CN106338542A - Method for detecting serum small molecule metabolites by using mass spectrometry - Google Patents

Abstract

The invention discloses a method for detecting serum small molecule metabolites by using a mass spectrometry. The method employs a micro nano granular material as a matrix, uses an internal standard substance for a quantitative analysis, can reach high sensitivity in a wide detection scope, and only requires to consume little micro biological sample; the method can remove the background interference and hot spot effect of the traditional matrix, and can realize the detection of molecule metabolites in a serum sample. Under prerequisite that enrichment and separating processes are not required, the method can rapidly and efficiently perform quantitative determination and analysis on the metabolite in serum.

Description

A method for detecting small molecule metabolites in serum by mass spectrometry

technical field

The invention relates to a molecular detection application technology based on matrix-assisted laser desorption ionization mass spectrometry, in particular to its application in the detection of serum small molecule metabolites.

Background technique

The traditional analysis methods for serum mainly involve proteins, genes and peptides, but there are still a lot of research gaps in the detection and analysis of metabolic molecules. Due to the complexity of the system and the low abundance of metabolic molecules, traditional detection methods are severely limited, thereby limiting their application in medical diagnosis. Compared with traditional detection technologies, such as electrochemical sensors and Raman spectroscopy, mass spectrometry has become a preferred means of detection and analysis due to its high throughput, sensitivity, and ability to carry out molecular identification and structural analysis.

The most commonly used mass spectrometry detection techniques are electrospray ionization and laser desorption ionization. Compared with electrospray ionization, laser desorption ionization has the characteristics of simple sample preparation and high analysis efficiency. But so far, laser analytical ionization still has certain defects in the detection and analysis of biological systems:

(1) Laser desorption ionization is usually time-consuming and labor-intensive in the analysis of biochemical samples, and a large number of steps are required to reduce the complexity of the system.

(2) The mass spectrometry quantitative technology based on laser desorption ionization still needs to further improve the sensitivity, accuracy and detection range, so that it can be better applied to the detection of a large number of molecules.

(3) The traditional organic matrix of mass spectrometry is easy to cause high point-to-point effect and low sample repeatability, and it is easy to generate strong background signal when detecting small molecules, which affects the detection effect.

(4) The use of inorganic substrates can well reduce the background signal and overcome the point-to-point effect, but there are still great limitations in selectivity, sensitivity, accuracy and clinical detection range.

Traditional matrices tend to generate background noise at the small molecular weight end (m/z<1000), which greatly interferes with the detection of small molecules. And in actual biological systems, biological samples are usually very complex. The existence of various biological macromolecules, as well as different pH and salt content will hinder the detection of small molecules. Therefore, traditional matrices are difficult to meet the needs of small molecule detection, and a new type of matrix material that can be used for the detection of biological systems and has certain anti-interference and salt tolerance needs to be developed urgently.

Contents of the invention

In view of the above-mentioned defects of the prior art, the present invention provides a novel particle-assisted mass spectrometry detection technology. By using micro-nano-scale particle materials as the matrix, it overcomes the defects of traditional matrices, and can quickly, high-throughput, and high-sensitivity quantify specific molecules in serum.

Technical scheme of the present invention is as follows:

A method for detecting serum small molecule metabolites by mass spectrometry, comprising the following steps:

Step 1: Preparation of instruments and reagents: laser desorption ionization mass spectrometry, using reflection mode, positive ion detection;

Step 2: preparing a metal oxide particle matrix, including the following steps;

Step 2.1: dissolving metal salt and trisodium citrate in ethylene glycol solution;

Step 2.2: Add sodium acetate to the above mixed solution, and sonicate at room temperature until the solution becomes a homogeneous system;

Step 2.3: The reaction is carried out in a Teflon autoclave, and reacted at 200 degrees Celsius for 10 hours to form metal oxide particles;

Step 2.4: The metal oxide particles obtained in step 2.3 were washed repeatedly with ethanol and deionized water, and finally dried at 60 degrees Celsius for use;

Step 2.5: resuspending the metal oxide particles in deionized water and using them as a matrix;

Step 3: Proportionally dilute the serum sample;

Step 4: Carry out sample preparation on the mass spectrometry target plate, and dry at room temperature;

Step 5: Quantitative analysis and detection of small molecules in serum samples by internal standard method;

Step 6: Analyze the mass spectrometry results and draw conclusions.

Preferably, the metal oxide particles are iron oxide particles.

Preferably, the iron oxide particles have a particle diameter of 50 nm˜1 μm and are uniform in size.

More preferably, the particle size of the iron oxide particles is 200nm-300nm.

Preferably, the iron oxide particles have a rough surface, and the rough surface is composed of nanospheres with a diameter of less than 50 nm.

Preferably, the iron oxide particles are Fe ₂ O ₃ , Fe ₃ O ₄ or a mixture thereof.

Preferably, the iron oxide particles have ultraviolet absorption.

Preferably, the dilution factor of the serum sample is less than or equal to 10000 times.

Preferably, the detection molecular weight range is less than 10000Da.

Preferably, the detected substances include carbohydrates and amino acids.

The beneficial effects of the present invention are: the preparation cost of the micro-nano-scale metal oxide particle matrix is low, it can be produced in large quantities, and the synthesis steps are simple; the micro-nano-scale metal oxide particle matrix can remove the background interference and hot spot effect of the traditional matrix, Has high salt tolerance. The present invention only needs to consume trace amounts of serum, and can quickly and efficiently detect and analyze metabolites in serum quantitatively without enrichment and separation operations; the entire detection process has simple steps, low cost, and high throughput; the obtained The quantitative results have high accuracy and can be applied in clinical practice.

The present invention will be further described below in conjunction with the accompanying drawings, in order to fully illustrate the purpose, technical features and technical effects of the present invention.

Description of drawings

Figure 1 is a characterization picture of iron oxide particles prepared in a preferred embodiment of the present invention, Figure 1a is a SEM characterization picture, and Figure 1b is a TEM characterization picture;

Fig. 2 is the mass spectrogram of glucose standard molecule detected by laser desorption ionization technology in specific embodiment 1;

Fig. 3 is the mass spectrogram of the small molecular weight end of serum detected by laser desorption ionization technology in specific embodiment 2.

detailed description

The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

The preparation of iron oxide particle matrix comprises the following steps:

Step 1: ferric chloride and trisodium citrate are dissolved in ethylene glycol solution;

Step 2: Add sodium acetate to the above mixed solution, and sonicate at room temperature for half an hour until the solution becomes a homogeneous system;

Step 3: The reaction will be carried out in a Teflon autoclave at 200 degrees Celsius for 10 hours to form iron oxide particles;

Step 4: Wash the iron oxide particles obtained in Step 3 with ethanol and deionized water repeatedly, and finally dry them at 60 degrees Celsius for use;

Step 5: Resuspend the iron oxide particles in deionized water and use as a matrix.

Characterization of the matrix:

The instruments used for characterization are: JEOL JEM-2100F instrument was used to obtain transmission electron microscope pictures, high-resolution transmission electron microscope pictures and selected area electron diffraction patterns; silicon wafers were used to prepare scanning electron microscope samples, and Hitachi S-4800 instrument was used to obtain scanning electron microscope pictures; materials at room temperature The ultraviolet absorbance value was obtained by AuCy UV1900 photometer; the hysteresis curve was obtained by vibrating sample magnetometer VSM, Quantum Design, PhysicalProperty Measurement System; the contact angle was obtained by EasyDrop device KRUSS GmbH, Germany; the nitrogen adsorption curve was measured by Micromeritics ASAP 2020M .

The characterization results are:

The obtained particulate material is spherical and has a size of about 250 nm. It can be seen from the transmission electron microscope pictures that the granular material has a subunit structure, and it can also be seen from the high-resolution transmission electron microscope picture obtained by selecting the edge area of the granular material that the regular lattice pattern further proves that the granular material is composed of many nanocrystals. According to the measured scanning electron microscope pictures, it can be seen that the surface of the granular material is rough and uniform in size. It can be known from the nitrogen adsorption curve that the specific surface area of the functional granular material is greater than 100m ² /g, the saturation magnetization is greater than 50emu/g, the surface is hydrophilic, and it has good absorption of ultraviolet rays in the 270-1100nm region. The above structure is an important condition for becoming a good matrix material for mass spectrometry detection of biomolecules.

The application of matrix-assisted laser desorption ionization mass spectrometry in the detection and analysis of serum metabolic molecules will be further clarified through several typical application examples below.

Embodiment 1: the detection of glucose standard substance

(1) Preparation of instruments and reagents: laser desorption ionization mass spectrometer, using reflection mode, positive ion detection; prepared micro-nano iron oxide particles.

(2) Configure a series of glucose standards with concentration gradients.

(3) Sample preparation was carried out on the mass spectrometer target plate and dried at room temperature.

(4) Analyze the obtained mass spectrum image, and the result is shown in FIG. 2 .

Example 2: Detection of metabolic small molecules in serum samples

(1) Preparation of instruments and reagents: laser desorption ionization mass spectrometer, using reflection mode, positive ion detection; prepared micro-nano iron oxide particles.

(2) Dilute the serum sample according to a certain ratio.

(3) Sample preparation was carried out on the mass spectrometer target plate and dried at room temperature.

(4) Analyze the obtained serum mass spectrum image, and the result is shown in FIG. 3 .

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 utilizing mass spectrometry to detect serum small molecule metabolites, is characterized in that, comprises the following steps: Step 1: Preparation of instruments and reagents: laser desorption ionization mass spectrometry, using reflection mode, positive ion detection; Step 2: preparing a metal oxide particle matrix, including the following steps; Step 2.1: dissolving metal salt and trisodium citrate in ethylene glycol solution; Step 2.2: Add sodium acetate to the above mixed solution, and sonicate at room temperature until the solution becomes a homogeneous system; Step 2.3: The reaction is carried out in a Teflon autoclave, and reacted at 200 degrees Celsius for 10 hours to form metal oxide particles; Step 2.4: The metal oxide particles obtained in step 2.3 were washed repeatedly with ethanol and deionized water, and finally dried at 60 degrees Celsius for use; Step 2.5: resuspending the metal oxide particles in deionized water and using them as a matrix; Step 3: Proportionally dilute the serum sample; Step 4: Carry out sample preparation on the mass spectrometry target plate, and dry at room temperature; Step 5: Quantitative analysis and detection of small molecules in serum samples by internal standard method; Step 6: Analyze the mass spectrometry results and draw conclusions. 2 . The method for detecting small molecule metabolites in serum by mass spectrometry according to claim 1 , wherein the metal oxide particles are iron oxide particles. 3 . 3 . The method for detecting small molecule metabolites in serum by mass spectrometry according to claim 2 , wherein the iron oxide particles have a particle diameter of 50 nm to 1 μm and are uniform in size. 4 . 4 . The method for detecting small molecule metabolites in serum by mass spectrometry according to claim 2 , wherein the particle diameter of the iron oxide particles is 200 nm to 300 nm. 5 . The method for detecting small molecule metabolites in serum by mass spectrometry according to claim 2 , wherein the iron oxide particles have a rough surface, and the rough surface is composed of nanospheres below 50 nm. 6 . 6 . The method for detecting small molecule metabolites in serum by mass spectrometry according to claim 2 , wherein the iron oxide particles are Fe ₂ O ₃ , Fe ₃ O ₄ or a mixture thereof. 7. A method for detecting small molecule metabolites in serum by mass spectrometry according to claim 2, wherein the iron oxide particles have ultraviolet absorption. 8. A method for detecting serum small molecule metabolites by mass spectrometry according to claim 1, characterized in that the dilution factor of the serum sample is less than or equal to 10000 times. 9. A method for detecting serum small molecule metabolites by mass spectrometry according to claim 1, characterized in that the detection molecular weight range is less than 10000Da. 10. A method for detecting serum small molecule metabolites by mass spectrometry according to claim 1, characterized in that the detected substances include carbohydrates and amino acids.