阅读说明：本技术 激光解吸附离子化质谱分析方法和系统 (Laser desorption ionization mass spectrometry method and system ) 是由 欧阳证 杨静 于 2019-11-04 设计创作，主要内容包括：本发明提出了激光解吸附离子化质谱分析方法,所述方法包括：(1)在固体材料表面形成聚多巴胺修饰层,以便得到载样基底；(2)将待测样品施加至所述载样基底的聚多巴胺修饰层表面,再使所述待测样品干燥；以及(3)将所述载样基底进行激光解吸附离子化质谱分析。采用聚多巴胺修饰的固体材料作为激光解吸附离子化的载样基底(靶板),只需一步靶板制备过程,无需添加基质,采用其进行质谱检测及质谱成像具有良好的信噪比、重复性及高空间分辨率,可以实现通用、简单、高效、快速、灵敏的质谱软电离检测及质谱成像分析。(The invention provides a laser desorption ionization mass spectrometry analysis method, which comprises the following steps: (1) forming a polydopamine modification layer on the surface of a solid material so as to obtain a sample-carrying substrate; (2) applying a sample to be detected to the surface of the polydopamine modified layer of the sample loading substrate, and drying the sample to be detected; and (3) carrying out laser desorption ionization mass spectrometry on the sample loading substrate. The solid material modified by polydopamine is used as a sample carrying substrate (target plate) for laser desorption ionization, only one step of the preparation process of the target plate is needed, no matrix is needed to be added, the mass spectrometry detection and mass spectrometry imaging performed by the solid material have good signal-to-noise ratio, repeatability and high spatial resolution, and the universal, simple, efficient, rapid and sensitive mass spectrometry soft ionization detection and mass spectrometry imaging analysis can be realized.)

1. A method of laser desorption ionization mass spectrometry comprising:

(1) forming a polydopamine modification layer on the surface of a solid material so as to obtain a sample-carrying substrate;

(2) applying a sample to be detected to the surface of the polydopamine modified layer of the sample loading substrate, and drying the sample to be detected; and

(3) and carrying out laser desorption ionization mass spectrometry on the sample loading substrate.

2. The method according to claim 1, wherein the polydopamine modifying layer has a thickness of 5 to 50 nm.

3. The method according to claim 1 or 2, wherein the forming of the polydopamine modifying layer is performed by:

and (3) soaking the solid material in a dopamine solution, taking out the solid material and drying.

4. The method according to claim 3, wherein the concentration of the dopamine solution is 0.25-5 mg/ml, and the solvent is selected from aqueous solutions with pH value of 8-9.

5. The method according to claim 3, wherein the soaking time is 1 to 24 hours.

6. The method according to claim 3, wherein the dopamine solution is continuously stirred at 200-500 rpm during the soaking process.

7. A method according to claim 3, wherein the solid material is removed and then washed with deionized water and then dried.

8. The method of claim 1, wherein the solid material comprises at least one of copper, aluminum, iron, stainless steel, polypropylene, polyethylene, polystyrene, and silicon.

9. The method of claim 1, wherein the sample to be tested comprises at least one of amino acids, saccharides, drug molecules, lipids, polymers and polypeptides, and the molecular weight of the sample to be tested is 100-3000 Da;

optionally, the sample to be tested is a lipid comprising at least one of phosphatidylcholine, phosphatidylethanolamine, triglyceride, diglyceride, cholesterol ester and lysophosphatidylcholine;

optionally, the sample to be tested is selected from a biological tissue or a biological fluid;

optionally, the thickness of the biological tissue is 3-50 μm.

10. A system for performing the laser desorption ionization mass spectrometry method of any one of claims 1 to 9, comprising:

a mass spectrometer equipped with a laser desorption ionization source; and

the sample loading substrate is provided with a polydopamine modification layer on the surface.

Technical Field

The invention relates to the technical field of mass spectrometry. In particular, the present invention relates to laser desorption ionization mass spectrometry methods and systems.

Background

Mass Spectrometry Imaging (MSI) is a molecular imaging technology based on mass spectrometry, and by performing mass spectrometry scanning on the surface of a biological tissue slice, spatial distribution and abundance information of various molecules (such as proteins, metabolites and lipids) on the surface of the slice can be obtained, so that the method has important significance in aspects of drug metabolism distribution research, pathological research, discovery of disease biomarkers and the like. Matrix-assisted laser desorption/ionization (MALDI) is the most widely used mass spectrometry imaging technique, and has been widely used in recent years for mass spectrometry analysis of molecules such as polypeptides, proteins, drugs, and lipids. In a conventional MALDI imaging analysis procedure, the basic steps are: tissue sections are prepared and transferred to a MALDI target plate, a suitable organic matrix is selected to be sprayed onto the tissue section surface and dried, and laser irradiation is then used to desorb and ionize the molecules on the tissue section surface. The outstanding problems with this procedure are inaccurate results, poor reproducibility, poor spatial resolution, etc. caused by the displacement, dilution and uneven matrix spray of the matrix solution on the analytes on the tissue sections. In general, MALDI imaging can achieve a spatial resolution of 20-100 μm. The matrix-free LDI mass spectrometry can eliminate the problems of background impurities, chemical information dilution displacement, complicated process and the like caused by the matrix.

Dopamine is catecholamine combined with a catechol group of levodopa and an amino functional group of lysine, and can form a polydopamine composite layer on the surfaces of various materials through spontaneous oxidative polymerization in a mild aqueous phase.

At present, no method for directly modifying polydopamine on a solid material for laser desorption ionization mass spectrometry has been reported.

Disclosure of Invention

The present invention aims to solve at least to some extent at least one of the technical problems of the prior art. Therefore, the invention provides a laser desorption ionization mass spectrum detection method and a system, wherein a solid material modified by polydopamine is used as a sample-carrying substrate (target plate) for laser desorption ionization, only one step of the preparation process of the target plate is needed, no matrix is needed to be added, the mass spectrum detection and mass spectrum imaging performed by the method have good signal-to-noise ratio, repeatability and high spatial resolution, and the universal, simple, efficient, rapid and sensitive mass spectrum soft ionization detection and mass spectrum imaging analysis can be realized.

To this end, in one aspect of the invention, a laser desorption ionization mass spectrometry method is provided. According to an embodiment of the invention, the method comprises: (1) forming a polydopamine modification layer on the surface of a solid material so as to obtain a sample-carrying substrate; (2) applying a sample to be detected to the surface of the polydopamine modified layer of the sample loading substrate, and drying the sample to be detected; and (3) carrying out laser desorption ionization mass spectrometry on the sample loading substrate.

According to the method, only one-time polydopamine modification is needed to be carried out on the solid material, the polydopamine modification layer can promote desorption and ionization of a sample to be detected, and a matrix is not needed to be added, so that the phenomenon of non-uniform co-crystallization with the matrix is avoided, and the repeatability of analysis is improved; the sample application process is simplified, the used matrix does not need to be optimized according to different analytes, and the universality is strong; the poly-dopamine based bionics chemistry has the advantages of convenient synthesis, stability, mildness, good biocompatibility and the like, can form poly-dopamine films on the surfaces of various solid materials, and has strong universality; the porous structure of the polydopamine film and the ultraviolet absorption characteristic near 300nm can effectively enhance the laser desorption ionization efficiency, and is beneficial to LDI mass spectrum imaging of lipid molecules on the surface of biological tissues; the omission of the matrix spraying process can greatly simplify the operation steps of mass spectrum detection and improve the spatial resolution of mass spectrum imaging; the laser desorption ionization analysis can be directly carried out on the biological sample without sample pretreatment and purification processes, and the rapid mass spectrum detection of the sample is realized. The solid material with the polydopamine modification layer has good signal-to-noise ratio, repeatability and high spatial resolution when used for mass spectrometry detection and mass spectrometry imaging, and can realize universal, simple, efficient, rapid and sensitive mass spectrometry soft ionization detection and mass spectrometry imaging analysis.

According to an embodiment of the present invention, the laser desorption ionization mass spectrometry method may further have the following additional technical features:

according to the embodiment of the invention, the thickness of the polydopamine modification layer is 5-50 nanometers. The inventor obtains the optimal thickness of the modified layer through a large number of experiments, so that desorption and ionization of a sample to be detected can be effectively promoted, and normal operation of subsequent mass spectrum detection is not influenced.

According to an embodiment of the present invention, the forming of the polydopamine modifying layer is performed by: and (3) soaking the solid material in a dopamine solution, taking out the solid material and drying. In an aerobic environment, dopamine can be oxidized and polymerized spontaneously to form polydopamine to be modified on the surface of a solid material.

According to the embodiment of the invention, the concentration of the dopamine solution is 0.25-5 mg/ml, wherein the solvent is selected from an aqueous solution with the pH value of 8-9. Therefore, the dopamine can form a polydopamine modification layer with proper thickness (5-50 nanometers) and good membrane uniformity on the surface of a solid material in a weakly alkaline environment, so that desorption and ionization of a sample to be detected can be effectively promoted.

According to the embodiment of the invention, the soaking time is 1-24 hours. Therefore, a poly-dopamine modifying layer with proper thickness (5-50 nanometers) and good membrane uniformity is formed on the surface of the solid material, so that desorption and ionization of a sample to be detected are effectively promoted.

According to the embodiment of the invention, in the soaking process, the dopamine solution is continuously stirred at the rotating speed of 200-500 rpm. Under the condition, the deposition of large polydopamine particles can be prevented, more uniform polydopamine particles can be formed, and better film uniformity is achieved.

According to an embodiment of the invention, the solid material is removed and then washed with deionized water and then dried. Thereby removing surface impurities and dopamine monomers.

According to an embodiment of the present invention, the surface of the solid material may be cleaned with an organic solvent before step (1), such as methanol, isopropanol, water, and the like, and then ultrasonically cleaned respectively.

According to an embodiment of the invention, the solid material contains at least one of copper, aluminum, iron, stainless steel, polypropylene, polyethylene, polystyrene and silicon. The polydopamine can modify the metal material or the nonmetal material, and has wide applicability. In order to further improve the performance of the polydopamine modified material in the field of mass spectrometry detection, polydopamine modification can be performed on the surface of a laser-etched metal sheet. The solid material is obtained by processing with ultrafast pulsed laser, and has a micro/nano composite structure with high light absorption, as disclosed in patent application CN 103627883.

According to the embodiment of the invention, the sample to be detected comprises at least one of amino acid, carbohydrate, drug molecule, lipid, polymer and polypeptide, and the molecular weight of the sample to be detected is 100-3000 Da. Thereby facilitating mass spectrometric detection.

According to an embodiment of the present invention, the sample to be tested is a lipid including at least one of phosphatidylcholine, phosphatidylethanolamine, triglyceride, diglyceride, cholesterol ester and lysophosphatidylcholine. Therefore, the mass spectrum imaging analysis can be carried out on the sample to be detected.

According to an embodiment of the invention, the sample to be tested is selected from a biological tissue or a biological fluid. The biological fluid mainly refers to blood, plasma, urine, sweat, etc. Aiming at biological fluid, the biological fluid can be directly dripped on the surface of the polydopamine modified layer; for biological tissue sections (e.g., mouse brain tissue, mouse liver tissue, etc.), the sections can be transferred to the surface of the polydopamine modified layer. Specifically, the tissue sample was sectioned using a cryomicrotome under cryogenic conditions, and the frozen tissue section was then transferred onto a target plate using a fiber brush and vacuum-dried for 30 min. The method provided by the embodiment of the invention can be used for widely performing mass spectrometry on the sample to be detected.

According to the embodiment of the invention, the thickness of the biological tissue is 3-50 μm. Thereby facilitating mass spectrometry imaging analysis.

In another aspect of the invention, the invention provides a system for performing the laser desorption ionization mass spectrometry method described above. According to an embodiment of the invention, the system comprises: a mass spectrometer equipped with a laser desorption ionization source; and a sample carrying substrate, wherein a polydopamine modifying layer is formed on the surface of the sample carrying substrate. By utilizing the system provided by the embodiment of the invention, a sample to be detected can be directly desorbed and ionized on the polydopamine modification layer, an organic matrix is not required to be added, the mass spectrometry detection and mass spectrometry imaging performed by adopting the system have the advantages of good signal-to-noise ratio, repeatability and high spatial resolution, and the universal, simple, efficient, rapid and sensitive mass spectrometry soft ionization detection and mass spectrometry imaging analysis can be realized.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 shows scanning electron microscope images of polydopamine modified stainless steel sheets at different modification times according to an embodiment of the present invention; wherein a is modified for 30min, b is modified for 1h, c is modified for 2h, d is modified for 5h, e is modified for 10h, and f is modified for 15 h;

FIG. 2 shows a scanning electron microscope image of a polydopamine modified laser etched stainless steel sheet according to an embodiment of the present invention; wherein a is introducing violent oxygen in the modification process, and b is continuously stirring the solution at the rotating speed of 300rpm in the modification process;

FIG. 3 shows a laser desorption ionization mass spectrum of phosphatidylcholine PC 16:0/18:1 using a polydopamine modified stainless steel sheet as a substrate according to one embodiment of the present invention;

FIG. 4 shows a laser desorption ionization mass spectrum of various analytes using a polydopamine modified laser etched stainless steel sheet as a substrate according to one embodiment of the present invention; wherein a is phosphatidylcholine PC 16:0/18:1, b is glucose, c is a mixture of polyethylene glycol PEG 600,1000,2000, and d is angiotensin II;

FIG. 5 shows a laser desorption ionization mass spectrum of human plasma and plasma extracts analyzed using a polydopamine modified laser etched stainless steel sheet as a substrate, according to one embodiment of the present invention; wherein a is human plasma extract and b is human plasma;

FIG. 6 shows the use of a polydopamine modified stainless steel sheet as a substrate for detecting drugs in urine, according to one embodiment of the present invention; wherein a is a primary mass spectrum and b is a secondary mass spectrum of the selected ions;

FIG. 7 shows an atmospheric pressure laser desorption ionization mass spectrum of rat brain tissue analyzed using a polydopamine modified stainless steel sheet, a laser etched stainless steel sheet, and a polystyrene sheet, respectively, as a substrate according to an embodiment of the present invention; wherein a is a stainless steel sheet modified by polydopamine, b is a laser-etched stainless steel sheet modified by polydopamine, and c is a polystyrene sheet modified by polydopamine;

FIG. 8 shows a graph of laser desorption ionization mass spectrometry images of rat brain tissue using a polydopamine modified stainless steel sheet and a laser etched stainless steel sheet as substrates, respectively, according to an embodiment of the present invention; wherein, a is a stainless steel sheet modified by polydopamine, and b is a laser etched stainless steel sheet modified by polydopamine.

Detailed Description

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.