Method for biosynthesizing 15N-labeled amino acid isotope standard substance by using yeast and application of 15N-labeled amino acid isotope standard substance

Abstract

The invention belongs to the technical field of biosynthesis, and particularly relates to a method for biosynthesizing a < 15 > N-labeled amino acid isotope standard substance by using yeast and application of the < 15 > N-labeled amino acid isotope standard substance. The full-spectrum 15N labeled amino acid can be efficiently and economically produced. The obtained labeled amino acid extract is used as a mixed internal standard, and high-precision and high-accuracy absolute quantification of multiple amino acids in a biological sample can be realized by combining an isotope dilution mass spectrometry. The method is simple in preparation process, low in cost, high in labeling efficiency and good in product biocompatibility, perfectly solves the problems that chemical synthesis isotope internal labels are high in price and limited in variety and possibly have biological interference, and provides a powerful tool for metabonomics research, disease marker discovery and clinical diagnosis.

Description

Technical Field

[0001] This invention belongs to the field of biosynthesis technology, specifically relating to a method for biosynthesizing 15N-labeled amino acid isotope standards using yeast and its application. Background Technology

[0002] Amino acids are fundamental molecules for life activities. They are not only monomers for protein synthesis but also participate in various physiological and pathological processes as signaling molecules, energy metabolism intermediates, and nitrogen storage forms. Precise absolute quantitative analysis of the composition and content of amino acids in biological samples is crucial for studying metabolic regulation mechanisms, discovering disease biomarkers, assessing nutritional status, and developing targeted drugs.

[0003] Currently, isotope dilution based on liquid chromatography-tandem mass spectrometry (LC-MS / MS) is widely recognized as the gold standard for quantitative analysis of small biological molecules. This method involves adding a known amount of an internal standard (e.g., a chemically identical standard but with a different isotopic composition) to the sample. 13 C or 15 (N-labeled) can effectively correct for pretreatment losses and differences in mass spectrometry ionization efficiency, thereby achieving high accuracy and high precision in absolute quantification.

[0004] However, current commercially available stable isotope-labeled amino acid standards mainly rely on chemical synthesis, which has the following significant drawbacks:

[0005] 1. Extremely high cost: The chemical synthesis process is complex, involving multiple organic reactions, intermediate purification and final product separation, which makes the price of isotope-labeled amino acids much higher than that of ordinary amino acids, greatly limiting their application in large-scale screening and routine testing.

[0006] 2. Incomplete coverage of label types: The types of commercially available labeled amino acids are limited, especially for some rare or specially modified amino acids, it is difficult to obtain corresponding isotope internal standards, which hinders the comprehensive quantitative analysis of related metabolic pathways.

[0007] 3. Potential biocompatibility issues: Residual organic solvents or byproducts introduced during chemical synthesis may have unpredictable effects on subsequent cell or animal experiments. For example, in metabolic flow animal experiments using isotope-labeled feed, animals may develop feeding preferences due to factors such as taste and odor, affecting the reliability of the experimental results. Biosynthesized markers, on the other hand, are chemically identical to their natural counterparts and may be more biocompatible.

[0008] Therefore, developing a low-cost, highly efficient, widely applicable, and biocompatible method for preparing isotope-labeled amino acids is a key technical problem that urgently needs to be solved in metabolomics and related research fields. Summary of the Invention

[0009] To address the problems mentioned in the background art, this invention proposes a method for biosynthesizing 15N-labeled amino acid isotope standards using yeast. This method utilizes yeast as a "cell factory" to synthesize inexpensive... 15 The conversion of inorganic nitrogen sources into high-value labeled amino acids avoids complex organic synthesis and purification steps, and the production cost can be reduced by more than an order of magnitude compared to chemical synthesis methods.

[0010] The technical solution adopted by this invention to solve its technical problem is: to provide a method for biosynthesizing 15N-labeled amino acid isotope standards using yeast, comprising the following steps:

[0011] S1, with 15 N-labeled inorganic nitrogen compounds were used as the sole nitrogen source in the culture medium to prepare a yeast synthesis medium.

[0012] S2. Inoculate a single yeast colony into the culture medium, allowing the yeast cells to utilize their own nitrogen assimilation and amino acid synthesis metabolic pathways to... 15 N-labeled nitrogen is integrated into all intracellular amino acid molecules;

[0013] S3. Culture the yeast cells to the mid-to-late logarithmic growth stage or early stationary stage, collect the cells, and use physical methods to break the cells and release intracellular metabolites.

[0014] S4. Extract the lysed cells using a pre-cooled organic solvent or solvent mixture, precipitate proteins, and extract water-soluble metabolites, said water-soluble metabolites containing... 15 N-labeled amino acids;

[0015] S5. After concentrating the extract supernatant by vacuum centrifugation, redissolve it with a suitable solvent and adjust it to a uniform concentration to obtain... 15 N-labeled amino acid mixed standard extract.

[0016] Further, in step S1, the 15 N-labeled inorganic nitrogen compounds include 15 N-Ammonium sulfate, 15 N-ammonium chloride, 15 N-urea, K 15 At least one of NO3.

[0017] Furthermore, the composition of the synthetic culture medium in step S1 includes: glucose, ( 15 NH4)2SO4, KH2PO4, MgSO4·7H2O, yeast nitrogen, and adjust the pH of the culture medium to 6.0 with KOH.

[0018] Further, in step S1, the yeast includes Saccharomyces cerevisiae, Pichia pastoris, or Yersinia lipolytica.

[0019] Furthermore, in step S3, the physical method for disrupting the cells includes liquid nitrogen grinding, ultrasonic disruption, or high-pressure homogenization.

[0020] Further, in step c), the pre-cooled organic solvent is methanol or acetonitrile, and the solvent mixture is methanol:acetonitrile:water = 4:4:2 (v / v / v); the extraction is carried out at 3-5°C or on ice, and the extract contains a stabilizer, which is formic acid or ammonia.

[0021] A sort of 15 The N-labeled amino acid isotope standard was prepared by the method described above.

[0022] A sort of 15 Application of N-labeled amino acid isotope standards in the preparation of diagnostic kits for metabolomics, proteomics, or clinical diagnostics.

[0023] An absolute quantification method for amino acids in a biological sample, comprising the following steps:

[0024] i) Add a known amount of [unspecified substance] to the biological sample to be tested. 15 N-labeled amino acid isotope standards were used as internal standards.

[0025] ii) The samples with internal standard added were processed and analyzed using liquid chromatography-tandem mass spectrometry;

[0026] iii) By comparing the amino acids to be tested ( 14 N) and its corresponding 15 The absolute concentration of the analyte amino acid in the sample was calculated by combining the ratio of the mass spectrometry response signal of the N-labeled internal standard with the pre-established isotope dilution calibration curve.

[0027] The liquid chromatography conditions included: a p-HILIC column (150×2.1 mm, 5 μm); a column temperature of 40–45 °C; mobile phase A of 20 mM ammonium carbonate (pH=9.0) and mobile phase B of 100% acetonitrile; a flow rate of 0.15 mL / min; an injection volume of 5 μL; and a total time of 30 min.

[0028] The gradient elution procedure is as follows:

[0029]

[0030] The mass spectrometry detection conditions include: an electrospray ion source, a positive ion mode, and a scan range of m / z 50-600; an ion source temperature of 350℃; a spray voltage of 3.5kV; an oxy-acetylene gas concentration of 40psi; an auxiliary heating gas concentration of 35psi; and a data-dependent MS / MS secondary scan.

[0031] Furthermore, the biological samples include blood, serum, plasma, urine, cell culture medium, tissue homogenate, or microbial fermentation broth.

[0032] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0033] 1. This invention utilizes yeast as a "cell factory" to produce inexpensive... 15 The conversion of inorganic nitrogen sources into high-value labeled amino acids avoids complex organic synthesis and purification steps, and the production cost can be reduced by more than an order of magnitude compared to chemical synthesis methods.

[0034] 2. The yeast of this invention possesses a complete amino acid biosynthesis network, capable of simultaneously producing all essential and non-essential amino acids with high labeling efficiency (typically >95%). 15 The N-labeled form enables the one-pot preparation of a broad-spectrum internal standard mixture, solving the problem of incomplete commercial product variety.

[0035] 3. Biosynthetic 15 N-labeled amino acids differ from their natural counterparts in chemical structure, optical rotation, and physicochemical properties. 14 The N) is identical in form, differing only in isotopic nucleus mass. Therefore, in subsequent biological experiments such as cell culture and animal feeding, it exhibits metabolic behavior indistinguishable from that of the natural molecule, avoiding potential interference from chemically synthesized products.

[0036] 4. The bio-fermentation process of the present invention is under mild conditions, mainly consuming carbon sources, nitrogen sources and inorganic salts, and the waste liquid produced is easy to treat and environmentally friendly.

[0037] 5. The mixed extract prepared by this invention is itself a "cocktail" type internal standard solution containing multiple amino acid internal standards, which can be directly added to the sample to be tested for absolute quantitative analysis of non-targeted or targeted amino acid omics, simplifying experimental operations. Attached Figure Description

[0038] Figure 1 As described in this invention 15 Overall flow chart for the preparation, qualitative and quantitative application of N-labeled amino acid standards;

[0039] Figure 2 For yeast in 14Schematic diagram of the primary chromatogram of arginine extract after culturing in N medium. Figure 1 ;

[0040] Figure 3 For yeast in 14 Schematic diagram of the primary mass spectrum of arginine extract after culturing in N medium. Figure 2 ;

[0041] Figure 4 For yeast in 14 Schematic diagram of the secondary mass spectrum of arginine extract after culturing in N medium. Figure 3 ;

[0042] Figure 5 For yeast in 15 Schematic diagram of the primary chromatogram of arginine extract after culturing in N medium. Figure 1 ;

[0043] Figure 6 For yeast in 15 Schematic diagram of the primary mass spectrum of arginine extract after culturing in N medium. Figure 2 ;

[0044] Figure 7 For yeast in 15 Schematic diagram of the secondary mass spectrum of arginine extract after culturing in N medium. Figure 3 ;

[0045] Figure 8 For yeast in 14 N and 15 Chromatograms of unlabeled and labeled amino acids after culturing in N medium. Detailed Implementation

[0046] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0047] Example 1: 15 Preparation of N-labeled amino acid mixed standards.

[0048] 1. Strains and culture medium:

[0049] Strain: Saccharomyces cerevisiae BY4741.

[0050] 15 N-labeled medium (1L): glucose 20g, ( 15 NH4)2SO4 (98% atom) 15 5g of glucose, 3g of KH₂PO₄, 0.5g of MgSO₄·7H₂O, 1mL of trace element solution, and 1mL of vitamin solution. Adjust the pH to 6.0 with KOH. Sterilize the glucose, trace element, and vitamin solutions separately before mixing.

[0051] 14 N control culture medium: except for ( 15 NH4)2SO4 was replaced with an equal mass of ordinary ( 14 Except for NH4)2SO4, the rest are exactly the same.

[0052] 2. Cultivation and Gains:

[0053] like Figure 1 As shown, a single colony was picked from a YPD plate and inoculated into 5 mL of the plate. 14 Seed culture was prepared by incubating the N control medium at 30°C and 200 rpm overnight with shaking.

[0054] The seed culture was transferred to 200 mL at a 1% inoculum volume. 15 N-labeled medium and 14 In N control medium.

[0055] Incubate at 30℃ and 200 rpm with shaking for approximately 16 hours, until OD reaches the target value. 600 Approximately 6.0 (late logarithmic growth stage).

[0056] Take the equivalent of 50 OD-mL (i.e., OD) 600 * Collect bacterial cells by centrifuging a culture medium (volume = 50 mL) at 4°C and 5000g for 5 minutes. Wash twice with pre-cooled physiological saline, then quickly freeze in liquid nitrogen and store at -80°C or extract immediately.

[0057] 3. Metabolite extraction:

[0058] The frozen bacterial cells were placed in a pre-cooled mortar and ground into a fine powder with liquid nitrogen.

[0059] Weigh approximately 50 mg of the ground powder into a pre-cooled 2 mL centrifuge tube.

[0060] Immediately add 1 mL of pre-cooled extraction solvent (methanol:acetonitrile:water = 4:4:2, v / v / v, containing 0.1% formic acid).

[0061] The vortex oscillates violently for 1 minute, followed by 10 minutes of ultrasound treatment on ice.

[0062] Let it stand at -20℃ for 1 hour to precipitate the protein.

[0063] Centrifuge at 4℃ and 13000g for 15 minutes.

[0064] Carefully transfer 800 μL of supernatant to a new centrifuge tube and evaporate the solvent at 30°C in a vacuum centrifuge.

[0065] The residue was redissolved in 100 μL of a reconstitution solution (water:acetonitrile = 2:3, v / v) and vortexed to mix.

[0066] The filtrate obtained after passing through a 0.22 μm organic nylon filter membrane is... 15 N-labeled amino acid mixed standard stock solution ( 15 N-Extract) and 14 N control extract ( 14 N-Extract), store at -80℃ for later use.

[0067] Example 2: 15 Qualitative analysis and labeling efficiency calculation of N-labeled amino acids.

[0068] 1. Mass spectrometry data acquisition:

[0069] Instruments: Ultra-high performance liquid chromatography (Shimadzu LC-30A system) tandem quadrupole time-of-flight mass spectrometry (Sciex 6600+ QTOF).

[0070] Chromatographic conditions: p-HILIC column (150×2.1 mm, 5 μm); column temperature 40~45 ℃; mobile phase A was 20 mM ammonium carbonate (pH=9.0), and mobile phase B was 100% acetonitrile; flow rate was 0.15 mL / min; injection volume was 5 μL; gradient elution.

[0071] Mass spectrometry conditions: electrospray ionization (ESI) source, positive ion mode; data-dependent acquisition (DDA) mode, scan range m / z 50-600.

[0072] 2. Qualitative analysis and labeling efficiency evaluation:

[0073] Separate injection analysis 14 N-Extract and 15 N-Extract.

[0074] Use software (such as MS-DIAL) to 14 N-Extract was used to identify non-targeted metabolites by comparing retention time, precise mass, and secondary mass spectra with standard databases (such as HMDB) to identify the main amino acid species.

[0075] for 15 N-Extract, utilizing its...14 The high consistency in retention times of identified amino acids in N-Extract allows for targeted analysis. Based on the number of nitrogen atoms (N_num) in the amino acid molecular formula, their... 15 The theoretical mass-to-charge ratio after N is fully labeled: m / z(15N) = m / z(14N) + N_num × (0.997035) 15 N and 14 (Poor quality of N).

[0076] exist 15 From the total ion chromatogram of N-Extract, the ion chromatographic peaks with the theoretical m / z mentioned above were extracted, and their retention times were related to... 14 If the N peaks are consistent and their shapes are symmetrical, then it can be confirmed as the corresponding peaks. 15 N-labeled amino acids.

[0077] Labeling efficiency calculation: Select a specific amino acid (e.g., arginine, which contains 4 nitrogen atoms) 15 The ratio of the intensity of the fully labeled N peak (M+4) to the total intensity of all isotope peaks (M+0, M+1, M+2, M+3, M+4) is used as an approximate labeling efficiency for the amino acid. More precise calculations can be performed using isotope distribution simulation software (such as IsoPro).

[0078] Result: As Figures 2 to 7 As shown in this embodiment, the prepared 15 The N-labeled amino acid mixed standard contains at least 20 common proteinogenic amino acids. Taking arginine as an example, its M+4 peak has a relative abundance exceeding 98%, indicating... 15 N-labeling is extremely efficient.

[0079] Example 3: Application and verification in absolute quantitative analysis of serum amino acids.

[0080] 1. IDMS calibration curve establishment:

[0081] Standard solutions: Prepare unlabeled solutions containing 15 different concentration gradients. 14 N) A mixture of amino acid standard solutions.

[0082] Internal standard addition: To each concentration gradient standard solution (100 μL), add an equal volume (e.g., 10 μL) of the solution prepared in Example 1. 15 N-Extract (as a mixed internal standard).

[0083] Mass spectrometry analysis: same instrument conditions as above for sample injection analysis.

[0084] Data processing: For each amino acid, integrate its... 14 N form (analyte) and 15The peak area of ​​the extracted ion chromatography in the N-form (internal standard) was used. A calibration curve was established by performing linear regression between the analyte concentration (x-axis) and the analyte / internal standard peak area ratio (y-axis).

[0085] 2. Quantitative analysis of actual samples:

[0086] Take 10 μL of human serum sample and add 90 μL of solution containing... 15 The N-Extract internal standard was used as the extraction solvent (same as in Example 1) to precipitate proteins and extract metabolites.

[0087] Centrifuge and collect the supernatant for analysis to obtain the analyte / internal standard peak area ratio for each amino acid.

[0088] Substitute this ratio into the corresponding IDMS calibration curve to calculate the absolute concentration of various amino acids in serum.

[0089] 3. Methodological Validation and Comparison:

[0090] Table 1 shows the data based on... 15 Example table of the performance of the amino acid quantification method of N-labeled yeast extract internal standard.

[0091]

[0092] Linearity and sensitivity: as shown in Table 1 and Figure 8 As shown, taking leucine as an example, the linear relationship is good within the concentration range of 0.1-500 μM, with a correlation coefficient R0. 2 > 0.999. The limit of detection (LOD, S / N=3) is less than 0.05 μM.

[0093] Accuracy and precision: Spiking recovery experiments were conducted by adding three known concentrations of amino acid standards (low, medium, and high) to serum samples. The recovery rates were between 92% and 108%, and the intra-day and inter-day precision (RSD) were both less than 8%.

[0094] Comparison with commercially available internal standards: Using the same serum sample, the standards prepared according to this invention were compared... 15 N-Extract and the commodification of purchases 13 C / 15 Quantification was performed using a single amino acid internal standard kit doubly labeled with N. Paired t-tests were performed on the quantification results of 10 key amino acids, showing no significant difference between the two groups (p > 0.05), demonstrating that the internal standard prepared in this invention is comparable to expensive commercial internal standards in terms of quantitative accuracy.

[0095] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for biosynthesizing 15N-labeled amino acid isotope standards using yeast, characterized in that, Includes the following steps: S1, with 15 N-labeled inorganic nitrogen compounds were used as the sole nitrogen source in the culture medium to prepare a yeast synthesis medium. S2. Inoculate a single yeast colony into the culture medium, allowing the yeast cells to utilize their own nitrogen assimilation and amino acid synthesis metabolic pathways to... 15 N-labeled nitrogen is integrated into all intracellular amino acid molecules; S3. Culture the yeast cells to the mid-to-late logarithmic growth stage or early stationary stage, collect the cells, and use physical methods to break the cells and release intracellular metabolites. S4. Extract the lysed cells using a pre-cooled organic solvent or solvent mixture, precipitate proteins, and extract water-soluble metabolites, said water-soluble metabolites containing... 15 N-labeled amino acids; S5. After concentrating the extract supernatant by vacuum centrifugation, redissolve it with a suitable solvent and adjust it to a uniform concentration to obtain... 15 N-labeled amino acid mixed standard extract.

2. The method for synthesizing 15N-labeled amino acid isotope standards using yeast biosynthesis according to claim 1, characterized in that: In step S1, the 15 N-labeled inorganic nitrogen compounds include 15 N-Ammonium sulfate, 15 N-ammonium chloride, 15 N-urea, K 15 At least one of NO3.

3. The method for synthesizing 15N-labeled amino acid isotope standards using yeast biosynthesis according to claim 1, characterized in that: The composition of the synthetic culture medium in step S1 includes: glucose, ( 15 NH4)2SO4, KH2PO4, MgSO4・7H2O, yeast nitrogen.

4. The method for synthesizing 15N-labeled amino acid isotope standards using yeast biosynthesis according to claim 1, characterized in that: In step S1, the yeast includes Saccharomyces cerevisiae, Pichia pastoris, or Yersinia lipolytica.

5. The method for synthesizing 15N-labeled amino acid isotope standards using yeast biosynthesis according to claim 1, characterized in that: In step S3, the physical methods for disrupting cells include liquid nitrogen grinding, ultrasonic disruption, or high-pressure homogenization.

6. The method for synthesizing 15N-labeled amino acid isotope standards using yeast biosynthesis according to claim 1, characterized in that, In step c), the pre-cooled organic solvent is methanol or acetonitrile, and the solvent mixture is methanol:acetonitrile:water = 4:4:2 (v / v / v). The extraction is carried out at 3-5°C or on ice, and the extract contains a stabilizer, which is formic acid or ammonia.

7. A 15N-labeled amino acid isotope standard, characterized in that: It is prepared by the method described in any one of claims 1-6.

8. The use of the 15N-labeled amino acid isotope standard as described in claim 7 in the preparation of detection kits for metabolomics, proteomics, or clinical diagnosis.

9. An absolute quantitative method for amino acids in biological samples, characterized in that, Includes the following steps: i) Add a known amount of the substance as described in claim 7 to the biological sample to be tested. 15 N-labeled amino acid isotope standards were used as internal standards. ii) The samples with internal standard added were processed and analyzed using liquid chromatography-tandem mass spectrometry; iii) By comparing the amino acids to be tested ( 14 N) and its corresponding 15 The absolute concentration of the analyte amino acid in the sample was calculated by combining the ratio of the mass spectrometry response signal of the N-labeled internal standard with the pre-established isotope dilution calibration curve. The liquid chromatography conditions included: a p-HILIC column (150×2.1 mm, 5 μm); a column temperature of 40–45 °C; mobile phase A of 20 mM ammonium carbonate (pH=9.0) and mobile phase B of 100% acetonitrile; a flow rate of 0.15 mL / min; an injection volume of 5 μL; and a total time of 30 min. The gradient elution procedure is as follows: ； The mass spectrometry detection conditions include: an electrospray ion source, a positive ion mode, and a scan range of m / z 50-600; an ion source temperature of 350℃; a spray voltage of 3.5kV; an oxy-acetylene gas concentration of 40psi; an auxiliary heating gas concentration of 35psi; and a data-dependent MS / MS secondary scan.

10. The absolute quantification method for amino acids in a biological sample according to claim 9, characterized in that: The biological samples include blood, serum, plasma, urine, cell culture medium, tissue homogenate, or microbial fermentation broth.

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