A method for metabolite detection based on enhanced multiple alkali metal ion addition

Abstract

The application discloses a kind of detection techniques of small molecule metabolite based on enhanced multiple alkali metal ion addition technology, it is related to the field of metabolomics, the detection method of small molecule metabolite, including adding alkali metal cation salt solution to the small molecule metabolite to be measured, then using mass spectrum to detect sample.The detection method disclosed in the application can 1) realize multiple alkali metal cation addition of small molecule metabolite significantly enhanced, significantly improve the detection and quantification performance of small molecule metabolite in laser desorption ionization mass spectrometry, improve small molecule metabolite detection capability;2) realize high-throughput and high repeatability detection of small molecule metabolite in biological fluid;3) and by simple comparison multiple alkali metal cation addition, realize isomer differentiation directly in primary mass spectrum.

Description

Technical Field

[0001] This invention relates to the field of metabolomics, and in particular to a detection technique for small molecule metabolites based on enhanced multiple alkali metal ion addition technology. Background Technology

[0002] Metabolomics is a technique for studying changes in the metabolic substances of organisms, reflecting real-time changes in the phenotype of biological systems. Mass spectrometry is one of the main tools in metabolomics research, offering advantages such as high sensitivity, high specificity, high resolution, and label-free identification. Laser desorption / ionization mass spectrometry (LASMS) is a solid-phase mass spectrometry technique that offers advantages over other mass spectrometry techniques, including simple sample preparation and fast analysis speed, and holds great promise for large-scale applications in clinical settings. Ionization is a key step in molecular detection during LASMS. Currently, H+ ionization of macromolecules has been achieved with the assistance of organic matrices. + Addition and detection. However, in the low molecular weight range (molecular weight less than 1000), the self-ionization of organic matrices can cause background interference, and poor homogeneity of co-crystallization with the sample can lead to poor detection reproducibility, limiting its development in the detection of small molecule metabolites. In contrast, the use of inorganic nanomaterials for the detection of small metabolites has advantages such as low background interference and high reproducibility, attracting widespread global attention.

[0003] H with organic matrix + The addition processes differ; inorganic nanomaterials primarily utilize alkali metal cation addition (Na+). + / K + This is for the detection of small molecule metabolites. This is because biological samples often have high concentrations of alkali metal cations (e.g., Na+ in serum). + and K + The concentrations (135-145 mM and 3.5-5.5 mM, respectively) are strong, and they exhibit strong affinity for N / O atoms in small molecule metabolites. Significant discoveries have been made regarding alkali metal cation addition during ionization, such as cation-metabolite affinity, multiple cation addition, and cation adduct formation. However, the detection of small molecule metabolites using alkali metal cation addition currently faces two main challenges: 1) difficulty in enhancing multiple alkali metal cation addition to improve its quantitative performance in small molecule metabolite detection; and 2) difficulty in achieving high-throughput and highly reproducible detection of small molecule metabolites in biological fluids using multiple alkali metal cation addition, and in enabling direct isomer differentiation in primary mass spectrometry, thereby improving the analytical capabilities of laser desorption / ionization mass spectrometry for small molecule metabolites in complex biological samples.

[0004] Therefore, those skilled in the art are dedicated to developing a high-performance detection method for small molecule metabolites based on enhanced multiple alkali metal ion addition technology. Summary of the Invention

[0005] In view of the above-mentioned deficiencies of the prior art, the technical problem to be solved by the present invention is to improve the analytical capability of laser desorption / ionization mass spectrometry for small molecule metabolites in complex biological samples.

[0006] To achieve the above objectives, the present invention provides a method for detecting small molecule metabolites, which includes adding an alkali metal cation salt solution to the small molecule metabolite to be tested, and then using mass spectrometry to detect the sample.

[0007] Preferably, the small molecule metabolite is an amino acid metabolite; more preferably, the amino acid is proline, glutamic acid, serine, threonine, arginine, alanine, isoleucine, phenylalanine, and methionine.

[0008] Preferably, the alkali metal cation is Na. + and / or K + .

[0009] Preferably, the alkali metal cation salt solution is a sodium chloride and / or potassium chloride solution.

[0010] Preferably, the mass spectrometry is matrix-assisted laser desorption / ionization Fourier transform ion cyclotron resonance mass spectrometry.

[0011] Preferably, the detection can quantify or distinguish isomers of the sample.

[0012] Preferably, the specific steps of the detection method for small molecule metabolites are as follows:

[0013] 1) Dissolve the small molecule metabolite to be tested in deionized water;

[0014] 2) Add sodium chloride and / or potassium chloride to the solution containing the small molecule metabolite to be tested obtained in step 1);

[0015] 3) Sample preparation was performed on the mass spectrometry target plate, with 1.5 μL of each sample spotted and dried at room temperature;

[0016] 4) Matrix preparation was performed on the mass spectrometry target plate, with 1.5 μL of each matrix sample spotted and dried at room temperature. The matrix was an inorganic nanomaterial.

[0017] 5) Mass spectrometry is performed on the sample to distinguish isomers. The mass spectrometry is matrix-assisted laser desorption / ionization Fourier transform ion cyclotron resonance mass spectrometry; optionally,

[0018] 6) Perform statistical analysis on the mass spectrometry detection results to obtain quantitative detection results and draw conclusions.

[0019] Preferably, the final concentration of the small molecule metabolites is 5 mM / L, the final concentration of sodium chloride and potassium chloride is 10 mM / L, and the final concentration of the inorganic nanomaterials is 1 mg / mL.

[0020] Preferably, in the quantitative performance assay, the concentration range of the small molecule metabolite is 0.32 μM / L-200 μM / L.

[0021] The detection technology of this invention can: 1) significantly enhance the addition of multiple alkali metal cations to small molecule metabolites, significantly improve the quantitative performance of small molecule metabolite detection in laser desorption / ionization mass spectrometry, and enhance the detection capability of small molecule metabolites; 2) achieve high-throughput and highly reproducible detection of small molecule metabolites in biological fluids; and 3) achieve direct isomer differentiation in primary mass spectrometry by simply comparing the addition of multiple alkali metal cations.

[0022] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0023] Figure 1 These are typical mass spectra of proline in a solution with added sodium chloride (A) and a solution without added sodium chloride (B).

[0024] Figure 2 It is a high-throughput (2364 mass-to-charge ratio features) detection spectrum of small molecule metabolites in 300 serum samples based on enhanced multiple alkali metal cation addition technology;

[0025] Figure 3 It is a detection spectrum based on enhanced multiple alkali metal cation addition technology, which has high reproducibility (median mass-to-charge ratio characteristic coefficient of variation in the range of 11.6-15.0%, and 74.4-77.6% mass-to-charge ratio characteristic coefficient of variation less than 30%) for small molecule metabolites in serum samples;

[0026] Figure 4 This is a first-order mass spectrometry spectrum of O-acetyl-L-serine and glutamic acid based on enhanced multiple alkali metal cation addition technology. Detailed Implementation

[0027] The following description, with reference to the accompanying drawings, illustrates several preferred embodiments of the present invention to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0028] Example 1

[0029] Preparation of instruments and reagents: matrix-assisted laser desorption / ionization Fourier transform ion cyclotron resonance mass spectrometry, small molecule metabolite standards (proline, glutamic acid, serine, threonine, arginine, alanine, isoleucine, phenylalanine and methionine), metal salts (sodium chloride and potassium chloride), deionized water, matrix (inorganic nanomaterials).

[0030] Step 1: Prepare standard molecules of small molecule metabolites (proline, glutamic acid, serine, threonine, arginine, alanine, isoleucine, phenylalanine, and methionine) in mixtures of different salt solutions (sodium chloride and potassium chloride) to enhance the addition of multiple alkali metal cations and improve their quantitative performance in the detection of small molecule metabolites. The final concentration of the small molecule metabolites is 5 mM / L, the final concentration of sodium chloride and potassium chloride is 10 mM / L, and the final concentration of the inorganic nanomaterials is 1 mg / mL. Specifically, in the quantitative detection, the concentration range of the small molecule metabolites is 0.32 μM / L–200 μM / L.

[0031] Step 2: Prepare samples on the mass spectrometry target plate, spotting 1.5 μL of each sample and drying at room temperature;

[0032] Step 3: Prepare the matrix on the mass spectrometry target plate, spotting 1.5 μL of each matrix and drying at room temperature;

[0033] Step 4: Mass spectrometry detection of different small molecules;

[0034] Step 5: Perform statistical analysis on the mass spectrometry detection results and draw conclusions.

[0035] Adding alkali metal cations (Na+) to small molecule metabolites + / K + Salt solutions significantly enhance the addition of multiple alkali metal cations, thereby significantly improving their quantitative performance in the detection of small molecule metabolites.

[0036] Table 1

[0037]

[0038]

[0039] As shown in Table 1, the addition of sodium chloride solution significantly enhanced the multiple alkali metal cation addition of different small molecule metabolites (p<0.05).

[0040] like Figure 1 As shown, compared with the solution without sodium chloride (B), proline in solution A with added sodium chloride (A) exhibits greater multiple alkali metal cation addition ([M+Na)). + [M+H+2Na] +The effect was significantly enhanced after the addition of sodium chloride solution (p<0.05).

[0041] For quantitative detection, the results are shown in Table 2:

[0042] Table 2

[0043]

[0044] The detection and quantification performance of different small molecule metabolites was significantly improved after the addition of sodium chloride solution (p<0.05).

[0045] Example 2

[0046] Preparation of instruments and reagents: matrix-assisted laser desorption / ionization Fourier transform ion cyclotron resonance mass spectrometry, standard serum, small molecule metabolite isomer standards (O-acetyl-L-serine and glutamate), metal salts (sodium chloride and potassium chloride), deionized water, matrix (inorganic nanomaterials).

[0047] By preparing complex biological fluids, standard serum is diluted 10 times and 10 mM / L sodium chloride and potassium chloride solutions are added to achieve high-throughput and highly reproducible detection of small molecule metabolites in biological fluids.

[0048] Based on enhanced multiple alkali metal cation addition, high-throughput and highly reproducible detection of small molecule metabolites in biological fluids can be achieved.

[0049] like Figure 2-3 As shown, based on enhanced multiple alkali metal cation addition, high-throughput (2364 mass-to-charge ratio features) and high reproducibility (median coefficient of variation of mass-to-charge ratio features in the range of 11.6-15.0%, and 74.4-77.6% of the mass-to-charge ratio features have a coefficient of variation of less than 30%) detection of small molecule metabolites in 300 serum samples were achieved.

[0050] Example 3

[0051] Preparation of instruments and reagents: matrix-assisted laser desorption / ionization Fourier transform ion cyclotron resonance mass spectrometry, standard serum, small molecule metabolite isomer standards (O-acetyl-L-serine and glutamate), metal salts (sodium chloride and potassium chloride), deionized water, matrix (inorganic nanomaterials).

[0052] Step 1: Prepare isomer standard molecules (O-acetyl-L-serine, glutamic acid, fumaric acid, and maleic acid) in mixtures of different salt solutions (sodium chloride and potassium chloride) to achieve direct isomer differentiation in primary mass spectrometry. The final concentration of the small molecule metabolites is 5 mM / L, the final concentration of sodium chloride and potassium chloride is 10 mM / L, and the final concentration of the inorganic nanomaterials is 1 mg / mL.

[0053] Step 2: Prepare samples on the mass spectrometry target plate, spotting 1.5 μL of each sample and drying at room temperature;

[0054] Step 3: Prepare the matrix on the mass spectrometry target plate, spotting 1.5 μL of each matrix and drying at room temperature;

[0055] Step 4: Mass spectrometry detection of different small molecules.

[0056] Isomers can be distinguished directly in primary mass spectrometry by simply comparing the addition of multiple alkali metal cations.

[0057] like Figure 4 As shown, isomers can be directly distinguished in primary mass spectrometry by simply comparing the addition of multiple alkali metal cations. For example, for the isomers O-acetyl-L-serine and glutamic acid, the difference between these two isomers can be achieved by simply comparing the difference in the number of addable alkali metal cations in their primary mass spectra (2 for O-acetyl-L-serine and 3 for glutamic acid).

[0058] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A method for small molecule metabolite detection based on enhanced multiple alkali metal ion addition, characterized by, The method comprises the following steps: 1) dissolving the small molecule metabolite to be detected in deionized water; 2) adding sodium chloride and potassium chloride to the solution containing the small molecule metabolite to be detected obtained in step 1), so that the final concentration of the small molecule metabolite is 5 mM / L and the final concentration of the sodium chloride and potassium chloride is 10 mM / L; 3) preparing samples on a mass spectrometry target plate, 1.5 μL of each sample is spotted and dried at room temperature; 4) preparing a matrix on the mass spectrometry target plate, 1.5 μL of each matrix is spotted and dried at room temperature, the matrix is an inorganic nanomaterial; 5) detecting the sample to be detected by using matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry, obtaining a primary mass spectrum; based on the number and peak intensity of the multiple alkali metal ion adducts formed by the small molecule metabolite in the primary mass spectrum, distinguishing the isomers thereof; by comparing the peak intensity of the multiple alkali metal ion adducts, quantitatively analyzing the small molecule metabolite; 6) statistically analyzing the mass spectrometry detection result, obtaining a quantitative detection result, and drawing a conclusion.

2. The method of claim 1, wherein, The small molecule metabolite is an amino acid metabolite; the amino acid is proline, glutamic acid, serine, threonine, arginine, alanine, isoleucine, phenylalanine or methionine.

3. The method of claim 1, wherein, In the quantitative detection, the concentration range of the small molecule metabolite is 0.32 μM / L-200 μM / L.

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