CN116429871A - A Method Based on Enhanced Multiple Alkali Metal Ion Addition for Metabolite Detection - Google Patents

Abstract

The invention discloses a small-molecule metabolite detection technology based on enhanced multiple alkali metal ion addition technology, and relates to the field of metabolomics. The detection method of the small-molecule metabolite includes adding Alkali metal cation salt solution, and then use mass spectrometry to detect the sample. The detection method of the present invention can 1) significantly enhance the addition of multiple alkali metal cations to small molecule metabolites, significantly improve the detection and quantification performance of small molecule metabolites in laser desorption ionization mass spectrometry, and improve the detection ability of small molecule metabolites; 2) realize biological High-throughput and high-reproducibility detection of small molecule metabolites in body fluids; 3) and through simple comparison of multiple alkali metal cation additions, it is possible to directly distinguish isomers in primary mass spectrometry.

Description

A Method Based on Enhanced Multiple Alkali Metal Ion Addition for Metabolite Detection

technical field

The invention relates to the field of metabolomics, in particular to a detection technology for small molecule metabolites based on enhanced multiple alkali metal ion addition technology.

Background technique

Metabolomics is a technique for studying changes in metabolites in organisms, which can reflect real-time changes in phenotypes of biological systems. Among them, mass spectrometry is one of the main tools in metabolomics research, which has the advantages of high sensitivity, high specificity, high resolution and label-free identification. Laser desorption ionization mass spectrometry is a solid-phase mass spectrometry technique. Compared with other mass spectrometry techniques, it has the advantages of simple sample preparation and fast analysis speed, and has great prospects in clinical and other large-scale applications. Ionization is a key step in molecular detection in laser desorption ionization mass spectrometry. Currently, the H ⁺ addition and detection of macromolecular species has been achieved with the assistance of organic substrates. However, in the low molecular weight range (molecular weight less than 1000), the self-ionization of the organic matrix will cause background interference, and its poor co-crystallization with the sample will lead to poor detection reproducibility, which limits its use in the detection of small molecule metabolites. development of. In contrast, the detection of small metabolites using inorganic nanomaterials has the advantages of low background interference and high reproducibility, which has attracted widespread attention worldwide.

Different from the H ⁺ addition of organic substrates, the addition of alkali metal cations (Na ⁺ /K ⁺ ) to inorganic nanomaterials is mainly used for the detection of small molecule metabolites. This is because in biological samples, the concentration of alkali metal cations is high (for example, Na ⁺ and K ⁺ in serum are 135-145 mM and 3.5-5.5 mM, respectively), and they are related to N/O in small molecule metabolites, etc. Atom affinity is strong. During ionization, many significant discoveries have been made regarding the addition of alkali metal cations, such as cation-metabolite affinity, multiple cation addition, and cation adduct formation processes. However, the current detection of small molecule metabolites using alkali metal cation addition still faces two major challenges, including 1) it is difficult to achieve enhanced multiple alkali metal cation addition to improve its quantitative performance in the detection of small molecule metabolites, and 2) It is difficult to use multiple alkali metal cation additions to achieve high-throughput and high-reproducibility detection of small molecule metabolites in biological fluids, and to achieve isomer distinction directly in the first-order mass spectrometer, so as to improve the laser desorption ionization mass spectrometry. Analysis capabilities for small molecule metabolites in complex biological samples.

Therefore, those skilled in the art are committed to developing a technology based on enhanced multiple alkali metal ion addition to achieve high-performance detection of small molecule metabolites.

Contents of the invention

In view of the above-mentioned defects in the prior art, the technical problem to be solved by the present invention is to improve the ability of laser desorption ionization mass spectrometry to analyze small molecule metabolites in complex biological samples.

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

Preferably, the small molecule metabolites are amino acid metabolites; more preferably, the amino acids are proline, glutamic acid, serine, threonine, arginine, alanine, isoleucine, Phenylalanine and methionine.

Preferably, the alkali metal cations are Na ⁺ and/or K ⁺ .

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

Preferably, the mass spectrometer is matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry.

Preferably, the assay is capable of quantifying or isomerically distinguishing the sample.

Preferably, the specific steps of the detection method of small molecule metabolites are:

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

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

3) Prepare samples on the mass spectrometer target plate, apply 1.5 μL of each sample, and dry at room temperature;

4) Prepare the matrix on the mass spectrometry target plate, apply 1.5 μL of each matrix, and dry at room temperature, and the matrix is an inorganic nanomaterial;

5) performing mass spectrometry detection on the sample to be tested to realize the distinction of isomers, and the mass spectrometry is matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry; optionally,

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

Preferably, the final concentration of 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 inorganic nanomaterials is 1 mg/mL.

Preferably, in the quantitative performance test, the concentration range of small molecule metabolites is 0.32 μM/L-200 μM/L.

The detection technology of the present invention can 1) significantly enhance the addition of multiple alkali metal cations to small molecule metabolites, significantly improve the detection and quantification performance of small molecule metabolites in laser desorption ionization mass spectrometry, and improve the detection ability of small molecule metabolites; 2) realize small molecule metabolites in biological fluids High-throughput and high-reproducibility detection of molecular metabolites; 3) and through simple comparison of multiple alkali metal cation additions, it is possible to directly distinguish isomers in primary mass spectrometry.

The idea, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings, so as to fully understand the purpose, features and effects of the present invention.

Description of drawings

Fig. 1 is that proline adds sodium chloride salt solution (A) and does not add sodium chloride solution (B) corresponding typical mass spectrogram;

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

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

Figure 4 is the primary mass spectrum of O-acetyl-L-serine and glutamic acid based on the enhanced multiple alkali metal cation addition technique.

Detailed ways

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

Example 1

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, iso leucine, phenylalanine and methionine), metal salts (sodium chloride and potassium chloride), deionized water, matrix (inorganic nanomaterials).

Step 1: Prepare small molecule metabolite standard molecules (proline, glutamic acid, serine, threonine, arginine, alanine, isoleucine) mixed with different salt solutions (sodium chloride and potassium chloride) acid, phenylalanine, and methionine) to achieve enhanced multiple alkali metal cation addition and improve its quantitative performance in the detection of small molecule metabolites. Among them, the final concentration of small molecule metabolites is 5mM/L, the final concentration of sodium chloride and potassium chloride is 10mM/L, and the final concentration of inorganic nanomaterials is 1mg/mL. In particular, in quantitative performance testing, the concentration range of small molecule metabolites is 0.32μM/L-200μM/L;

Step 2: Prepare samples on the mass spectrometer target plate, apply 1.5 μL of each sample, and dry at room temperature;

Step 3: Prepare the matrix on the mass spectrometry target plate, apply 1.5 μL of each matrix, and dry at room temperature;

Step 4: Perform mass spectrometry detection on different small molecular substances;

Step 5: Perform statistical analysis on the mass spectrometry detection results to draw conclusions.

Adding alkali metal cation (Na ⁺ /K ⁺ ) salt solution to small molecule metabolites can significantly enhance the addition of multiple alkali metal cations, and significantly improve its quantitative performance in the detection of small molecule metabolites:

Table 1

As shown in Table 1, the addition of multiple alkali metal cations of different small molecule metabolites was significantly improved after adding sodium chloride salt solution (p<0.05).

As shown in Figure 1, the addition of multiple alkali metal cations ([M+Na] ⁺ , [M+H +2Na] ⁺ ) was significantly increased (p<0.05) after adding sodium chloride solution.

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

Table 2

After adding sodium chloride salt solution, the detection and quantification performance of different small molecule metabolites was significantly improved (p<0.05).

Example 2

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 glutamic acid), metal salts (sodium chloride and potassium chloride), deionized water, matrix (inorganic nanomaterials).

Prepare complex biological fluids, dilute standard serum 10 times, and add 10mM/L sodium chloride and potassium chloride solutions to achieve high-throughput and high-reproducibility detection of small molecule metabolites in biological fluids.

Based on the enhanced addition of multiple alkali metal cations, high-throughput and high-reproducibility detection of small-molecule metabolites in biological fluids is achieved.

As shown in Figure 2-3, based on the enhanced addition of multiple alkali metal cations, high throughput (2364 features of mass-to-charge ratio) and high reproducibility (in the coefficient of variation of features of mass-to-charge ratio) of small molecule metabolites in 300 serum samples The number of digits is 11.6-15.0%, and the characteristic coefficient of variation of the mass-to-charge ratio of 74.4-77.6% is less than 30%).

Example 3

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 glutamic acid), metal salts (sodium chloride and potassium chloride), deionized water, matrix (inorganic nanomaterials).

Step 1: Prepare isomeric standard molecules (O-acetyl-L-serine, glutamic acid, fumaric acid, and maleic acid) mixed in different salt solutions (sodium chloride and potassium chloride) to achieve direct Isomer differentiation was performed in primary mass spectrometry. Among them, the final concentration of small molecule metabolites is 5mM/L, the final concentration of sodium chloride and potassium chloride is 10mM/L, and the final concentration of inorganic nanomaterials is 1mg/mL.

Step 2: Prepare samples on the mass spectrometer target plate, apply 1.5 μL of each sample, and dry at room temperature;

Step 3: Prepare the matrix on the mass spectrometry target plate, apply 1.5 μL of each matrix, and dry at room temperature;

Step 4: Perform mass spectrometry detection on different small molecular substances.

By simple alignment of multiple alkali metal cation additions, isomeric differentiation can be achieved directly in the first-order mass spectrometer.

As shown in Figure 4, by simply comparing the addition of multiple alkali metal cations, it is possible to distinguish isomers directly in the primary mass spectrometer. For example, for isomers O-acetyl-L-serine and glutamic acid, by simply comparing the number of alkali metal cations that can be added in their primary mass spectra (O-acetyl-L-serine is 2, glutamic acid is 2 3), the distinction between these two isomers can be realized.

The preferred specific embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make many modifications and changes according to the concept of the present invention without creative efforts. 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 experiments on the basis of the prior art shall be within the scope of protection defined by the claims.

Claims (10)

1. A detection method of a small molecule metabolite is characterized by comprising the steps of adding an alkali metal cation salt solution into the small molecule metabolite to be detected, and detecting a sample by using mass spectrometry.

2. The method of claim 1, wherein the small molecule metabolite is an amino acid metabolite.

3. The method according to claim 2, wherein the amino acid is proline, glutamic acid, serine, threonine, arginine, alanine, isoleucine, phenylalanine or methionine.

4. The method according to claim 1, wherein the alkali metal cation is Na ⁺ And/or K ⁺ 。

5. The method according to claim 1, wherein the alkali metal cation salt solution is sodium chloride and/or potassium chloride solution.

6. The method of claim 1, wherein the mass spectrum is matrix-assisted laser desorption ionization fourier transform ion cyclotron resonance mass spectrum.

7. The method of claim 1, wherein the assay is capable of quantifying or isomerically differentiating the sample.

8. The method for detecting a small molecule metabolite according to any one of claims 1 to 7, characterized by comprising the steps of:

1) Dissolving a small molecule metabolite to be detected in deionized water;

2) Adding sodium chloride and/or potassium chloride into the solution containing the small molecule metabolite to be detected obtained in the step 1);

3) Sample preparation is carried out on a mass spectrum target plate, each sample is spotted by 1.5 mu L, and the sample is dried at room temperature;

4) Preparing matrixes on a mass spectrum target plate, wherein each matrix is spotted by 1.5 mu L and dried at room temperature, and the matrixes are inorganic nano materials;

5) Carrying out mass spectrum detection on a sample to be detected to realize the discrimination of isomers, wherein the mass spectrum is matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrum; optionally, the composition may be used in combination with,

6) And carrying out statistical analysis on the mass spectrum detection result to obtain a quantitative detection result, and obtaining a conclusion.

9. The method according to claim 8, wherein the final concentration of the small molecule metabolite is 5mM/L, the final concentration of the sodium chloride and potassium chloride is 10mM/L, and the final concentration of the inorganic nanomaterial is 1mg/mL.

10. The method according to claim 8, wherein the concentration of the small molecule metabolite is in the range of 0.32. Mu.M/L to 200. Mu.M/L in the quantitative performance test.

Images