57 results about "Stable Isotope Labeling" patented technology

The invention belongs to the technical field of teenager depression marker detection, and particularly relates to a teenager depression marker detection kit and a teenager depression marker detection method, the kit comprises an extraction liquid, an internal standard substance, a quality control substance and a mobile phase additive, the internal standard substance comprises a stable isotope labeled metabolic marker, the quality control substance comprises a standard metabolic marker, and the mobile phase additive comprises a mobile phase. The detection method comprises the following steps: S1, dissolving an internal standard substance by using a methanol-containing aqueous solution to prepare an internal standard stock solution; s2, methanol is added into the internal standard stock solution for dilution, uniform mixing is performed, and an extraction working solution is prepared; s3, taking a quality control product / sample, adding an extraction working solution, carrying out oscillation incubation, centrifuging, and taking supernate as a to-be-detected sample; s4, the sample to be detected is detected through a liquid chromatograph-mass spectrometer, various biomarkers related to depression in the body of the teenager can be rapidly and accurately detected, and an objective and reliable basis is provided for early diagnosis and illness state evaluation of the depression of the teenager.

The invention discloses a method for preparing deuterated methane from CO2RR, and relates to the technical field of deuterated methane. The method comprises the following steps: adding an electrolyte solution taking D2O as a solvent into a three-electrode flow cell, continuously introducing CO2 gas with the purity of more than 99.999%, and electrolyzing at normal temperature and normal pressure under the action of a catalyst. The preparation method provided by the invention realizes efficient synthesis of deuterated methane with high added value in a safe, low-cost and environment-friendly manner. By means of mild reaction conditions, simplified process design and successful application of a non-noble metal catalyst, a new normal form is provided for large-scale production of stable isotope labeled compounds, and the method particularly has wide application prospects in the fields of medicine synthesis, nuclear magnetic resonance tracing and the like needing high-purity deuterated methane.

The present application relates to a kind of detection methods of isotopic abundance in acetic acid, comprising the following steps: stable isotope labeled acetic acid is decomposed under vacuum and high temperature conditions, generates stable isotope labeled methane or carbon dioxide, can determine 13 C labeled in carboxyl site acetic acid CH3 13 COOH in 13 C isotopic abundance, 13 C labeled in methyl site acetic acid 13 CH3COOH in 13 C isotopic abundance, 18 O labeled acetic acid CH3C 18 O 18 OH in 18 O isotopic abundance, methyl deuterium isotopic abundance in deuterium labeled methyl site acetic acid CD3COOH;Compared with prior art, the present application has the advantages that the isotopic abundance in acetic acid labeled by stable isotope in different sites is detected, can be used for deuterium, 13 C or 18 O labeled acetic acid isotopic abundance determination, test data accuracy and precision etc.

The invention provides fusion protein containing snake venom protein and application of the fusion protein in biosynthesis of stable isotope labeled snake venom protein, and belongs to the technical field of genetic engineering and protein preparation. The fusion protein comprises a 6-poly-histidine tag, a fusion tag, a restriction enzyme cutting site of WELQ protease and snake venom protein which are connected in sequence. According to the fusion tag, the solubility of the snake venom protein fusion protein can be improved; the 6-poly-histidine tag and the fusion tag can be removed through enzyme digestion of WELQ protease, the enzyme digestion removal effect is good, and the natural structure and activity of the snake venom protein can be guaranteed; besides, a culture medium containing a unique nitrogen source or a unique carbon source labeled by an isotope is used for carrying out induction culture on recombinant bacteria for expressing the fusion protein, the fusion protein containing the target snake venom protein is subjected to induction expression, purification, WELQ protease digestion and the like are carried out, and the stable isotope labeled snake venom protein with the purity reaching 95% or above and the labeling efficiency exceeding 91% can be obtained.

Provided herein are methods for measuring bioavailability of nutrients in a subject after a meal, modeling nutrient metabolism in a subject in need thereof and monitoring nutrient requirements in a patient. The methods utilize a combination of stable isotope-labeled pulse tracers and compartmental analysis to determine at least bioavailability of amino acids in the meal or intracellular amino acid responses to meal components. Also provided is a profile of nutrient metabolism produced by the methods.

The application discloses a kind of detection methods and purposes of SOS1 protein content, belong to biological medicine technical field.The method includes the following steps: sample to be tested enzymolysis, obtain the enzymolysis product comprising the characteristic peptide segment of SOS1 protein, the amino acid sequence of characteristic peptide segment is: FEIPEPEPTEADR and QLTLLESDLYR, stable isotope-labeled SOS1 protein characteristic peptide segment is used as internal standard, the signal value of characteristic peptide segment and internal standard peptide segment is detected using liquid chromatography-tandem mass spectrometry, the relative content of SOS1 protein is calculated by peak area ratio, and the absolute content is obtained by substituting calibration curve linear equation.The method can be used for the quantitative detection of SOS1 protein in tumor sample and other tissues, cells, blood sample, with the advantages of high sensitivity, good accuracy and the like.

The invention belongs to the technical field of biomarker detection, and particularly relates to a method for synchronously and quantitatively detecting urotoxin p-cresol sulfate and indoxyl sulfate based on LC-MS / MS of an alternative analyte method and application. According to the method, stable isotope labeled pCS-d4 and INDS-d4 are creatively adopted as alternative analytes, pCS-d7 and INDS-13C6 are adopted as internal labels, a protein binding state target object is released through serum protein precipitation, a free state target object is obtained through an ultrafiltration method, a universal reversed-phase C18 chromatographic column is adopted for gradient elution and separation, and the detection result is accurate. An organic solvent containing ammonium acetate, formic acid aqueous solution-methanol and the like is used as a mobile phase, and a dual specific detection system for the total content and the free state content of the target object in the serum is established through a multi-reaction monitoring (MRM) mode of LC-MS / MS. The problem of accurate quantification of the nephropathy biomarker in a biological sample under the condition of no blank matrix is solved in a breakthrough manner, and key data support is provided for evaluating pathological toxicity risk of urotoxin.

The present disclosure provides a peptide library comprising stable isotope-labeled (SIL) peptides, wherein the peptides comprise or consist of the sequences according to Table A, Table B, or Table C.

The invention relates to the technical field of high-resolution mass spectrum determination, and discloses a complete 15N stable isotope labeled protein abundance determination method based on high-resolution mass spectrometry, which comprises the following steps: collecting and calculating a mass spectrum signal of a sample to be detected to obtain the actually measured relative isotope peak intensity of the sample at m / z = i; calculating to obtain predicted relative isotope peak intensity of m / z = i under different 15N isotope abundance; according to a formula 1, respectively calculating correlation coefficients r under different 15N isotope abundance; the correlation coefficients r calculated under different 15N isotope abundance are compared, and the r value closest to 1 is the 15N stable isotope labeled protein abundance value. The use amount of required samples is small, pretreatment such as derivatization is not needed, and operation is easy and convenient; the method is high in resolution, small in calculation workload, and good in stability and accuracy.

Provided herein are methods for determining a metabolic rate for at least one endogenous short-chain fatty acid in a subject, for diagnosing a pulmonary disease in a subject and for diagnosing a neurological disorder in a subject. Generally in the methods stable isotope labeled short-chain fatty acid are pulsed and metabolic rates are calculated in a first baseline blood sample and in a series of second blood samples obtained at intervals. The metabolic rates provide information about fatty acid metabolism and production.

Dried blood spots (DBS) collected on substrate materials are used to quantify amino acids, acylcarnitines, organic acids, and numerous other small molecules. One of their main application areas is newborn screening. To properly quantify the target analyte small molecule in DBS, a stable isotope-labeled internal standard (typically a deuterium- or carbon-13-labeled version of the desired target analyte to be quantified) is preloaded onto the substrate before blood collection. For example, to quantify phenylalanine, phenylalanine, which has six carbon-13 atoms, is used as F.

This method for quantifying hemoglobin F includes a step for mixing a sample containing hemoglobin F with a stable isotope-labeled polypeptide having a partial sequence of the α-chain polypeptide of hemoglobin F and a stable isotope-labeled polypeptide having a partial sequence of the γ-chain polypeptide of hemoglobin F, a step for applying trypsin treatment to the mixture, a step for analyzing the result of the trypsin-treatment by LC-MS or LC-MS / MS, and a step for measuring the amounts of the α-chain polypeptide and the γ-chain polypeptide in the sample by comparison with the signals of each of the stable isotope-labeled polypeptides, and determining the amount of hemoglobin F in the sample on the basis of the measured values.

The application relates to the technical field of high-resolution mass spectrometry, and discloses a complete N stable isotope labeling protein abundance determination method based on high-resolution mass spectrometry 15 A method for determining the abundance of N stable isotope labeled protein comprises the following steps: collecting and calculating the mass spectrum signal of a sample to be measured to obtain the actual measured relative isotope peak intensity of the sample at m / z=i; calculating the predicted relative isotope peak intensity of m / z=i under different N isotope abundances; calculating the correlation coefficient r under different N isotope abundances according to formula 1; and comparing the correlation coefficients r calculated under different N isotope abundances, wherein the r value closest to 1 is the N stable isotope labeling protein abundance value. 15 15 15 15 The application has the advantages of small sample consumption, no need for pretreatment such as derivatization, simple operation, high resolution, small calculation workload, good stability and accuracy.​​​

The invention provides a tea tree nitrogen efficient phenotype identification method and application, and discloses a calculation method for quantifying the tea tree nitrogen efficient phenotype, stable isotope 15N marking time capable of indicating a woody plant tea tree nitrogen storage migration rule, a marking method and the tea tree nitrogen storage migration rule for the first time. The phenotypes of the nitrogen utilization efficiency of different tea tree varieties can be rapidly and efficiently evaluated on the basis of not destroying the assimilation level of background nitrogen of the tea tree and the normal growth and development state of the tea tree by applying the method to the tea tree varieties with different genotypes. The invention provides a rapid and efficient nitrogen high-efficiency phenotype identification method for the field of tea tree breeding, has a wide application prospect in the current seed industry science and technology period, and can accelerate the breeding process especially for tea trees with a long breeding period.

The present invention provides a method based on high resolution mass spectrometry. 13 C. 15 The method for determining the abundance of N-stable isotope labeled polypeptides belongs to the field of high-resolution mass spectrometry. The determination method of the present invention comprises the following steps: selecting a certified reference material to calculate the high-resolution mass spectrometer system deviation δ1; calculating according to formula 2 13 C. 15 N stable isotope labeled peptides 13 C. 15 The theoretical relative deviation of the N isotope is δ2; the mass spectrum signal of the sample to be tested is collected and recorded, and the actual measured relative deviation δ3 of the sample to be tested is calculated; when δ2>δ3>δ1, the corresponding isotope labeling abundance value is calculated according to Formula 3 and Formula 4. The method of the present invention uses high-resolution mass spectrometry to directly detect the sample by flow injection, which has a fast detection speed, ensures high resolution, and saves detection time. The analysis of the mass spectrum only requires the response intensity and m / z value of the target ion peak, and the operation is simple.

The invention discloses an intracellular nucleic acid quantification method taking stable isotope labeled escherichia coli as an internal standard, which comprises the following steps: preparing stable isotope labeled escherichia coli, adding the stable isotope labeled escherichia coli as the internal standard into a sample to be detected, and extracting intracellular nucleic acid; the number of internal standard bacteria is determined through a plate counting method, the copy number of single copy genes of the internal standard bacteria in the extracted nucleic acid is detected through real-time fluorescent quantitative PCR, and the intracellular nucleic acid recovery rate is calculated; after the extracted nucleic acid is subjected to enzymolysis, monomers which are not labeled by isotopes are quantitatively analyzed by adopting a liquid chromatography-tandem mass spectrometry technology, and the concentration of the extracted nucleic acid is obtained; and finally, calculating the actual concentration of the intracellular nucleic acid in the sample by combining the extraction concentration and the recovery rate. By introducing the stable isotope labeling internal label, the problem of variability in the extraction process of the intracellular nucleic acid is effectively solved, and the bottleneck of accurately quantifying the concentration of the intracellular nucleic acid is broken through.

The application belongs to the field of environmental analysis chemistry and mass spectrometry, and particularly relates to a carboxylic acid compound targeted-non-targeted collaborative analysis method based on stable isotope labeled aniline derivative reagent and double library system. The method uses aniline and d5-aniline as derivative reagents to perform amide derivative treatment on carboxylic acid compounds, and realizes high-sensitivity detection in combination with liquid chromatography-mass spectrometry. Further, a self-built double library system containing a derivative spectrum library and a non-derivative spectrum library is constructed, and in combination with targeted quantification and non-targeted screening, the collaborative identification and quantitative analysis of known and unknown carboxylic acid compounds are realized. The actual PM2.5 sample is analyzed by using the method, 52 kinds of carboxylic acid compounds are detected, 13 of which are known carboxylic acids with standard samples, and 39 of which are carboxylic acids without standard samples but verified and confirmed by the double library collaborative verification, which verifies the effectiveness of the application.

The invention relates to the technical field of ecological system evaluation, and discloses a large water surface ecological system food web evaluation method based on an Ecopath model, which comprises the steps of model construction and parameter functionalization: constructing a food web topological structure of an ecological system, and establishing a core balance equation based on the Ecopath model; defining at least one key process parameter in the core balance equation as a dynamic parameter response function related to an environmental factor, wherein the dynamic parameter response function comprises a response function coefficient to be solved; a data acquisition step: in the ecological system, monitoring an environmental factor corresponding to the dynamic parameter response function to obtain dynamic monitoring data of the environmental factor; meanwhile, pulse type stable isotope labeling is carried out on primary producers of the ecological system. Key process parameters in an Ecopath model are innovatively defined as dynamic parameter response functions related to environmental factors, and the fundamental limitation that a traditional model can only conduct static snapshot type evaluation is overcome.

The application discloses a method for distinguishing the carbon assimilation pathway type characteristics of methanotrophs. 13 CH4 stable isotope labeling and collecting single-cell Raman full spectrum data; based on the PCA-LDA discriminant model of the reference strains 13 CH4 labeled single-cell Raman full spectrum data to establish a PCA-LDA discriminant model; a plurality of single-cell Raman full spectrum data of a to-be-tested methanotroph sample are input into the previously established PCA-LDA discriminant model, the corresponding I-type or II-type prediction category of each single-cell Raman spectrum is obtained, and it is determined that the to-be-tested sample is closer to the carbon assimilation pathway characteristics of I-type methanotrophs or the carbon assimilation pathway characteristics of II-type methanotrophs. The method can distinguish different carbon assimilation pathway types of methanotrophs.

The application belongs to the technical field of stable isotope labeled compound preparation, and specifically discloses a carbon 13 labeled carbonate and a one-step preparation method thereof. Under an inert atmosphere, carbon 13 urea is mixed with water to obtain a carbon 13 urea solution; metal hydroxide is mixed with water to obtain a metal hydroxide dispersion; then under the inert atmosphere, the carbon 13 urea solution is added dropwise into the metal hydroxide dispersion to perform a reaction, and a slurry containing carbon 13 labeled carbonate is obtained; after post-treatment, the carbon 13 labeled carbonate is obtained. The application can be widely adapted to various metal hydroxide raw materials, realize the large-scale preparation of a series of carbon 13 labeled carbonate products, and efficiently realize the high-value utilization of low-abundance unqualified carbon 13 urea, effectively solving the industry pain points such as large carbon 13 abundance loss, complicated preparation steps, serious equipment corrosion and easy pollution by environmental CO2 in the existing process.

The invention discloses a method for detecting the content of SOS1 protein and application, and belongs to the technical field of biological medicine. The method comprises the following steps that a sample to be detected is subjected to enzymolysis, an enzymolysis product containing SOS1 protein characteristic peptide fragments is obtained, the amino acid sequences of the characteristic peptide fragments are FEIPEPEPTEADR and QLTLLESDLYR, the SOS1 protein characteristic peptide fragments labeled by stable isotopes serve as internal labels, signal values of the characteristic peptide fragments and the internal label peptide fragments are detected through liquid chromatography-tandem mass spectrometry, and the characteristic peptide fragments and the internal label peptide fragments are determined according to the signal values of the characteristic peptide fragments and the internal label peptide fragments. Calculating the relative content of the SOS1 protein through the peak area ratio, and substituting the relative content into the calibration curve linear equation to obtain the absolute content. The method can be used for quantitative detection of SOS1 protein in tumor samples and other tissues, cells and blood samples, and has the advantages of high sensitivity, good accuracy and the like.

A method for synthesizing a stable isotope-labeled toltrazuril-D3 belongs to the field of organic synthesis technology. This method uses methylamine hydrochloride-D3 as the stable isotope labeling source, reacting it with p-trifluoromethylthiophenol and 1-chloro-2-methyl-5-nitrobenzene as starting materials, undergoing sequential condensation and reduction to obtain an aminodiphenyl ether intermediate. Simultaneously, methylamine hydrochloride-D3 reacts with sodium cyanate to generate methylurea-D3, which then reacts with carbonyl diimidazole to obtain an activated intermediate. Finally, the aminodiphenyl ether intermediate condenses with the activated intermediate, followed by base-catalyzed cyclization to obtain the target product, toltrazuril-D3. This invention features a rationally designed synthetic route, readily available raw materials, mild reaction conditions, simple operation, and easy product separation and purification. The obtained product achieves a chemical purity and isotope abundance of over 99%.

The invention discloses a method for screening AD biomarkers in blood based on a liquid chromatography-mass spectrometry technology, and relates to the technical field of biological analysis. The method comprises the following steps: S1, sample pretreatment: collecting a blood sample of a subject, and separating serum or plasma; the method comprises the following steps: adding an internal standard mixed solution containing a stable isotope label into a sample, adding an organic solvent to precipitate protein, carrying out vortex mixing, centrifuging, and taking a supernatant; s2, derivatization treatment: dividing the supernate obtained in S1 into a first sample and a second sample which are equal in volume; adding a chiral derivatization reagent into the second sample to carry out pre-column derivatization reaction to generate a diastereoisomer derivative; s3, carrying out liquid chromatography-mass spectrometry analysis; and S4, data processing. Through a 13C stable isotope internal standard correction strategy, the matrix effect of a blood sample is effectively eliminated, especially for quantification of D-amino acid, the detection limit can reach the nanomole level, the quantitative repeatability error is controlled between 3% and 8%, and the method is remarkably superior to a traditional method.

The invention provides a method for preparing high-purity stable isotope labeled polypeptide through biosynthesis and application of the high-purity stable isotope labeled polypeptide, and relates to the technical field of genetic engineering. The preparation method of the polypeptide comprises the following steps: transforming a polypeptide sequence added with a fusion tag into escherichia coli; culturing the recombinant Escherichia coli, when the recombinant Escherichia coli grows to a logarithmic phase, collecting and transferring the recombinant Escherichia coli to a trace element culture medium taking 15NH4Cl as a sole nitrogen source or 13C-glucose as a sole carbon source, and inducing gene expression isotope labeled fusion protein for coding the fusion protein; purifying the fusion protein by nickel ion affinity chromatography, and performing enzyme digestion to obtain free polypeptide; and separating and purifying the free polypeptide to obtain the high-purity isotope labeled polypeptide. The stable isotope labeled polypeptide can be obtained through a biosynthesis route, and the purity of the polypeptide can reach 95% or above.

The application discloses a kind of synthetic biological fermentation metabolite isotopic internal standard detection method, belong to analytical detection technical field.The method is by to fermentation process carries out multi-point sampling, constructs metabolic fingerprint and monitors chiral evolution, determines the harvest window of target and directional purification strategy;In harvest window period, corresponding stable isotope-labeled substrate of target biosynthesis path is introduced to fermentation system, and biological self-source isotopic-labeled internal standard consistent with target structure is prepared by microorganism metabolism;After the same purification strategy is handled target and internal standard respectively, by mass balance method and isotopic internal standard-quantitative nuclear magnetic resonance method and / or isotopic internal standard-liquid chromatography tandem mass spectrometry method, combined value is determined;Comprehensive evaluation uncertainty obtains standard value.The application is with process monitoring-internal standard preparation-closed loop strategy of combined value to eliminate the system deviation caused by replacement internal standard, applicable to high-precision purity of microbial fermentation product value.

A plurality of standard curve samples each containing a stable isotope-labeled compound B of a target compound A having a different concentration and a predetermined concentration, an actual sample containing a compound B having a predetermined concentration and having an unknown concentration of the compound A, and a correction sample containing a compound A having a predetermined concentration and having no compound B added thereto are used. The peak area SA of the mass chromatogram of the compound A and the peak area SB of the mass chromatogram of the compound B are acquired (106), the SB / SA value of the calibration sample is calculated as a calibration coefficient r (107), and the peak area SB (108) of each of the standard curve samples and the actual sample is calibrated using the calibration coefficient r. A standard curve (109) indicating the relationship between the concentration ratio of the compound A and the compound B and the peak area ratio SA / SC is created using the corrected peak area SC of each standard curve sample, and the concentration of the compound A in the actual sample is calculated from the standard curve, the peak intensity ratio SA / SC in the actual sample, and the concentration of the compound B in the actual sample (110).

The invention discloses a non-stable isotope-labeled amino acid derivatization reagent pair and its preparation method and application, wherein the amino acid derivatization reagent pair is synthesized by a two-step method, wherein L-phenylalanine and paraacetaldehyde or paraformaldehyde are first refluxed to synthesize N, N-diethylphenylalanine and N, N-dimethylphenylalanine, and then the product of the first step is reacted with N-hydroxysuccinimide to synthesize N, N-dimethylphenylalanine succinimide ester and N, N-diethylphenylalanine succinimide ester. The synthesis step of the present invention is simple, low cost, no stable isotope internal standard is required, and the detection cost of chiral amino acids can be greatly reduced; in addition, the present invention can also achieve D- / L-split and internal standard quantification of more than 40 kinds of amino acids, with high sensitivity, good stability and other advantages, and can be used for splitting and detecting D- / L-amino acids in complex samples.

This invention relates to the field of detection, specifically to an in-situ method for determining the sulfate reduction rate of marine sediments. The in-situ determination method provided by this invention involves injecting a certain amount of... 34 S stable isotope labeled SO4 2‑ (in 34 S abundance was much higher than that of SO4 under natural conditions. 2‑ middle 34 The abundance of S was 4.2%, when SO4 2‑ When reduced, SO4 is marked 2‑ Unlabeled SO4 2‑ Converted proportionally to S 2‑ The S generated at this time 2‑ middle 34 The abundance of S will increase. Determination of labeled SO4 2‑ Before (control CK) and marked SO4 2‑ - S in the sediment after a period of time (ΔT) 2‑ of 34 By measuring the sulfate abundance difference and other relevant parameters, the sulfate reduction rate can be calculated. This invention enables in-situ measurement of sulfate reduction rates in marine sediments, yielding results that conform to objective laws and are highly accurate. The measurement process is simple, safe, and stable.

The invention relates to a preparation method of a stable isotope labeled pencycuron internal standard reagent for food safety detection, and belongs to the field of research and development of standard substances for food safety detection. According to the preparation method of the stable isotope labeled pencycuron internal standard reagent, commercially available starting raw materials and stable isotope labeled halogenated benzene are adopted as raw materials, and stable isotope labeled pencycuron is obtained through two-step reaction. The method has the advantages of easily available raw materials, simple operation, no use of highly toxic intermediates, mild reaction conditions, short reaction route, high final product total yield, easy separation and purification, and no dilution of isotope abundance. The chemical purity of the final product stable isotope labeled pencycuron is greater than 98%, and the isotope abundance is greater than 99%. The stable isotope labeled pencycuron internal standard reagent prepared by the preparation method can meet the technical requirements of the stable isotope labeled internal standard reagent required by a liquid chromatography-tandem mass spectrometry method for food safety detection.