CN113358733B - Method for detecting new biosynthesis pathway in cell - Google Patents
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Abstract
The invention discloses a method for detecting a new biosynthesis pathway in a cell, which comprises the following steps: s1, preparing a sample slice, and injecting an isotope solution into the sample slice to generate a target sample slice; s2, carrying out MS detection on the target sample slice to obtain a metabolic pathway of the chemical substances in the sample slice, wherein the S2 comprises the following steps: s21, carrying out MS detection on the target sample slice to obtain MS data corresponding to the target sample slice and analyzing the MS data; s22, obtaining a metabolic pathway of the chemical substances in the sample slice according to the analysis result of the MS data; the invention can standardize the ion intensity value of the metabolite of the marked isotope, avoid the comparability difference between different samples, ensure the effective isotope tracking and obtain the complete synthetic path of chemical substances.
Description
Technical Field
The invention relates to the technical field of human biosynthesis, in particular to a method for detecting a novel intracellular biosynthesis pathway.
Background
With the development of science and technology, the detection of biosynthesis pathways is more and more important, especially new biosynthesis pathways in cells, so that researchers can more deeply understand the synthesis of chemical substances, and thus, some problems in the biological field can be overcome.
At present, most of researches on synthesis paths are based on isotope tracking, and different compounds generated by isotopes know the biosynthesis paths, so that isotope tracking processing is needed to be researched by technicians in the field to ensure the accuracy of the biosynthesis paths, but in the prior art, MS calculation related to the isotope tracking process is calculated through original data, the accuracy of the MS calculation is influenced, and effective isotope tracking cannot be ensured to obtain the complete synthesis paths of chemical substances.
Disclosure of Invention
In order to solve the problems in the prior art, the ion intensity value of the labeled isotope metabolite can be standardized, the poor comparability among different samples is avoided, and effective isotope tracking is ensured so as to obtain a complete synthetic path of chemical substances; the embodiment of the invention provides a method for detecting a novel biosynthesis pathway in a cell, which comprises the following steps:
s1, preparing a sample slice, and injecting an isotope solution into the sample slice to generate a target sample slice;
s2, carrying out MS detection on the target sample slice to obtain a metabolic pathway of chemical substances in the sample slice;
wherein, S2 comprises the following steps:
s21, carrying out MS detection on the target sample slice to obtain MS data corresponding to the target sample slice and analyzing the MS data;
s22, obtaining a metabolic pathway of the chemical substances in the sample slice according to the analysis result of the MS data, wherein the analysis result of the MS data adopts the following formula:
wherein the content of the first and second substances,
is marked by 13 The ratio of the metabolites of C is such that,
means not marked 13 C metabolite ratio.
Further, S1 further comprises the following steps of determining the sample slice:
taking a brain tissue sample of a target subject;
placing the brain tissue sample in a slicing solution for culture treatment, wherein the slicing solution comprises the following components: sucrose, naCl, naHCO 3 、MgCl 2 Glucose, KCl and NaH 2 PO 4 ；
And slicing the cultured brain tissue sample to obtain the sample slice.
Further, sucrose, naCl, naHCO 3 、MgCl 2 Glucose, KCl and NaH 2 PO 4 Comprises the following chemical components: 200mM, 30mM, 26mM, 1mM, 10mM, 4.5mM and 1.2mM, the contents being in order of chemical composition.
Further, the step S1 further includes the steps of determining a target sample slice: preparing a preformed solution, wherein the preformed solution is a diluted solution of a labeled isotope histidine;
under the condition of room temperature, the sample slice is incubated in the prepared solution for a preset time;
cleaning the incubated sample slice, and obtaining a single neuron sample on the cleaned sample slice;
and injecting a preset isotope solution into the single neuron sample to obtain the target sample slice.
Further, the isotope may be used 13 C。
Further, before analyzing the MS data in S21, the method further includes: carrying out calibration processing on a UCA numerical curve in MS data, wherein the calibration processing comprises the following steps:
preparing a plurality of calibration solutions with different concentrations according to a standard sample, wherein each calibration solution is an intracellular solution;
and sequentially calibrating the UCA numerical curve according to the concentration based on each calibration solution.
Further, the chemical composition of the intracellular solution is: potassium gluconate, naCl, EGTA, HEPES, caCl 2 、CsOH、MgCl 2 Na-ATP and Na-GTP.
Further, potassium gluconate, naCl, EGTA, HEPES, caCl 2 、CsOH、MgCl 2 The contents of Na-ATP and Na-GTP are respectively as follows: 130mM, 6mM, 10mM, 1mM, 4mM, 1mM, 2mM, and 0.2mM, wherein the order of contents is in order of chemical composition.
Further, the diluted solution of isotopic histidine was subjected to dilution and washing treatment by cerebrospinal fluid.
Further, in S22, obtaining a metabolic pathway of the chemical substance in the sample slice according to the analysis result of the MS data, further comprising the steps of:
comparing the increment corresponding to the metabolite ion strength of the labeled isotope with a preset threshold, wherein the increment is an increment value of the metabolite ion strength in percentage of the mass number of the substance;
and when the corresponding increment of the ion intensity of the metabolite of the labeled isotope is larger than the threshold value, tracking the metabolic path formed by the chemical substance of the labeled isotope.
The method for detecting the new biosynthesis pathway in the cell has the following technical effects:
according to the invention, based on a formula of MS data, the ion intensity value of the metabolite to be detected is obtained accurately when the isotope is tracked, and then the proportion change value between the metabolite marked with the isotope and the metabolite not marked with the isotope is obtained according to the ion intensity value of the metabolite to be detected, so that the complete synthesis path of the chemical substance can be effectively obtained according to the tracked isotope, the accurate chemical substance can be obtained according to the synthesis path of the chemical substance, the inaccuracy of a detection result is avoided, and further the judgment on the chemical substance in the cell is influenced, and a researcher can predict the metabolic state of the cell according to the chemical substance in the cell.
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In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is a flow chart of a method for detecting a novel biosynthetic pathway in a cell according to an embodiment of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
As shown in FIG. 1, the present example provides a method for detecting a novel biosynthetic pathway in a cell, the method comprising the steps of:
s1, preparing a sample slice, and injecting an isotope solution into the sample slice to generate a target sample slice;
s2, carrying out MS detection on the target sample slice to obtain a metabolic pathway of chemical substances in the sample slice;
wherein, S2 includes the following steps:
s21, carrying out MS detection on the target sample slice to obtain MS data corresponding to the target sample slice and analyzing the MS data;
s22, obtaining a metabolic pathway of the chemical substances in the sample slice according to the analysis result of the MS data, wherein the analysis result of the MS data adopts the following formula:
wherein, the first and the second end of the pipe are connected with each other,
is marked by 13 The ratio of the metabolites of C is such that,
means not marked 13 Ratio of C metabolites.
Specifically, S1 further comprises the following steps of determining the sample slice:
taking a brain tissue sample of a target subject;
placing the brain tissue sample in a slicing solution for culture treatment, wherein the slicing solution comprises the following components: sucrose, naCl, naHCO 3 、MgCl 2 Glucose, KCl and NaH 2 PO 4 ；
And slicing the cultured brain tissue sample to obtain the sample slice.
Further, the target object refers to an experimental object to be subjected to the metabolic pathway assay of a compound, for example, an experimental mouse or an experimental monkey, etc., wherein the target object is in a normal biologically-recognized dead state.
Further, sucrose, naCl, naHCO 3 、MgCl 2 Glucose, KCl and NaH 2 PO 4 Comprises the following chemical components in percentage by weight: 200mM, 30mM, 26mM, 1mM, 10mM, 4.5mM and 1.2mM, the content sequence is ordered according to the sequence of chemical components, which can facilitate the tracing of isotopes of different compounds for cultured sample slices and improve the accuracy of isotope analysis.
Further, the slicing solution corresponds to a temperature of 0-10 ℃.
Further, the slicing solution corresponds to a pH of 7.4.
Further, the specimen slice is a coronal slice of 250 μm.
Specifically, the step S1 further includes the steps of determining a target specimen slice:
preparing a preformed solution, wherein the preformed solution is a diluted solution of a labeled isotope histidine;
under the condition of room temperature, the sample slice is incubated in the prepared solution for a preset time;
cleaning the incubated sample slice, and obtaining a single neuron sample on the cleaned sample slice;
and injecting a preset isotope solution into the single neuron sample to obtain the target sample slice.
Specifically, dilute the diluted solution with isotope histidine through the cerebrospinal fluid, wherein, the cerebrospinal fluid still is used for the cleaning process, can avoid the introduction of other chemicals on the one hand, leads to the inaccuracy that MS detected, and on the other hand adopts the cerebrospinal fluid to reduce the cost to can cyclic utilization cerebrospinal fluid.
Further, the content of the pre-prepared solution was 4mg/ml.
Further, the preset time is 1 hour.
Further, the isotope may be used 13 C, in some embodiments, the isotope may be used 15 N。
Specifically, S22 further includes:
the MS data corresponding to the target sample slice is preprocessed, wherein the preprocessing refers to the fact that the metabolite ion strength and the total ion current (I/TIC) in the MS data are compared to realize standardization, so that the difference of the total ion current of mass spectrum detection signals of different samples can be avoided, comparability among the samples is poor, and the comparability of the signals among the samples and the accuracy of analysis results are improved; wherein the normalized error value ranges from 10ppm.
Specifically, before analyzing the MS data in S21, the method further includes: carrying out calibration processing on a UCA numerical curve in MS data, wherein the calibration processing comprises the following steps:
preparing a plurality of calibration solutions with different concentrations according to a standard sample, wherein each calibration solution is an intracellular solution;
and sequentially calibrating the UCA numerical curve according to the concentration based on each calibration solution.
Specifically, the chemical composition of the intracellular solution is: potassium gluconate, naCl, EGTA, HEPES, caCl 2 、CsOH、MgCl 2 Na-ATP and Na-GTP.
Further, potassium gluconate, naCl, EGTA, HEPES, caCl 2 、CsOH、MgCl 2 The contents of Na-ATP and Na-GTP are respectively as follows: 130mM, 6mM, 10mM, 1mM, 4mM, 1mM, 2mM, and 0.2mM, wherein the order of contents is in order of chemical composition.
Further, the intracellular solution corresponds to a PH of 7.4.
Further, the concentrations of the calibration solutions were 2, 20, 200 and 400 μ M, respectively.
Further, in S22, obtaining a metabolic pathway of the chemical substance in the sample slice according to the analysis result of the MS data, further comprising the steps of:
comparing the increment corresponding to the metabolite ion strength of the labeled isotope with a preset threshold, wherein the increment is an increment value of the metabolite ion strength in percentage of the mass number of the substance;
and when the corresponding increment of the ion intensity of the metabolite of the labeled isotope is larger than the threshold value, tracking the metabolic path formed by the chemical substance of the labeled isotope.
In one specific example, data from blood and cerebrospinal fluid with and without isotope are compared as shown in Table 1.
TABLE 1
In a specific example, the results of the MS analysis data analysis corresponding to the target sample sections incubated with and without the isotope are compared, as shown in table 2.
TABLE 2
From the data in tables 1 and 2, it can be observed that the ratio of histidine, urocanic acid and glutamic acid is significantly different when the sample sections are incubated and not incubated, and the ratio can be traced by isotope, and the product of histidine, urocanic acid and glutamic acid is the metabolite synthesis path, so that the metabolite synthesis path can be obviously characterized.
The method for detecting a new intracellular biosynthesis pathway provided by the embodiment can be based on a formula of MS data, so that when an isotope is tracked, an accurate ion intensity value of a metabolite to be detected is obtained, and then a ratio change value between the metabolite with the isotope labeled and the metabolite without the isotope labeled is obtained according to the ion intensity value of the metabolite to be detected, a complete synthesis pathway of a chemical substance can be effectively obtained according to the tracked isotope, so that an accurate chemical substance is obtained according to the synthesis pathway of the chemical substance, the inaccuracy of a detection result is avoided, and the judgment of the chemical substance in the cell is further influenced, and a researcher can predict the metabolic state of the cell according to the chemical substance in the cell.
Claims (7)
1. A method for detecting a new biosynthetic pathway in a cell, comprising the steps of:
s1, preparing a sample slice, and injecting an isotope solution into the sample slice to generate a target sample slice;
s2, carrying out MS detection on the target sample slice to obtain a metabolic pathway of chemical substances in the sample slice;
wherein, S2 comprises the following steps:
s21, carrying out MS detection on the target sample slice to obtain MS data corresponding to the target sample slice and carrying out MS detection on the target sample slice
Analyzing MS data;
s22, obtaining a metabolic pathway of the chemical substances in the sample slice according to the analysis result of the MS data, wherein the analysis result of the MS data adopts the following formula:
wherein the content of the first and second substances,
is marked by 13 The ratio of the metabolites of C is such that,
means not marked 13 The ratio of metabolites of C, I/TIC is the ratio of metabolite ion intensity to total ion current;
s1 further comprises the steps of determining the sample slice:
taking a brain tissue sample of a target subject;
placing the brain tissue sample in a slicing solution for culture treatment, wherein the slicing solution comprises the following components:
sucrose, naCl, naHCO 3 、MgCl 2 Glucose, KCl and NaH 2 PO 4 ；
Slicing the cultured brain tissue sample to obtain a sample slice;
the sucrose, naCl and NaHCO 3 、MgCl 2 Glucose, KCl and NaH 2 PO 4 Comprises the following chemical components: 200mM, 30mM, 26mM, 1mM, 10mM, 4.5mM and 1.2mM, the contents being in order of chemical composition.
2. The method for detecting a new biosynthetic pathway in a cell according to claim 1, wherein the step S1 further comprises the steps of determining a target sample section:
preparing a preformed solution, wherein the preformed solution is a diluted solution of a labeled isotope histidine;
under the condition of room temperature, the sample slice is incubated in the prepared solution for a preset time;
cleaning the incubated sample slice, and obtaining a single neuron sample on the cleaned sample slice;
and injecting a preset isotope solution into the single neuron sample to obtain the target sample slice.
3. The method for detecting a new biosynthesis pathway in a cell according to claim 1, wherein the step of analyzing the MS data in S21 further comprises: carrying out calibration processing on a UCA numerical curve in MS data, wherein the calibration processing comprises the following steps:
preparing a plurality of calibration solutions with different concentrations according to a standard sample, wherein each calibration solution is an intracellular solution;
the UCA numerical curve is sequentially calibrated based on each of the calibration solutions according to the magnitude of the concentration.
4. The method for exploring a new intracellular biosynthetic pathway of claim 3, wherein the chemical composition of said intracellular solution is: potassium gluconate, naCl, EGTA, HEPES, caCl2, csOH, mgCl2, na-ATP, and Na-GTP.
5. The method for detecting a novel biosynthetic pathway of claim 4, wherein the kit comprises potassium gluconate, naCl, EGTA, HEPES, caCl 2 、CsOH、MgCl 2 The contents of Na-ATP and Na-GTP are respectively as follows: 130mM, 6mM, 10mM, 1mM, 4mM, 1mM, 2mM, and 0.2mM, wherein the order of contents is in order of chemical composition.
6. The method for detecting a new biosynthesis pathway in a cell according to claim 2, wherein the diluted solution of isotopic histidine is subjected to a dilution and washing treatment by cerebrospinal fluid.
7. The method for detecting a novel intracellular biosynthesis pathway according to claim 1, wherein in S22, a metabolic pathway of a chemical substance in a sample slice is obtained based on an analysis result of the MS data, and further comprising the steps of:
comparing the increment corresponding to the metabolite ion strength of the labeled isotope with a preset threshold, wherein the increment is an increment value of the metabolite ion strength in percentage of the mass number of the substance;
and when the corresponding increment of the ion intensity of the metabolite of the labeled isotope is larger than the threshold value, tracking the metabolic path formed by the chemical substance of the labeled isotope.
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