CN112697894A - Quantitative analysis method of amino acid isotope label - Google Patents
Quantitative analysis method of amino acid isotope label Download PDFInfo
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Abstract
本发明属于氨基酸分析方法技术领域，具体公开了一种氨基酸同位素标记物的定量分析方法，包括以下步骤：包括以下步骤：S1、配制13C培养基和12C培养基；S2、分别采用S1得到的13C培养基和S1得到的12C培养基培养细胞；S3、细胞收集：弃去12C培养基和13C培养基，得到13C细胞和12C细胞进行衍生化前准备，得到13C样本和12C样本；S4、MCF衍生化；S5、采用GC‑MS对衍生化溶液进行分析；S6、数据挖掘获得氨基酸的碎片的荷质比、保留时间、13C可取代氨基酸的位置及个数。本发明通过实验得到衍生化代谢物可能的特征离子荷质比，进一步扩展了GC‑MS的SIAM的数据库，用于基于GC‑MS的13C同位素标记物的定位和定量分析，能够直观观测在不同环境或疾病的条件下细胞的代谢的变化情况，准确反映细胞代谢与环境或疾病之间的关系具有重要意义。
The invention belongs to the technical field of amino acid analysis methods, and specifically discloses a quantitative analysis method for amino acid isotope markers, comprising the following steps: comprising the following steps: S1, preparing a 13 C medium and a 12 C medium; S2, using S1 to obtain respectively The 13 C medium and the 12 C medium obtained from S1 were used to culture cells; S3, cell collection: discard the 12 C medium and the 13 C medium to obtain 13 C cells and 12 C cells for preparation before derivatization to obtain 13 C Samples and 12 C samples; S4, MCF derivatization; S5, GC-MS analysis of the derivatized solution; S6, data mining to obtain the charge-to-mass ratio, retention time, position and number of 13 C-substitutable amino acids of amino acid fragments number. The present invention obtains possible characteristic ion charge-to-mass ratios of derivatized metabolites through experiments, further expands the SIAM database of GC-MS, and is used for localization and quantitative analysis of 13 C isotope labels based on GC-MS, and can intuitively observe It is of great significance to accurately reflect the relationship between cell metabolism and the environment or disease to accurately reflect the changes in cell metabolism under different environmental or disease conditions.
Description
Technical Field
The invention belongs to the technical field of amino acid analysis methods, and particularly relates to a quantitative analysis method for an amino acid isotope marker.
Background
Amino acid is the basic constituent unit of protein, contains amino functional group and carboxyl functional group, and participates in multiple metabolic pathways of human body, so that the analysis technology aiming at amino acid has important significance for the research of protein chemistry, biochemistry and even whole life science.
In Stable Isotope Assisted Metabonomics (SIAM) and by utilizing a compound (a labeled compound) labeled by a stable isotope tracer, the substance conversion or change rule is researched, and the specific principle is as follows: since the labeled compound and its corresponding non-labeled compound (substance to be tracked) have the same chemical and biological properties, and the chemical changes and biological processes occurring in the organism are completely the same, the content of the same non-labeled compound is measured by the change of the isotopic abundance or ratio after mixing the two.
In the case of amino acids, the stable isotope labeled amino acid is one or more elements of a common amino acid stabilized isotope (e.g.13C、15N or2H) The amino acid marked by the stable isotope is introduced into an organism, and the physicochemical process of metabolism in the organism or cells can be uncovered by measuring the change of the stable isotope abundance of the amino acid and the metabolic products thereof in the metabolic process, so that the metabolic rule of the organism can be known.
The technical platform for SIAM is mainly liquid chromatography-mass spectrometry (LC-MS for short), but LC-MS has the following problems: (1) the co-elution matrix component causes ion inhibition (the matrix refers to a component except for an analyte in a sample, and the co-elution matrix refers to a matrix component which has a peak together with a target substance), so that the generation efficiency and the ionic strength of a tracked substance are reduced, a detection signal is weakened, and a detection result is biased; (2) the substrate may be affected by the microbial species and pretreatment process, and the dilute sample method may eliminate the substrate effect but may reduce the sensitivity to low abundance metabolites, due to: dilution is the simplest sample pretreatment technology, a sample only needs to be diluted by an LC-MS compatible solvent, but all components in the sample enter an LC-MS system, so that the sensitivity to low-abundance metabolites is reduced, and therefore, the sample dilution method is only suitable for the situation that the matrix effect is not large and the sensitivity problem does not exist.
GC-MS is less useful for SIAM and can only analyze volatile compounds, which require amino acid derivatization first to reduce the boiling point of the amino acid. Common methods of derivatization of amino acids are Methyl Chloroformate (MCF) and Ethyl Chloroformate (ECF).
Because the chemical properties of amino acids are greatly different due to different R groups of the amino acids, for example, the amino acids can be divided into acidic amino acids, basic amino acids and neutral amino acids according to the pH value at the isoelectric point, the determination of the amino acids by adopting a gas chromatography-mass spectrometry (GC-MS for short) has certain difficulty, an amino acid database established based on the GC-MS at present is not complete enough, and the limitation is large for uncovering the physicochemical process of organism or intracellular metabolism and knowing the metabolism rule of the organism.
Disclosure of Invention
The invention aims to provide a quantitative analysis method of an amino acid isotope marker, which aims to solve the technical problems that an amino acid database established based on GC-MS is not complete enough, and the method has great limitation on uncovering the physicochemical process of organism or intracellular metabolism and understanding the metabolism rule of an organism.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a method for quantitative analysis of an amino acid isotope label, comprising the steps of:
s1, preparation13C Medium and12c, culture medium;
s2 samples obtained by S113C Medium and obtained in S112C, culturing the cells in a culture medium;
s3, cell collection: discard12C Medium and13c medium, obtaining13C cells and12c cells are prepared prior to derivatization to give13C sample and12c, sampling;
s4 and MCF derivatization;
s5, analyzing the derivatization solution by adopting GC-MS, wherein the flow rate of the helium flow is 0.5-1.5 mL/min; the temperature of the sample inlet is 270-300 ℃; the column temperature program was as follows: the GC oven is set to be 42-48 ℃, the temperature is raised to 185 ℃ at the speed of 5-9 ℃/min after being maintained for 1-2min, the temperature is raised to 230 ℃ at the speed of 35-45 ℃/min after being maintained for 4-8min, the temperature is raised to 250 ℃ at the speed of 30-50 ℃/min after being maintained for 4-8min, the temperature is raised to 290 ℃ at the speed of 30-50 ℃/min after being maintained for 10-15min, and the temperature is maintained for 1-4 min; the temperature of the mass spectrum ion source is 140-160 ℃; the mass spectrum quadrupole temperature is 225-235 ℃; the temperature of the connecting rod is 245-255 ℃;
s6, obtaining the charge-to-mass ratio, retention time and mass ratio of the fragments of the amino acid by data mining,13C positions and numbers of substitutable amino acids, see table below:
the principle and the beneficial effects of the technical scheme are as follows:
(1) according to the technical scheme, firstly, amino acid is derivatized, then, peaks of all amino acids are fully separated by strictly controlling a column temperature rise program, the flow rate of helium gas flow and the temperature of a sample inlet, so that the characteristic ion charge-to-mass ratio of a derivatized metabolite is obtained, a database of a SIAM of a GC-MS is further expanded, the change condition of cell metabolism can be further visually observed, the cell metabolism change can be further accurately reflected, and the method has important significance for uncovering a physical and chemical process of organism or intracellular metabolism and knowing the metabolism rule of an organism.
(2) The technical scheme is realized by calculation13The content difference of the C isotope in different metabolites is more accurate and is calculated13The utilization rate and the conversion rate of the C-labeled metabolites and the analysis of metabolic flux provide better basis.
(3) The technical scheme has high precision, and RSD is less than 10%.
Drawings
FIG. 1 is a schematic diagram of derivatization in example 1 of the present invention;
FIG. 2 is a mass spectrum of alanine in example 1 of the present invention;
FIG. 3 is a mass spectrum of glycine in example 1 of the present invention;
FIG. 4 is a mass spectrum of valine in example 1 of the present invention;
FIG. 5 is a mass spectrum of leucine in example 1 of the present invention;
FIG. 6 is a mass spectrum of isoleucine in example 1 of the present invention;
FIG. 7 is a mass spectrum of proline in example 1 of the present invention;
FIG. 8 is a mass spectrum of threonine in example 1 of the present invention;
FIG. 9 is a mass spectrum of aspartic acid of example 1 of the present invention;
FIG. 10 is a mass spectrum of glutamine in example 1 of the present invention;
FIG. 11 is a mass spectrum of histidine in example 1 of the present invention;
FIG. 12 is a mass spectrum of tyrosine in example 1 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 accompanying drawings of 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 derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The embodiment provides a quantitative analysis method of an amino acid isotope label, which specifically comprises the following steps:
s1, preparation13C Medium and12c, culture medium: adding into the culture medium13C glucose, in the culture medium13C accounts for 20-70% to obtain13C Medium, preferably13In C medium13C is 20-50%, and more preferably13C accounts for 30 percent; adding to the culture medium13Equal amount of C glucose12C glucose to obtain12And C, a culture medium.
For example: if it is not13In C medium12The concentration of C is 17mM, of which12C accounts for 70% of the total concentration, so that the total concentration of the finally prepared culture was 17 mM/70% -24.3 mM, and the content of C in the culture was estimated to be 30%13The concentration of C was 24.3mM × 30% ═ 7.3 mM. When preparing 100ml13C medium, it is necessary to add13Mass of C is 0.1L × 7.3mM × 181g/mol is 132mg12C added to the culture medium12Mass of C was 0.1L × 7.2mM × 180g/mol — 131 mg. Preferably in a culture medium12The concentration of C is set between 7.3 and 34mM, preferably in the culture medium13The concentration of C was set between 7.3-34 mM.
S2, cell culture: respectively obtained by S113C Medium and obtained in S112C culture medium (this experiment adopts the experiment of normal stem cell LO2, other types of cells can be used for isotope labeling experiment, and the same method is adopted),13c Medium and12the culture conditions of the two groups of the C culture medium are completely the same, and the two groups of the C culture medium are cultured in culture dishes with the same specification.
S3, cell collection: discard12C Medium and13c medium, obtaining13C cells and12c cells. Respectively cleaning with physiological saline13C cells and12after C cells, adding liquid nitrogen to promote cell metabolism arrest; then respectively to13C cells and12adding methanol-chloroform mixed extract into C cells, repeatedly scraping cells with cell scraper for 1-3mim (avoiding scraping for too long time to volatilize methanol-chloroform mixed extract), sucking methanol-chloroform mixed extract into EP tube, centrifuging at 10000rpm/min for 5min, placing supernatant in condensing and draining machine, condensing and draining to obtain the final product13C sample and12samples C, stored at-20 ℃ for derivatization.
S4, MCF derivatization: at room temperature to S313C sample and12to the sample C, 200. mu.L of 1M sodium hydroxide was added, followed by shaking for 10 seconds, and then 167. mu.L of methanol (M: 32, density: 0.7918 g/cm)3) And 34 μ L pyridine (M79, density 0.9819 g/cm)3) Then, 20. mu.L of MCF (M: 94.5, density: 1.22 g/cm) was added3) Oscillating for 30s, then adding 20 mu L of MCF, oscillating for 30s, adding 400 mu L of chloroform, oscillating again and mixing evenly for 10 s; and finally adding 400 mu L of 50mM sodium bicarbonate, centrifuging for 5min at the rotating speed of 1000r/min, discarding the upper layer liquid, adding anhydrous sodium sulfate to absorb excessive water, and absorbing the lower layer derivatization solution. The sample injection volume was 1ul of derivatization solution.
In the step, MCF derivatization is used for leading amino acid and non-amino acid organic matters to form volatile ester or carbamate, firstly, in MCF derivatization reaction, oxygen in carboxyl and nitrogen in primary or secondary amino groups can be used as a substrate to replace chloride ions. In carboxylic acid, the intermediate product, namely acid anhydride, is attacked by electron pair to release one molecule of methyl carbonate and generate one molecule of ester; in the amino group, methyl carbonate binds to the nitrogen, depriving it of a proton, forming a carbamate group. After MCF derivatization, the molecular mass of each carboxyl group in the metabolite increased by 14 mass units and the molecular mass of each primary or secondary amino group increased by 58 mass units, see fig. 1 for the specific principles.
Derivatization of S4 is rapid, the obtained product is single, and the obtained derivatization product has stable structure.
S5, analyzing the derivatization solution by GC-MS, wherein the preferable chromatographic column is ZB-1701(30m × 250 μm × 0.15 μm, 5m protective column); the mobile phase is helium; the sample injection volume is 0.5-1.5 muL, and the preferred sample injection volume is 1 muL; the flow rate of the helium flow is 0.5-1.5mL/min, and the preferred flow rate is 1 mL/min; a pressure of 0.2 to 0.6bar, preferably a pressure of 0.5 bar; the temperature of the injection port is 270-300 ℃, and the temperature of the injection port is 290 ℃.
The column temperature program was as follows: the GC oven is set to be 42-48 ℃, the temperature is maintained for 1-2min, the temperature is raised to 185 ℃ at the rate of 5-9 ℃/min, the temperature is raised to 230 ℃ at the rate of 35-45 ℃/min after the temperature is maintained for 4-8min, the temperature is raised to 250 ℃ at the rate of 30-50 ℃/min (preferred) after the temperature is maintained for 4-8min, the temperature is raised to 290 ℃ at the rate of 30-50 ℃/min after the temperature is maintained for 10-15min, the temperature is maintained for 1-4min, the cumulative time of the whole column temperature raising program is 40-57min, preferably 43min, metabolites with close retention time can be separated in the time, and the mass spectrum column is cleaned.
A further preferred column temperature program is as follows: the GC oven was set at 45 ℃ and maintained for 2min, then ramped up to 180 ℃ at a rate of 9 ℃/min, maintained for 5min, ramped up to 220 ℃ at a rate of 40 ℃/min, maintained for 5min, ramped up to 240 ℃ at a rate of 40 ℃/min, maintained for 11.5min, ramped up to 280 ℃ at a rate of 40 ℃/min, and maintained for 2 min.
The commonly used temperature-raising procedure in the prior art is as follows: the temperature is raised to 260-280 ℃ at the speed of 10 ℃/min, the peak overlapping of the amino acid of less than 80 percent can be generated in the temperature raising program, the quantitative analysis of the amino acid is not facilitated, and the temperature raising program of the embodiment can separate the peaks of more than 90 percent of the amino acid.
The electron impact ionization energy was 70 eV. The mass scan range is 38-550m/z, the preferred mass scan range is 38-280m/z, and the scan speed is 1.562 μ/s.
The temperature of the mass spectrum ion source is 140-160 ℃, preferably 150 ℃, and the ionization efficiency can be ensured to be more than 95%.
The mass spectrum quadrupole temperature can influence the ion flight speed, and in the embodiment, the mass spectrum quadrupole temperature is 225-235 ℃, and is further preferably 230 ℃;
the temperature of the connecting rod is controlled to 245 ℃ and 255 ℃ in the embodiment, and the temperature is preferably controlled to 250 ℃. The temperature of the connecting rod is too low or too high, and the conduction efficiency of gas to the mass spectrum can be ensured to be more than or equal to 98% when the temperature of the connecting rod is 250 ℃.
In order to detect low concentrations of metabolites without unduly contaminating the meteorological column, S5 employs a pulsed split approach.
S6, data mining: extracting mass spectrum data by adopting AMDIS software and MassOmics software to obtain charge-to-mass ratios of fragments of 14 amino acids and mass ratios13The position and the number of the substituted amino acid C can be found in the following table:
the above quantitative analysis method leads to the following conclusions:
in the normal group of cell culture group, the growth of the cells promotes the growth of leucine, proline and lysine containing 2 carbon bonds13The highest percentage of C substitution; valine, threonine, and phenylalanine 113The percentage of C is highest; 1-2 of alanine, glycine, serine, aspartic acid, methionine and tyrosine13The percentage of C is highest; isoleucine contains 1 or 313The percentage of C is highest; glutamic acid contains 1-313The highest percentage of C; 2 or 4 of glutamic acid amide13The percentage of C is highest; 2 or 6 of histidine13The percentage of C is highest.
Example 2 calculation13Enrichment of C-labeled metabolites
The embodiment can be realized by calculation13C distribution to determine the dominant mass fraction13C and12c ratio, thereby calculating13Enrichment of C-labeled metabolites.
First of all, the first step is to,13the identification of C-labeled metabolites relies on the retention time of the chromatogram obtained from the AMDIS software; each one of which is13The C-labeled electron fragments are calculated based on13Fragments of C-tag and their correspondence12C-marked fragment difference.
Finally, the process is carried out in a batch,13enrichment of C-labeled metabolites equal to13Total percentage of C-labeled fragments minus those naturally occurring in nature13The percentage of C metabolites is specifically as follows:
m represents a metabolite12C ion concentration; n represents the nuclear-to-cytoplasmic ratio higher than M; m + n represents among the metabolites13C ion concentration; total enrichment13C represents a metabolite extracted from the cells of the experimental group and has13C, marking; nature of nature13C represents a metabolite extracted from cells of a control group and has13And C, marking.
The calculation method is described in detail below by taking fig. 2 as an example:
the specific calculation formula is as follows:
comprises a13C enrichment ratio (%) -10/20 x 100% to 5/20 x 100%;
comprises two13The enrichment ratio (%) of C is 5/20 × 100% to 1/20 × 100%.
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.
Claims (7)
1. The quantitative analysis method of the amino acid isotope label is characterized by comprising the following steps:
s1, preparation13C Medium and12c, culture medium;
s2 samples obtained by S113C Medium and obtained in S112C, culturing the cells in a culture medium;
s3, cell collection: discard12C Medium and13c medium, obtaining13C cells and12c cells are prepared prior to derivatization to give13C sample and12c, sampling;
s4 and MCF derivatization;
s5, analyzing the derivatization solution by adopting GC-MS, wherein the flow rate of the helium flow is 0.5-1.5 mL/min; the temperature of the sample inlet is 270-300 ℃; the column temperature program was as follows: the GC oven is set to be 42-48 ℃, the temperature is raised to 185 ℃ at the speed of 5-9 ℃/min after being maintained for 1-2min, the temperature is raised to 230 ℃ at the speed of 35-45 ℃/min after being maintained for 4-8min, the temperature is raised to 250 ℃ at the speed of 30-50 ℃/min after being maintained for 4-8min, the temperature is raised to 290 ℃ at the speed of 30-50 ℃/min after being maintained for 10-15min, and the temperature is maintained for 1-4 min; the temperature of the mass spectrum ion source is 140-160 ℃; the mass spectrum quadrupole temperature is 225-235 ℃; the temperature of the connecting rod is 245-255 ℃;
s6, obtaining the charge-to-mass ratio, retention time and mass ratio of the fragments of the amino acid by data mining,13The positions and numbers of the C substitutable amino acids are shown in the following table:
2. the method for quantitative analysis of an amino acid isotope label according to claim 1, wherein in S1,13in C medium13C accounts for 20-70%.
3. The method for quantitative analysis of an amino acid isotope label according to claim 1 or 2, wherein the mixed extract in S3 is a methanol-chloroform mixed extract, and the volume ratio of methanol to chloroform is 9: 1.
4. The method for quantitative analysis of an amino acid isotope-labeled substance according to claim 1 or 2, wherein the labeling reaction product obtained at S4 in S3 is analyzed at room temperature13C sample and12to the sample C, 200. mu.L of 1M sodium hydroxide was added, followed by shaking for 10 seconds, then 167. mu.L of methanol and 34. mu.L of pyridine were added, and then 20. mu.L of MCF (M: 94.5, density: 1.22 g/cm) was added3) Oscillating for 30s, then adding 20 mu L MCF, oscillating for 30s, adding 400 mu L chloroform, oscillating again and mixing evenly for 10 s; and finally adding 400 mu L of 50mM sodium bicarbonate, centrifuging for 5min at the rotating speed of 1000r/min, discarding the upper layer liquid, adding anhydrous sodium sulfate to absorb excessive water, and absorbing the lower layer derivatization solution.
5. The method for quantitative analysis of an amino acid isotope labeled substance according to claim 3, wherein the quantitative analysis is performed on S3 under ambient temperature conditions in S413C sample and12to the sample C, 200. mu.L of 1M sodium hydroxide was added, followed by shaking for 10 seconds, then 167. mu.L of methanol and 34. mu.L of pyridine were added, and then 20. mu.L of MCF (M: 94.5, density: 1.22 g/cm) was added3) Oscillating for 30s, then adding 20 mu L MCF, oscillating for 30s, adding 400 mu L chloroform, oscillating again and mixing evenly for 10 s; and finally adding 400 mu L of 50mM sodium bicarbonate, centrifuging for 5min at the rotating speed of 1000r/min, discarding the upper layer liquid, adding anhydrous sodium sulfate to absorb excessive water, and absorbing the lower layer derivatization solution.
6. The method for quantitative analysis of an amino acid isotope label according to claim 1, 2 or 5, wherein the scanning speed in S5 is 1.562. mu.s.
7. The method for quantitative analysis of an amino acid isotope label according to claim 1, 2 or 5, wherein S5 is divided by pulsed split method.
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