CN113447560B - Small molecule metabolite fragmentation control method based on metal ion addition and application - Google Patents

Abstract

The invention discloses a small molecule metabolite fragmentation control method based on metal ion addition, which comprises the steps of mixing a small molecule metabolite solution with a metal ion solution, and realizing small and medium size in the mixed solution through laser desorption ionizationAnd performing metal ion addition on the molecular metabolites, and performing tandem mass spectrometry on the small molecular metabolites added by different metal ions, thereby obtaining the types and the number of the small molecular metabolite fragments added by different metal ions. Metal ion (Li) by alteration of small molecule metabolite adduction ⁺ /Ag ⁺ /Na ⁺ /K ⁺ /Rb ⁺ /Cs ⁺ ) Species, the manner and extent of fragmentation of small molecule metabolites can be controlled, particularly for Li ⁺ And Ag ⁺ And for the addition of two metal ions, the fragment information of the small molecule metabolite is remarkably improved. The invention also discloses application of the metal ion addition small molecule metabolite fragmentation control method in identifying small molecule metabolites, and accurate identification of common small molecule metabolites in complex biological body fluid is realized.

Description

A Fragmentation Control Method and Application of Small Molecule Metabolites Based on Metal Ion Addition

technical field

The invention relates to the field of biochemical detection, in particular to a method and application for controlling fragmentation of small molecule metabolites based on metal ion addition.

Background technique

Mass spectrometry (MS) characterizes molecules directly according to their mass-to-charge ratio (m/z). As one of the most important analytical methods, it has important applications in the fields of disease diagnosis, environmental monitoring, and analytical chemistry. Currently, laser desorption ionization (LDI) and electrospray ionization (ESI) are the most commonly used and most advanced ionization mechanisms. In actual detection applications, compared with electrospray ionization mass spectrometry system (gas/liquid-gas conversion), laser desorption ionization mass spectrometry has the advantages of high throughput and simple sample pretreatment due to its unique solid-gas conversion method. Laser desorption ionization mass spectrometry has been successfully applied to the detection of biological macromolecules (proteins, polypeptides and nucleic acids, etc.). In particular, the application of laser desorption ionization mass spectrometry to small molecule metabolites still attracts a large number of research groups worldwide. However, both experimental and theoretical understanding of the main process of laser desorption ionization of small molecule metabolites is very limited. Therefore, a comprehensive study will greatly advance the development of mass spectrometry in small molecule metabolites, similar to its successful application in large molecules such as proteins and nucleic acids.

As the final product of life activities, small molecule metabolites can reflect the state of living systems in real time. Metabolite profiling is a technique that simultaneously analyzes all small molecule metabolites in actual clinical samples, and has the potential to accurately monitor and even control complex physiological and pathological processes. MS and tandem MS (MS/MS) are basic tools for the analysis of small molecule metabolites, facing the following challenges: I) Establishing the cation-metabolite affinity and multi-cation addition rules during the charge transfer process of cation addition ; II) Characterize the reaction pathway of specific group loss during the fragmentation of adducts; III) Control the way and degree of fragmentation of small molecule metabolites to enhance the identification ability of mass spectrometry in metabolite analysis. Since existing work so far has only partially addressed the above aspects, exploring solutions for all core processes may enhance the analytical capabilities of MS and MS/MS.

At present, there are very few studies, especially systematic studies, on the cation-metabolite affinity during the charge transfer process of cation addition, the law of multi-cation addition, and the reaction pathway of specific group loss during the fragmentation of small molecule metabolites. The exploration of these aspects will help to deepen the understanding of the adduct formation process and secondary fragmentation process of small molecule metabolites. In tandem mass spectrometry, the traditional methods to control the fragmentation of small molecule metabolites mainly include: I) changing the collision particles (argon/carbon dioxide/nitrogen/electron); II) changing the dissociation mode, such as collision-induced dissociation (CID) , Electron capture dissociation (ECD) and other methods; III) Extending the second-order mass spectrometry to multi-stage mass spectrometry and other methods to increase the fragmentation information of small molecule metabolites. However, it has disadvantages such as complex operation, large-scale instrument assistance, difficulty in precisely controlling the degree of fragmentation of small molecule metabolites, and insufficient fragmentation information. Especially in complex biological fluids (such as serum), its fragmentation information may be further reduced, so it is very difficult to accurately identify small molecule metabolites. Therefore, how to simplify the operating procedures and operating devices, control the fragmentation of small molecule metabolites, and realize the effective identification of small molecule metabolites is an urgent technical problem in this field.

Contents of the invention

In view of the above-mentioned defects in the prior art, the present invention realizes the detection of small molecule metabolites by changing the types of metal ions added to the small molecule metabolites (Li ⁺ /Ag ⁺ /Na ⁺ /K ⁺ /Rb ⁺ /Cs ⁺ ). Fragmentation mode and degree of control. Among them, especially for the addition of Li ⁺ and Ag ⁺ metal ions, the fragmentation information of small molecule metabolites has been significantly improved, which effectively solves the defects of small molecule metabolite fragmentation information and difficulty in controlling fragmentation.

In order to achieve the above object, the present invention provides a method for controlling the fragmentation of small molecule metabolites based on metal ion addition, which mainly includes the following steps:

Step 1: Prepare the instrument: set the matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometer to positive ion mode;

Step 2: Prepare at least one small molecule metabolite solution and at least one metal ion solution respectively; at least one of the at least one metal ion solution is selected from lithium ion solution, silver ion solution, sodium ion solution, potassium ion solution solution, a group consisting of rubidium ion solution and cesium ion solution;

Step 3: Mix each small molecule metabolite solution with each metal ion solution to obtain various mixed solution samples;

Step 4: Carry out sample preparation on the mass spectrometer target plate, apply samples to each mixed solution sample, and dry at room temperature;

Step 5: using iron oxide inorganic nanomaterials as a matrix, performing matrix spotting on the dried mass spectrometer target plate with mixed solution samples, mixing the matrix with each mixed solution sample, and drying at room temperature;

Step 6: Add metal ions to the small molecule metabolites in each mixed solution sample by laser desorption ionization, and then perform tandem mass spectrometry detection on the small molecule metabolites added with different metal ions to obtain a tandem mass spectrum;

Step 7: Analyze the tandem mass spectrogram to obtain the fragments and number of small molecule metabolites added with different metal ions.

Further, at least one of the at least one small molecule metabolite in step 2 is selected from glutamine, asparagine, isoleucine, lysine, proline, threonine, methionine, Phenylalanine, Leucine, Valine, Alanine, Arginine, Glucose, Citric Acid, Guanine, Vitamin B6, Cellobiose, Ferulic Acid, Folic Acid, Creatine, _Picolinic Acid, Semi Lactose, group consisting of lactose and uridine.

Further, in the mixed solution sample in step 3, the concentration of the small molecule metabolite is 1 mg/mL.

Further, in the mixed solution sample described in step 3, the concentration of the metal ion is 0.2 mg/mL.

Further, the concentration of the matrix in step 5 is 1 mg/mL.

Further, the sample volume of each mixed solution sample in step 4 is 1 uL, and the sample volume of each matrix in step 5 is 1 uL.

The present invention also provides an application of a metal ion-added small molecule metabolite fragmentation control method in the identification of small molecule metabolites, the application comprising the following steps:

Step 1: Prepare the instrument: set the matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometer to positive ion mode;

Step 2: preparing at least one metal ion solution; at least one of the at least one metal ion solution is selected from lithium ion solution, silver ion solution, sodium ion solution, potassium ion solution, rubidium ion solution and cesium ion solution group;

Step 3: providing a sample containing small molecule metabolites to be detected, and diluting the sample to an appropriate concentration;

Step 4: Mix the diluted sample with the at least one metal ion solution respectively to obtain various mixed solution samples;

Step 5: Spot each mixed solution sample on the mass spectrometer target plate and dry it at room temperature;

Step 6: using iron oxide inorganic nanomaterials as a matrix, performing matrix spotting on the dried mass spectrometer target plate with mixed solution samples, mixing the matrix with each mixed solution sample, and drying at room temperature;

Step 7: Realize metal ion addition of small molecule metabolites in each mixed solution sample by laser desorption ionization, and then perform tandem mass spectrometry detection on small molecule metabolites added with different metal ions to obtain a tandem mass spectrogram;

Step 8: Analyze the tandem mass spectrogram and compare it with the mass spectrogram of the metal ion-added small molecule metabolite of the standard, so as to determine the small molecule metabolite in the sample.

Further, the steps 1, 2 and 3 are in no particular order.

Further, in the mixed solution sample described in step 4, the concentration of the metal ion is 0.2 mg/mL.

Further, the concentration of the substrate in step six is 1 mg/mL.

Further, the sample volume of each mixed solution sample in step 5 is 1uL, and the sample volume of each matrix in step 6 is 1uL.

Further, the samples containing small molecule metabolites to be detected include but not limited to serum, urine, cerebrospinal fluid, aqueous humor, saliva, tears and other common body fluids.

Technical effect of the present invention

1. The present invention can control the mode and degree of its fragmentation by changing the type of metal ion (Li ⁺ /Ag ⁺ /Na ⁺ /K ⁺ /Rb ⁺ /Cs ⁺ ) added to the small molecule metabolite, especially, For the addition of Li+ and Ag+ metal ions, the fragmentation information of small molecule metabolites has been significantly improved, effectively solving the defects of small molecule metabolite fragmentation information and difficulty in controlling fragmentation;

2. The present invention realizes the precise identification of common small molecule metabolites in complex biological fluids, and solves the problem that traditional tandem mass spectrometry techniques cannot effectively identify small molecular metabolites in complex biological fluids;

3. Compared with the traditional technology, this technology is simple to implement, and it is expected to realize large-scale application in different laboratories and industrial production.

Description of drawings

Figure 1 is the results of identification of galactose by different metal ion addition metabolites in simulated serum;

Figure 2 is the result of identifying glucose by adding metabolites of different metal ions in standard serum.

detailed description

A number of preferred embodiments of the present invention are introduced below 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

A method for controlling fragmentation of small molecule metabolites based on metal ion addition, comprising the following steps:

Step 1: Set matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry to positive ion mode;

Step 2: Prepare various small molecule metabolites (glutamine, asparagine, isoleucine, lysine, proline, threonine, methionine, phenylalanine, leucine, valine acid, alanine, arginine, glucose, citric acid, guanine, vitamin B ₆ , cellobiose, ferulic acid, folic acid, galactose, lactose and uridine) solutions, and various metal salts ( Lithium chloride, silver nitrate, sodium chloride, potassium chloride, rubidium chloride, cesium iodide) solution;

Step 3: Mix each small-molecule metabolite solution with each metal salt solution in step 2 to obtain various mixed solution samples; in various mixed solution samples, the final concentration of small-molecule metabolites is 1mg /mL, the concentration of metal salt is 0.2mg/mL;

Step 4: spot each mixed solution sample on the mass spectrometer target plate, spot 1 μL of each sample, and dry at room temperature;

Step 5: Use iron oxide inorganic nanomaterials as a matrix with a concentration of 1 mg/mL, and perform matrix spotting on the dried mass spectrometer target plate with mixed solution samples, mix the matrix with each mixed solution sample, and each matrix Spot 1 μL and dry at room temperature;

Step 6: Add metal ions to the small molecule metabolites in each mixed solution sample by laser desorption ionization, and then perform tandem mass spectrometry detection on the small molecule metabolites added with different metal ions to obtain a tandem mass spectrum;

Step 7: Analyze the tandem mass spectrogram to obtain the fragments and number of small molecule metabolites added with different metal ions.

Table 1. The number of fragments produced by different metabolite small molecules under different metal ion addition conditions

Table 2. Fragments of metabolite small molecules produced by addition of different cations

From Table 1 and Table 2, it can be known that the types and degrees of fragmentation of metal ions (Li ⁺ /Ag ⁺ /Na ⁺ /K ⁺ /Rb ⁺ /Cs ⁺ ) adducted by small molecule metabolites are different. Especially for the addition of Li ⁺ and Ag ⁺ metal ions, compared with other metal ions, the fragmentation information of small molecule metabolites has been significantly improved, which can effectively solve the problem of small molecule metabolite fragmentation information. , problems such as difficulty in controlling fragmentation.

Example 2

This example provides a simple mixed system that simulates complex biological fluids, and realizes accurate identification of small molecule metabolites in the mixed system, including the following steps:

Step 1: Set matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry to positive ion mode;

Step 2: Prepare a simple mixed system, mix five small molecular metabolites of glutamic acid, creatine, galactose, cellobiose and picolinic acid, the concentration of each small molecular metabolite is 1mg/mL; add chloride Sodium (0.2mg/mL), potassium chloride (0.2mg/mL) and bovine serum albumin (5mg/mL) to simulate the high-salt and high-protein environment in complex biological fluids;

Step 3: preparing solutions of various metal salts (lithium chloride, silver nitrate, sodium chloride, potassium chloride, rubidium chloride, cesium iodide);

Step 4: Mix the simple mixing system in step 2 with each metal salt solution in equal proportions to obtain various mixed solution samples; in each mixed solution sample, the concentration of the metal salt is 0.2 mg/mL;

Step 5: Spot each mixed solution sample on the mass spectrometer target plate, spot 1 μL of each sample, and dry at room temperature;

Step 6: Use iron oxide inorganic nanomaterials as a matrix with a concentration of 1mg/mL, and perform matrix spotting on the dried mass spectrometer target plate with mixed solution samples, mix the matrix with each mixed solution sample, and each matrix Spot 1 μL and dry at room temperature;

Step 7: Metal ion addition of small molecule metabolites in each mixed solution sample is achieved by laser desorption ionization, and then tandem mass spectrometry is performed on the small molecule metabolites added with different metal ions to obtain a tandem mass spectrogram;

Step 8: Analyze the tandem mass spectrum and compare it with the mass spectrum of the metal ion-added small molecule metabolites of the standard, so as to determine the small molecule metabolites in the sample.

In a simple mixed system (simulated serum), precise identification of small molecule metabolites can be achieved by controlling the addition of metal ions. The mass spectra of metal ion-added galactose measured in simulated serum (lower part of the mass spectrum in Figure 1a-f) are shown in Figure 1a-f compared with the mass spectra of metal ion-added galactose in the standard (The upper part of the mass spectra in Fig. 1a-f) corresponded perfectly, from which it can be deduced that there are small molecule metabolites of galactose in the simulated serum.

In addition, in simulated serum, Li ⁺ /Ag ⁺ plus parent ions (187.08, 286.97) produced more fragments (>10), and they corresponded well to the corresponding fragments of galactose standard, which can realize the Accurate identification of galactose. However, the addition of Na ⁺ /K ⁺ /Rb ⁺ /Cs ⁺ parent ions (203.05, 219.03, 264.97, 312.97) produces fewer fragments (<2), and it is relatively difficult to accurately identify galactose in simulated serum .

Example 3

This embodiment provides an application to realize the precise identification of small molecule metabolites in serum, including the following steps:

Step 1: Set matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry to positive ion mode;

Step 2: Provide standard serum and dilute the standard serum 10 times;

Step 3: preparing solutions of various metal salts (lithium chloride, sodium chloride, potassium chloride, rubidium chloride);

Step 4: Mix the diluted serum in step 2 with each metal salt solution in equal proportions to obtain various mixed solution samples; in each mixed solution sample, the concentration of the metal salt is 0.2 mg/mL;

Step 5: Spot each mixed solution sample on the mass spectrometer target plate, spot 1 μL of each sample, and dry at room temperature;

Step 6: Use iron oxide inorganic nanomaterials as a matrix with a concentration of 1mg/mL, and perform matrix spotting on the dried mass spectrometer target plate with mixed solution samples, mix the matrix with each mixed solution sample, and each matrix Spot 1 μL and dry at room temperature;

Step 7: Metal ion addition of small molecule metabolites in each mixed solution sample is achieved by laser desorption ionization, and then tandem mass spectrometry is performed on the small molecule metabolites added with different metal ions to obtain a tandem mass spectrogram;

Step 8: Analyze the tandem mass spectrum and compare it with the mass spectrum of the metal ion-added small molecule metabolites of the standard, so as to determine the small molecule metabolites in the sample.

In the actual complex system (standard serum), the precise identification of small molecule metabolites can be achieved by controlling the addition of metal ions. As shown in Figure 2a-d, the mass spectrum of the metal ion-added glucose measured in the standard serum (the lower part of the mass spectrum of Figure 2a-d) is compared with the mass spectrum of the metal ion-added glucose of the standard (Fig. The upper half of the 2a-d mass spectrograms) are completely corresponding, so it can be inferred that there are glucose small molecule metabolites in the standard serum.

In addition, in the standard serum, the number of fragments generated by the addition of Li ⁺ parent ion (187.08) is more (>10), and it can better correspond to the corresponding fragments of the glucose standard, which can realize the accurate identification of glucose in the actual system. However, Na ⁺ /K ⁺ /Rb ⁺ added parent ions (203.05, 219.03, 264.97) produced fewer fragments (<2), and it was relatively difficult to accurately identify glucose in simulated serum.

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 method for controlling fragmentation of small molecule metabolites based on metal ion addition, the method comprising the steps of:

step 1: preparing an instrument: setting the matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrum into a cation mode;

and 2, step: respectively preparing at least one small molecule metabolite solution and at least one metal ion solution; at least one of the at least one metal ion solution is selected from the group consisting of a lithium ion solution, a silver ion solution, a sodium ion solution, a potassium ion solution, a rubidium ion solution, and a cesium ion solution;

and 3, step 3: mixing each small molecule metabolite solution with each metal ion solution to obtain various mixed solution samples;

and 4, step 4: preparing samples on a mass spectrum target plate, spotting each mixed solution sample, and drying at room temperature;

and 5: taking an iron oxide inorganic nano material as a matrix, carrying out matrix spotting on a dried mass spectrum target plate with mixed solution samples, mixing the matrix with each mixed solution sample, and drying at room temperature;

step 6: metal ion addition of small molecule metabolites in each mixed solution sample is realized through laser desorption ionization, and then tandem mass spectrometry detection is carried out on the small molecule metabolites added with different metal ions to obtain a tandem mass spectrogram;

and 7: and analyzing the cascade mass spectrogram to obtain fragments and the number of small molecule metabolites added by different metal ions.

2. The method for controlling fragmentation of a small molecule metabolite based on metal ion addition according to claim 1, wherein at least one of the at least one small molecule metabolite in step 2 is selected from the group consisting of glutamine, asparagine, isoleucine, lysine, proline, threonine, methionine, phenylalanine, leucine, valine, alanine, arginine, glucose, citric acid, guanine, vitamin B ₆ Cellobiose, ferulic acid, folic acid, creatine, picolinic acid, galactose, lactose and uridine.

3. The method for controlling fragmentation of a small molecule metabolite based on metal ion addition according to claim 1, wherein the concentration of the small molecule metabolite in the mixed solution sample in step 3 is 1mg/mL.

4. The method for controlling fragmentation of a small molecule metabolite based on metal ion addition according to claim 1, wherein in step 3 the concentration of the metal ion in the sample of the mixed solution is 0.2mg/mL.

5. The method for controlling fragmentation of a small molecule metabolite based on metal ion addition according to claim 1, wherein the amount of each mixed solution sample spotted in step 4 is 1uL, and the amount of each substrate spotted in step 5 is 1uL.

6. Use of a method for controlling fragmentation of small molecule metabolites based on metal ion addition according to claim 1 for identifying small molecule metabolites, comprising the steps of:

the method comprises the following steps: preparing an instrument: setting the matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrum into a cation mode;

step two: preparing at least one metal ion solution; at least one of the at least one metal ion solution is selected from the group consisting of a lithium ion solution, a silver ion solution, a sodium ion solution, a potassium ion solution, a rubidium ion solution, and a cesium ion solution;

step three: providing a sample to be detected containing a small molecule metabolite, and diluting the sample to a proper concentration;

step four: mixing the diluted sample with the at least one metal ion solution respectively to obtain various mixed solution samples;

step five: spotting each mixed solution sample on a mass spectrum target plate, and drying at room temperature;

step six: taking an iron oxide inorganic nano material as a matrix, carrying out matrix spotting on a dried mass spectrum target plate with mixed solution samples, mixing the matrix with each mixed solution sample, and drying at room temperature;

step seven: metal ion addition of small molecule metabolites in each mixed solution sample is realized through laser desorption ionization, and then tandem mass spectrometry detection is carried out on the small molecule metabolites added by different metal ions to obtain a tandem mass spectrogram;

step eight: and analyzing the cascade mass spectrogram, and comparing the mass spectrogram with the mass spectrogram of the small molecule metabolite added by the metal ions of the standard substance, thereby determining the small molecule metabolite in the sample.

7. The use of claim 6, wherein steps one, two and three are not in sequential order.

8. The use of claim 7, wherein in step four the concentration of said metal ions in said sample of mixed solution is 0.2mg/mL.

9. The use of claim 7, wherein the amount of spotting of each mixed solution sample in step five is 1uL and the amount of spotting of each substrate in step six is 1uL.

10. The use of claim 7, wherein the sample containing small molecule metabolites to be detected comprises serum, urine, cerebrospinal fluid, aqueous humor, saliva and tears.

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