpr (Boc-Aoa) -OH 250mg, HBTU 190mg, HOBt 80mg, 625. mu. L of DEEP A/DMF (TFA), after shaking for 2 hours, after shaking for 30 minutes to remove Fmoc group, adding PITA 2% solution containing Psol 2% of TFA + DMF, after shaking for 30 minutes to remove Fmoc group, adding PITA 2% solution of P10% TFA, after shaking for 30 minutes to a reaction vessel, after shaking for 30 minutes, adding PILAT-2% TFA, after shaking for 30 minutes to remove Fmoc group, adding PITA-2% DMF, after shaking for 30 minutes to a TFA, after shaking for 30 minutes to a supernatant, after shaking for 30 minutes to obtain P1.35% solution, after purification, adding PIT-2% TFA, adding PIT-DMF, after shaking for 10% TFA, after shaking for 2, after shaking for 3% TFA, adding PIT-DMF, after shaking for removing TFA, after shaking for 3% TFA, precipitating, after shaking for removing TFA, after centrifugation, adding TFA, after shaking for 2, precipitating as a TFA, after centrifugation, adding TFA, precipitating as a pale yellow solid, the theoretical 10% TFA, adding TFA, the filtrate containing TFA, the theoretical 10% TFA, the filtrate containing TFA, the filtrate, the supernatant 2-10% TFA, the filtrate, after shaking for 2-DMF 2-7% TFA, after shaking for 10% TFA, the filtrate, after shaking for 10% solution containing TFA, the filtrate, after shaking for 10% TFA, after adding TFA, the filtrate.
As shown in fig. 3, the specific synthesis preparation process is as follows:
Example two: CILAT-2PLex derivatized sample preparation procedure and LC-MS assay conditions
1 2human aortic endothelial cells were cultured for 7 consecutive days in lipid growth medium (EGM) containing 2% Fetal Bovine Serum (FBS), growth factors and 5mM (low sugar) or 30mM (high sugar) glucose, respectively, after the culture was completed, the cells were rapidly washed twice with PBS solution and then frozen with a dry ice/ethanol bath (-70 ℃), then, the cells were lysed using an ultrasonoscope, and the resulting mixture was centrifuged at 14000rpm at 4 ℃ for 10 minutes, the supernatant was collected for CILAT labeling. briefly, 20. mu.l of 20mM CILAT-2Plex114 or CILAT-2Plex115 was added to the low sugar or high sugar samples, respectively, Aniline (100mM) was added to the reaction to accelerate the labeling reaction rate, after the reaction was carried out for 3 hours, the two samples were mixed, 4- [3- (perfluorooyl) -propyl-1-oxy ] benz-aldehydide was added to react with excess CILAT-2Plex, the product was extracted with 500. mu.L of ethyl acetate, and the resulting mixture was subjected to extraction of streptavidin-immobilized on both sides of the streptavidin-aldehyde-labeled compound using a washing solution, after the removal of the streptavidin-labeled compound, and after the addition of the streptavidin-labeled compound, the mixture, and the addition of the streptavidin-labeled compound was removed with 500. mu.3. mu.L-labeled compound, and the addition of the streptavidin-labeled compound was removed, and the streptavidin-labeled compound was.
CILAT-labeled samples were first separated on an Orbitrap mass spectrometer equipped with Agilent 1200HPLC and nano-spray electrochemical interface using a self-contained C18 capillary column of 50 μm I.D. (Solvent A,10mM FA in H 2 O, Solvent B,10mM FA in MeOH,0-30min, 0-70% Solvent B,30-45min, 70-95% Solvent B). The capillary was heated at 200 deg.C, spray voltage 5kV, MS/MS HCD collision energy 45%, resolution 15000, instrument control, data acquisition and data analysis were set by the Xcalibur software.
Example three: signal enhancement in mass spectrometry of aldehyde ketone compounds derivatized with CILAT-2Plex
Several standards of the aldehyde ketone compounds were analyzed using the sample processing procedure of example two. Each standard was analyzed in labeled and unlabeled form at a concentration of 5. mu.M. Many in vivo aldehyde ketone compounds are electrically neutral, so that ionization is difficult to realize in a positive mode and a negative mode of a mass spectrum, and detection sensitivity is influenced. The sensitivity is obviously increased due to the introduction of a tertiary amine group by CILAT labeling. In the full scan MS mode, CILAT derivatization typically produced a 5-10 fold increase in signal-to-noise ratio (Table one).
Table one: signal to noise ratio of CILAT-2Plex labeled and unlabeled aldehyde ketone Compounds
example four: accuracy of CILAT-2Plex derivatization quantitation
The eight aldone compound standards were indexed to three different concentrations (0.5, 1, or 5 μ M) of each mixture, where the concentrations of the standards were the same, then each mixture was reacted with CILAT-2Plex114 or CILAT-2Plex115, respectively after labeling, the mixtures of different concentrations and different labels were further mixed to obtain various ratios, such as 5 μ M/0.5 μ M, 0.5/5 μ M, 5 μ M/1 μ M, 1 μ M/5 μ M, 1 μ M/0.5 μ M, 1 μ M/1 μ M, then the mixtures were analyzed using the procedure in example two, the results show that the linear fit coefficient (R 2) for theoretical and measured values in the range of 1:5-5:1 was greater than 0.97, R 2 in the range of 1:10-10:1 was greater than 0.95 (Table two).
Table two: linear Range of CILAT-2Plex quantitation
Example five: quantitative comparison of aldo-keto compounds in human aortic endothelial cells at low and high sugar levels
Human aortic endothelial cells were cultured at low or high sugar levels and their aldone compounds were analyzed as in example two. A multiple greater than 2.0 or less than 0.5 is considered a significantly varying threshold. On average, 16% or 280. + -.60 aldone compounds were increased 2-fold in the case of high sugars compared to the case of low sugars and 2% or 35+2 aldone compounds were decreased 2-fold in the case of high sugars compared to the case of low sugars. By matching with the metabolite database Metlin, 26 fold-increased (table three) and 6 fold-decreased aldone compounds (table four) were identified.
Table three: aldehyde ketone compounds with significantly increased concentrations at high sugar
Table four: aldone compounds with significantly reduced concentrations at high sugar
EXAMPLE VI Simultaneous assay of CILAT-10Plex derivatizing reagent for 10 biological samples in the solid phase synthesis of example I, IBT-NHS was the key step in the synthesis of CILAT-2Plex since IBT-NHS has been successfully used for the quantitation of proteomics for up to 10 biological samples (IBT-10Plex), the same reagent can also be used for the synthesis of CILAT-10Plex, which has the following specific structure (FIG. seven). the chemical structure of the derivatized aldone compound generated according to the procedure of example II is the same as that formed after CILAT-2Plex labeling, but contains different 13 C and 15 N isotopes.
The technical means disclosed in the invention scheme are not limited to the technical means disclosed in the above embodiments, but also include the technical scheme formed by any combination of the above technical features. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and such improvements and modifications are also considered to be within the scope of the present invention.
table five: molecular formula and exact mass of CILAT-10 Plex-generated reporter ions
Claims (10)
1. A novel derivatizing agent characterized by: the derivatization reagent comprises the following three parts: 1) an aminooxy functional group capable of reacting specifically with a carbonyl group to form a covalent bond; 2) a photocleavable biotin group for affinity enrichment of the labeled aldehyde ketone compound; 3) an isobaric label containing a stable isotope label is used for quantitative analysis of aldehyde ketone compounds.
2. A novel derivatizing reagent according to claim 1, wherein: the novel derivatization reagent comprises 2-10 molecules with completely identical chemical structures, and 13C or 15N isotopes are contained at different positions of isobaric labels in each molecule.
3. A novel derivatizing reagent according to claim 2, wherein: the structure of the isobaric label comprises a reporter group and a balancing group, wherein the reporter group and the balancing group are connected through a MS/MS shearing bond; the mass of each reporter group of the isobaric label is different, the different masses of the reporter groups are compensated by the balancing group, and the mass of the reporter group plus the balancing group in each reagent is the same.
4. a novel derivatizing reagent according to claim 2, wherein: the reporter group and the counter group are connected by a mass MS/MS cleavable bond and the intensity of the reporter ion formed by cleavage in tandem mass spectrometry is used for quantitative analysis.
5. A novel derivatizing reagent according to claim 1 or 2, wherein: the derivatization reagent is a derivatization reagent comprising 10 molecules, and the structure comprises CILAT-10Plex114, CILAT-10Plex115C, CILAT-10Plex116C, CILAT-10Plex117C, CILAT-10Plex118C, CILAT-10Plex119, CILAT-10Plex115N, CILAT-10Plex116N, CILAT-10Plex117N and CILAT-10Plex 118N; the structure of 10 molecular derivatization reagents is as follows:
6. The novel derivatization reagent of claim 1 or 2 wherein the derivatization reagent is a derivatization reagent comprising two molecules, the structure comprises two molecules, CILAT-2Plex114 and CILAT-2Plex115, CILAT-2Plex114 has position 1 of 13 C, position 2 of 14 N, CILAT-2Plex115 has position 1 of 12 C, position 2 of 15 N, the mass-to-charge ratio of the reporter ion is 114.12 and 115.12,
the structure of the derivatizing agent is as follows:
7. The use of a novel derivatizing reagent for qualitative and quantitative analysis for the determination of aldehyde ketone compounds in biological samples.
8. A derivatization method for measuring an aldehyde ketone compound in a biological sample using the novel derivatization reagent according to any one of claims 1 to 6, wherein: and (3) respectively reacting the derivatization reagents with aldehyde ketone compounds in a sample to be detected, mixing the reaction products together, enriching molecules containing the same amount of ectopic labels by using strong acting force between biotin and streptavidin, and finally cutting off the biotin part by using ultraviolet light to generate molecules which can be analyzed by using high-resolution mass spectrometry LC-MS/MS.
9. use of a novel derivatizing agent for determining the absolute content of an aldehyde ketone compound in a biological sample.
10. The use of a novel derivatizing agent as claimed in claim 9, wherein: the structure of the derivatization reagent comprises two molecules of CILAT-2Plex114 and CILAT-2Plex115, and the method for determining the absolute content of the aldehyde ketone compound in a biological sample by using the derivatization reagent comprises the following steps: CILAT-2PLex115 marks the aldehyde ketone compound in the biological sample and CILAT-2PLex116 marks the corresponding standard for the precisely determined concentration of the aldehyde ketone compound.
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| CN112961071A (en) * | 2021-02-08 | 2021-06-15 | 苏州大学 | Solid-phase marker and preparation method thereof, solid-phase carrier multichannel polypeptide or glycopeptide and preparation method thereof |
| CN114573659A (en) * | 2021-12-17 | 2022-06-03 | 南京谱利健生物技术有限公司 | Solid phase synthesis method of high-throughput proteome quantitative reagent |
| CN114835727A (en) * | 2022-06-07 | 2022-08-02 | 南京农业大学 | Design, preparation method and application of caffeic acid-biotin small-molecule probe |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112961071A (en) * | 2021-02-08 | 2021-06-15 | 苏州大学 | Solid-phase marker and preparation method thereof, solid-phase carrier multichannel polypeptide or glycopeptide and preparation method thereof |
| CN114573659A (en) * | 2021-12-17 | 2022-06-03 | 南京谱利健生物技术有限公司 | Solid phase synthesis method of high-throughput proteome quantitative reagent |
| CN114835727A (en) * | 2022-06-07 | 2022-08-02 | 南京农业大学 | Design, preparation method and application of caffeic acid-biotin small-molecule probe |
| CN114835727B (en) * | 2022-06-07 | 2023-05-23 | 南京农业大学 | Design and preparation method and application of caffeic acid-biotin micromolecular probe |
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Application publication date: 20191210 |
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