76 results about "Stable Isotope Labeling" patented technology

The invention relates to a novel thaumatin mutant as well as a preparation method and application thereof. The invention provides a thaumatin mutant. The thaumatin mutant comprises an amino acid sequence of SEQ ID NO: 5 and a 6His tag sequence. The invention also provides a construction method of pichia pastoris engineering bacteria for expressing the thaumatin mutant and a preparation method of the thaumatin mutant. The inventor constructs a novel thaumatin mutant through genetic engineering, and large-scale expression of the thaumatin mutant is realized by using a pichia pastoris expression system. Sensory evaluation proves that the novel thaumatin mutant has high sweetness, and his-tag has little influence on the sweetness of protein. Stable isotope labeling of the novel thaumatin mutant is realized through a stable isotope labeling technology and adjustment of culture medium conditions. A 1H-15N HSQC spectrogram is utilized to prove that the his-tag basically has no influence on the protein conformation.

The invention provides a 13C and 15N stable isotope labeled polypeptide abundance determination method based on a high-resolution mass spectrum, and belongs to the technical field of high-resolution mass spectrum determination. The determination method comprises the following steps: selecting a certified standard substance to calculate the system deviation delta 1 of the high-resolution mass spectrometer; calculating a theoretical relative deviation delta 2 of 13C and 15N isotopes in the 13C and 15N stable isotope labeled polypeptide according to a formula 2; collecting and recording a mass spectrum signal of a to-be-measured sample, and calculating to obtain an actual measurement relative deviation delta 3 of the to-be-measured sample; and when delta2 > delta3 > delta1, calculating the abundance value of the corresponding isotope label according to a formula 3 and a formula 4. According to the method disclosed by the invention, the flow injection sample is directly detected by adopting the high-resolution mass spectrometry, so that the detection speed is high, the high resolution is ensured, and the detection time is saved; only the response intensity and the m / z value of a target ion peak are needed for the analysis of a mass spectrum, and the operation is simple and convenient.

The invention belongs to the field of environmental analytical chemistry, and provides a method for preparing micro / nano plastic based on stable isotope labeling and application of the micro / nano plastic. Specifically, an initiator, a pH buffer solution, ultrapure water, an emulsifier and a deuterated styrene monomer are mixed and emulsified through an emulsion polymerization method, a polymerization reaction is performed after emulsification, and then the deuterated polystyrene microspheres are obtained through cleaning and drying. According to the method, high sensitivity, high accuracy and safety of deuterium labeling are utilized, and a new research thought is provided for deeply researching the migration and transformation rule of the micro / nano plastic in the environment and long-term influence of the micro / nano plastic on organisms. The method is expected to promote the wide application of a hydrogen stable isotope labeling quantitative technology in the field of environmental science, and provides a scientific basis for evaluating the influence of the micro / nano plastic on the ecological environment and human health.

The invention discloses a chemoautotrophic bacteria detection method based on a single cell Raman spectrum and 13C stable isotope labeling. 13C stable isotope is added into a bacterial culture solution or an environmental sample for enrichment culture, centrifugation is performed, cells are collected and used for Raman spectrum detection, and according to the deviation of two characteristic spectrum peaks, namely, the cytochrome c peak deviates from 747 cm <-1 >, 1112 cm <-1 >, 131 cm <-1 > and 1584 cm <-1 > to 725 cm <-1 >, 111 cm <-1 >, 1300 cm <-1 > and 1536 cm <-1 > under exciting light with the wavelength of 532 nm; the peak of phenylalanine shifts from 1002cm <-1 > to 965cm <-1 > under exciting light with the wavelength of 532nm, and chemoautotrophic bacteria can be identified. According to the detection method, the limitation of pure culture is overcome, the bottleneck that the metagenome cannot characterize the carbon sequestration activity is solved, and the single-cell and non-destructive detection of the chemoautotrophic bacteria in the natural environment is realized.

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 invention relates to the technical field of stable isotope tracing, and particularly discloses a temperature and humidity control 13C continuous stable isotope labeling device and method.The temperature and humidity control 13C continuous stable isotope labeling device comprises a labeling chamber, a concentration control assembly is arranged on one side of the labeling chamber, and a concentration monitoring assembly is arranged on one side of the concentration control assembly; a humidification assembly is arranged on the side, away from the concentration control assembly, of the concentration monitoring assembly, a dehumidification assembly is arranged on the side, away from the concentration monitoring assembly, of the humidification assembly, and an air pressure balance assembly is arranged on the side, away from the humidification assembly, of the dehumidification assembly. According to the device, accurate comprehensive regulation and control of multiple environmental factors such as gas concentration, temperature, humidity and air pressure in the device are realized, an ideal and accurate experimental environment is created for researching interaction of a plant-soil system under different environmental conditions, and the problems of poor temperature and humidity regulation effect and the like in the prior art are effectively solved.

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.

The invention relates to a preparation method of a stable isotope labeled fluridone internal standard reagent for food safety (pesticide residue) detection. The intermediate and stable isotope raw materials required by the preparation method are simple, easy to obtain and low in price, the reaction condition is mild, only two reaction steps are needed, and the atom economy of the stable isotope labeling raw materials is high. According to the present invention, the chemical purity of the target product stable isotope labeled fluridone can achieve more than 99%, the isotope abundance can achieve more than 99%, the final product yield is high, the isotope abundance is not diluted, and the product cost is effectively reduced. The stable isotope labeled fluridone 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 (pesticide residue) detection.

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.

The invention discloses a quantitative analysis method and device for stable isotope labeled recombinant human growth hormone, and belongs to the technical field of quantitative analysis of protein, the method comprises the following steps: S1, preliminarily measuring the purity of stable isotope labeled recombinant human growth hormone based on chromatography or electrophoresis; s2, on the basis of preliminarily determining the purity of the stable isotope labeled recombinant human growth hormone in the step S1, measuring the stable isotope labeled amino acid replacement efficiency through amino acid analysis-isotope dilution mass spectrometry; and S3, based on the stable isotope labeled amino acid replacement efficiency, measuring the content of the stable isotope labeled recombinant human growth hormone through peptide fragment quantitative analysis-isotope dilution mass spectrometry. According to the method, the isotope dilution mass spectrometry is adopted to eliminate system errors possibly introduced in the pretreatment step and the measurement process, and the accuracy and credibility of the measurement result can be remarkably improved. According to the method, traceable quantitative analysis of the content of the stable isotope labeled recombinant human growth hormone can be realized, and the measurement result can be traced to the international system of units.

The invention provides a method for determining the influence of micro-plastics on the nitrogen utilization efficiency of plants. The method comprises the following steps: 1) adding micro-plastics: mixing the micro-plastics with a preset concentration with soil without micro-plastic pollution; 2) plant planting and marking: planting a target plant in the soil added with the micro-plastic, and irrigating with a nitrogen fertilizer marked by a 15N stable isotope in the early growth stage; 3) sample collection: collecting the sample after the target plant grows; and 4) sample treatment and analysis: drying and grinding the collected sample, and carrying out determination, calculation and statistical analysis on the treated sample. According to the method, the influence of the micro-plastic on the nitrogen utilization efficiency of the plant is determined by utilizing a 15N stable isotope nitrogen labeling technology, the specific influence of the micro-plastic on the nitrogen absorption and utilization of the plant can be effectively revealed, and a scientific basis is provided for reasonably controlling micro-plastic pollution and protecting agricultural production and nutrient circulation of an ecological system.

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 a method for measuring the enthalpy value of stable isotope labeled ammonium nitrate by using a differential scanning calorimeter, which comprises the following steps of: calibrating the sensitivity of the differential scanning calorimeter by using a standard sample, filling stable isotope labeled ammonium nitrate to be measured by using a high-pressure crucible, and measuring the enthalpy value of the stable isotope labeled ammonium nitrate by using the differential scanning calorimeter. And correcting the enthalpy value of the stable isotope labeled ammonium nitrate by using the calibrated sensitivity. Compared with the prior art, the method is good in precision, RSD is smaller than 5%, and the method can be used for measuring the enthalpy value of the stable isotope labeled ammonium nitrate.

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 present invention relates to a method for target peptide quantification based on mass spectrometry using internal standard peptides labeled with a stable isotope, in which, for quantification of a target peptide with a known sequence, standard peptides having the same sequence as the target peptide and including various amino acids substituted with an isotope are synthesized, and then mixed with the target peptide at various concentrations, and through LC-MS analysis of the mixtures, an internal standard curve is generated and, at the same time, the target (endogenous) peptide is analyzed, by single analysis of the mixed samples, and the amount of the target (endogenous) peptide is accurately quantified from each of the peak areas obtained through the analysis.

The invention discloses an analysis method for determining the concentration of sitagliptin and metformin in human plasma. The analysis method comprises the following steps: S1, preparing a matrix, a standard working solution, an internal standard working solution and a quality control working solution in advance; s2, setting parameters of liquid chromatography and mass spectrometry; and S3, detecting and analyzing data. According to the method, the human plasma is extracted by adopting a protein precipitation method, and the K2EDTA retarder is added into the plasma, so that background impurity interference is small during detection. Sitagliptin and metformin labeled by stable isotopes are adopted as internal standards, and can be used as effective internal standards of objects to be detected for accurate quantitative analysis. In the analysis and detection process, a dark brown glass bottle is adopted for storage, and ethanol is adopted as a solvent, so that the stability of the sample can be improved, the property is not changed in the detection process, and the detection accuracy deviation is controlled to be less than 15%. According to the method, sitagliptin and metformin can be detected at the same time, the detection speed is high, and the detection efficiency is high.

The invention relates to a synthesis method of stable isotope 15N, 2H and 18O labeled ammonium nitrate, which comprises the following steps: adding D218O into 15N labeled ammonium nitrate in a stirring state, adding an ionic liquid supported metal catalyst, stirring for reaction, filtering, concentrating and drying to obtain stable isotope 15N, 2H and 18O labeled ammonium nitrate solid powder. Compared with the prior art, the method can solve the problems of tedious process steps, low isotope utilization rate and the like in the prior art.