A MALDI-TOF mass spectrometer integrated with a matrix coating function and its usage method

Abstract

The present invention relates to the technical field of mass spectrometry analysis, and particularly relates to a MALDI-TOF mass spectrometer integrated with a matrix coating function and a method for using the same. The MALDI-TOF mass spectrometer integrated with a matrix coating function includes: a sample inlet and outlet area; an ion flight detection area for detecting samples; a matrix spraying area located between the ion flight detection area and the sample inlet and outlet area for spraying a matrix on samples; an optical system for providing detection laser for the ion flight detection component; a vacuum system for providing a vacuum environment for the ion flight detection area and the matrix spraying chamber; and a data acquisition and imaging system electrically connected to the ion flight detection component for processing mass spectrometry data and imaging. The present invention integrates the three steps of matrix coating, vacuum drying, and mass spectrometry detection and imaging into one, which can effectively reduce the pre-treatment time of samples, reduce the pollution probability of sample transfer between different devices, enable more accurate detection, and has a simple method for using the device.

Description

Technical Field

[0001] The present invention relates to the technical field of mass spectrometry analysis, and particularly relates to a MALDI-TOF mass spectrometer with an integrated matrix coating function and a method for using the same. Background Art

[0002] Mass spectrometry imaging (MSI) is a new technology developed in the field of molecular imaging in recent years. It can not only retain the spatial structure information of tissues, but also detect biomolecules in tissues without labeling in situ, such as proteins, polypeptides, lipids, amino acids, nucleotides, drugs and their metabolites, etc. The working principle of MSI is to apply a suitable ion source to the surface of the sample for desorption ionization, obtain the ion intensities of various molecules in each pixel of the sample, and then use specific imaging software to reconstruct the spatial in-situ distribution information of various molecules in the sample, so as to visualize the mass spectrometry signal. Compared with traditional imaging techniques, mass spectrometry imaging technology has the characteristics of in-situ analysis, no need for labeling, high sensitivity, strong molecular specificity, and the ability to simultaneously detect thousands of biomolecules from tissue samples at one time.

[0003] Since the Caprioli group first reported the use of matrix-assisted laser desorption ionization (MALDI) on biological tissues in 1997, MALDI has become the most widely used soft ionization technology in MSI experiments and has been successfully applied to basic research, drug metabolism, plant biology, food analysis, microbiology, disease diagnosis, and the discovery of clinical biomarkers. MALDI-TOF mainly consists of three parts: an ion source, a time-of-flight mass analyzer, and a detector. Among them, the principle of MALDI is to form a co-crystalline thin film of a trace amount of sample and an excessive amount of small molecule matrix on the target plate. Under the action of a pulsed laser, the matrix absorbs energy from the laser and transfers it to the biomolecule, while transferring a proton to the biomolecule or obtaining a proton from the biomolecule, thereby ionizing and vaporizing the biomolecule. The principle of TOF is that ions are accelerated to fly through the flight tube under the action of an electric field. According to the fact that the flight speed of ions in the flight tube is proportional to the mass-to-charge ratio (m / z) of the ions, the flight times of sample molecules with different m / z values reaching the detector are different, so as to realize the identification of sample molecules with different mass-to-charge ratios. Among them, an environment condition for the free flight of sample ions in the time-of-flight mass analyzer is required, and a vacuum system is needed to provide a vacuum environment for the mass spectrometer.

[0004] The typical workflow of a MALDI-TOF mass spectrometer consists of three steps: preparing tissue sections, acquiring mass spectrometry data, and analyzing imaging data. Among them, in MALDI-TOF mass spectrometry imaging analysis, it is usually required that the matrix can form good co-crystals with the surface molecules of the tissue section. Therefore, the preparation of tissue samples is the basis of MALDI-TOF mass spectrometry imaging technology. In addition, the coverage method of the matrix is one of the keys to obtaining high-quality spectra. Therefore, it is necessary to ensure that there is no diffusion or displacement of the analyte molecules on the tissue section during matrix coverage. Currently, for the detection of a single sample, the spotting method is generally adopted, that is, the matrix solution is directly spotted on the surface of the sample by means of pipette operation. This method is simple and has a low cost, but it is easily affected by human factors, resulting in uneven matrix spraying. For the detection of mass spectrometry imaging samples, a matrix sprayer is often used at present. After spraying, drying treatment is required to ensure the stability of co-crystallization. Finally, it is manually transferred to the mass spectrometer for detection. The co-crystals on the sample surface are easily affected by air oxidation, and there is a risk of artificial contamination during the transfer process. Summary of the Invention

[0005] The purpose of the present invention is to provide an MALDI-TOF mass spectrometer integrated with a matrix coating function and its usage method, which can integrate the three functions of matrix coating, vacuum drying, and mass spectrometry detection into one. Therefore, it can effectively reduce the pre-treatment time of samples, reduce the contamination probability of samples during transfer between different devices, and the detection is more accurate.

[0006] An MALDI-TOF mass spectrometer integrated with a matrix coating function provided by the present invention includes: a working chamber, an optical system, a vacuum system, and a data acquisition and imaging system. The working chamber includes an ion flight detection area and a sample inlet and outlet area. A sample moving stage is provided at the bottom of the working chamber. The working chamber also includes a matrix spraying area provided between the ion flight detection area and the sample inlet and outlet area. An ion flight detection component is provided in the ion flight detection area. A sample inlet and outlet is provided at the position of the sample inlet and outlet area of the working chamber. A matrix spraying chamber is provided in the matrix spraying area. A first spraying chamber inlet and outlet is provided on one side of the matrix spraying chamber close to the sample inlet and outlet area. A second spraying chamber inlet and outlet is provided on one side of the matrix spraying chamber close to the ion flight detection area. The matrix spraying chamber is connected to the vacuum system. The data acquisition and imaging system is electrically connected to the ion flight detection component. The optical system is used to provide detection laser for the ion flight detection component.

[0007] Existing MALDI-TOF mass spectrometers only have detection functions. For the pre-treatment process of imaging samples such as cryosectioning, matrix coating, and vacuum drying, each requires unique equipment. Specifically, for the detection of a single sample, the spotting method is often used currently, which requires manual operation with a pipette. Therefore, it is easily affected by human factors and the matrix spraying is uneven. For the detection of mass spectrometry imaging samples, a matrix spraying instrument is often used. After spraying, the sample needs to be dried. After ensuring co-crystallization, the sample is then transferred to the MALDI-TOF mass spectrometer. Therefore, the sample is easily contaminated during the spraying and even transfer processes, which in turn affects its final imaging, and the detection steps are too many and the operation is complex.

[0008] An MALDI-TOF mass spectrometer integrated with a matrix coating function according to the present invention greatly improves the sample detection efficiency and reduces the pollution probability of sample transfer between different devices by means of the technical method of internally arranging a matrix spraying chamber in the MALDI-TOF mass spectrometer.

[0009] Secondly, since the ion flight detection region in a traditional MALDI-TOF mass spectrometer is a vacuum environment, therefore, setting a matrix spraying chamber for matrix spraying in this environment requires overcoming vacuum technical problems. Specifically, the present invention provides a technical solution of respectively arranging a first spraying chamber inlet and outlet and a second spraying chamber inlet and outlet on both sides of the matrix spraying chamber. When the sample enters the matrix spraying chamber, the vacuum environment is blocked, providing an atmospheric pressure environment for good matrix spraying.

[0010] In addition, the present invention realizes the vacuum state of the matrix spraying chamber after the sample treatment is completed through the technical solution of connecting the matrix spraying chamber with the vacuum system, preparing for the detection of the sample entering the ion flight detection region.

[0011] Preferably, a spraying assembly for matrix spraying the sample is provided in the matrix spraying chamber.

[0012] More preferably, the spraying assembly includes an ultrasonic nozzle.

[0013] The ultrasonic nozzle of the present invention can achieve spot spraying and continuous spraying, so it can be applied to the preparation of different samples and has a wide application range.

[0014] More preferably, the spraying assembly further includes a driving clamping member for moving the ultrasonic nozzle.

[0015] Through the above technical solutions, the present invention can achieve the overall uniform coating function of mass spectrometry imaging samples.

[0016] Preferably, a heating and drying component is further provided in the matrix spraying chamber, and the heating and drying component comprises a heating rod and a heating probe, wherein the heating rod is used to heat the sample, and the heating probe is used to control the temperature in the matrix spraying chamber.

[0017] The present invention adopts the technical solution of built-in heating rods and heating probes in the matrix spraying chamber, and can set a suitable temperature for drying according to the requirements of the sample, so the device has a wide range of applications and is more flexible in operation.

[0018] Preferably, a first vacuum degree detection module is provided in the matrix spraying chamber, and the first vacuum degree detection module is connected to the vacuum system.

[0019] Preferably, the ion flight detection area is provided with a second vacuum degree detection module, and the second vacuum degree detection module is connected to the vacuum system.

[0020] As a further preference, the ion flight detection component includes an ion source, an ion flight tube, and an ion flight detection module, and the ion flight detection module is used to detect mass spectrum data of different ions in the ion flight tube.

[0021] A method for using a MALDI-TOF mass spectrometer with integrated matrix coating function is also provided. Based on the above-mentioned MALDI-TOF mass spectrometer with integrated matrix coating function, the method comprises the following steps:

[0022] S1. The sample enters the sample inlet and outlet area through the sample inlet and outlet, and is moved into the matrix spraying room through the sample moving stage;

[0023] S2, closing the inlet and outlet of the first spray chamber near the sample inlet and outlet area, and the ultrasonic nozzle performs matrix coating on the sample;

[0024] S3. After coating, the sample is vacuum dried by a vacuum system and a heating and drying component;

[0025] S4. After vacuum drying is completed, the inlet and outlet of the second spray chamber near the ion flight detection area are opened, the sample is transferred to the ion flight detection area, and the detection is started through the ion flight detection component;

[0026] S5. After the test is completed, the sample is transferred back to the matrix spraying room and the inlet and outlet of the second spraying room are closed. The vacuum system is used to unload the vacuum in the matrix spraying room. When the matrix spraying room returns to normal pressure, the inlet and outlet of the first spraying room are opened, and the sample is moved back to the sample inlet and outlet;

[0027] S6. The data acquisition imaging system performs imaging based on the mass spectrometry data obtained by the ion flight detection module.

[0028] Preferably, the vacuum operation in the vacuum drying operation in step S3 specifically includes the steps of:

[0029] S3.1. The first vacuum degree detection module detects the first vacuum degree value in the matrix spraying chamber;

[0030] S3.2. The second vacuum degree detection module detects the second vacuum degree value in the ion flight detection area;

[0031] S3.3. The vacuum system performs vacuum operation on the matrix spraying chamber according to the first vacuum degree value and the second vacuum degree value.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] (1) The present invention integrates three functions of matrix coating, vacuum drying and mass spectrometry detection imaging into one, so it can effectively reduce the sample pretreatment time, reduce the pollution probability of sample transfer between different devices, and the detection is more accurate;

[0034] (2) The present invention integrates a closed and fully automated matrix spraying chamber in front of the ion source, realizing the functions of matrix spraying and heating drying of the sample. The matrix spraying is uniform, the imaging effect is good, and the applicable sample range is wide and the operation is flexible;

[0035] (3) The detection of the present invention is more efficient and the equipment operation is simple. Description of the Drawings

[0036] In order to more clearly illustrate the embodiments of the present invention, the drawings will be briefly introduced below:

[0037] Figure 1 It is a schematic structural diagram of a MALDI-TOF mass spectrometer integrating a matrix coating function according to the present invention;

[0038] Figure 2 It is a flowchart of the usage method of a MALDI-TOF mass spectrometer integrating a matrix coating function according to the present invention. Detailed Embodiments

[0039] The present invention will be further described below in conjunction with the drawings of the specification and specific embodiments. Those of ordinary skill in the art will be able to implement the present invention based on these descriptions. In addition, the embodiments of the present invention described below are usually only a part of the embodiments of the present invention, rather than all of the embodiments. Therefore, all other embodiments obtained by those of ordinary skill in the art without creative work based on the embodiments of the present invention shall fall within the protection scope of the present invention.

[0040] Embodiment 1:

[0041] Refer to Figure 1As shown in the figure, a MALDI-TOF mass spectrometer integrating matrix coating function in this embodiment includes a working chamber, an optical system, a vacuum system, and a data acquisition and imaging system. The working chamber includes an ion flight detection area and a sample inlet and outlet area. A sample moving stage is provided at the bottom of the working chamber. In addition, the working chamber further includes a matrix spraying area provided between the ion flight detection area and the sample inlet and outlet area. An ion flight detection component is provided in the ion flight detection area. A sample inlet and outlet is provided at the position of the sample inlet and outlet area of the working chamber. A matrix spraying chamber is provided in the matrix spraying area. A first spraying chamber inlet and outlet is provided on the side of the matrix spraying chamber close to the sample inlet and outlet area, and a second spraying chamber inlet and outlet is provided on the side of the matrix spraying chamber close to the ion flight detection area. The matrix spraying chamber is connected to the vacuum system. The data acquisition and imaging system is electrically connected to the ion flight detection component. The optical system is used to provide detection laser for the ion flight detection component.

[0042] In this embodiment, through the technical method of integrating a matrix spraying chamber, the sample detection efficiency is greatly improved. The sample slices without matrix spraying can be directly placed into the mass spectrometer of this embodiment, and the matrix spraying chamber in the mass spectrometer performs unified spraying of the samples, which reduces the processing time of the samples in the pre-treatment process and also reduces the pollution probability of the samples during transfer between different devices in the pre-treatment process. Therefore, the detection results are more accurate.

[0043] Secondly, in this embodiment, through the technical solutions of setting the first spraying chamber inlet and outlet and the second spraying chamber inlet and outlet, the requirements of the matrix spraying chamber for the matrix spraying environment are solved. At the same time, through the technical solution of connecting the matrix spraying chamber to the vacuum system, vacuum preparation is made for the samples to enter the ion flight detection area in a vacuum state, and the equipment operation is simple.

[0044] Specifically, a spraying component for performing matrix spraying on the samples is provided in the matrix spraying chamber. The spraying component includes an ultrasonic nozzle and a driving clamping member for moving the ultrasonic nozzle. The ultrasonic nozzle has a wide range of applicable samples and can be used for matrix spraying of different samples. The driving clamping member can realize the overall uniform coating function of the ultrasonic nozzle for mass spectrometry imaging samples.

[0045] A heating and drying component is further provided in the matrix spraying chamber. The heating and drying component includes a heating rod and a heating probe. The heating rod is used to heat the samples, and the heating probe is used to control the temperature in the matrix spraying chamber. It can heat and dry the samples after matrix spraying. The equipment operation is simple and the detection is convenient.

[0046] A first vacuum degree detection module is provided in the matrix spraying chamber. The first vacuum degree detection module is connected to the vacuum system and is used to detect the vacuum degree in the matrix spraying chamber.

[0047] The ion flight detection area is provided with a second vacuum degree detection module, which is connected to the vacuum system and used to detect the vacuum degree of the ion flight detection area.

[0048] The ion flight detection component includes an ion source, an ion flight tube, and an ion flight detection module. The ion flight detection module is used to detect the mass spectrometry data of different ions in the ion flight tube.

[0049] Embodiment 2:

[0050] Referring to Figure 2 As shown, this embodiment provides a MALDI-TOF mass spectrometry imaging method integrating matrix coating function. Based on the MALDI-TOF mass spectrometer integrating matrix coating function described in the above Embodiment 1, it includes the following steps:

[0051] S1. The sample enters the sample inlet and outlet area through the sample inlet and outlet, and is moved into the matrix spraying chamber through the sample moving stage;

[0052] S2. Close the first spraying chamber inlet and outlet, and the ultrasonic nozzle coats the sample with the matrix;

[0053] S3. After the coating is completed, vacuum-dry the sample through the vacuum system and the heating and drying component;

[0054] S4. After the vacuum drying is completed, open the second spraying chamber inlet and outlet, transfer the sample to the ion flight detection area by the sample moving stage, and start detection through the ion flight detection component;

[0055] S5. After the detection is completed, transfer the sample back to the matrix spraying chamber by the sample moving stage and close the second spraying chamber inlet and outlet. Vacuum the matrix spraying chamber through the vacuum system. Wait until the normal pressure is restored in the matrix spraying chamber, open the first spraying chamber inlet and outlet, and move the sample back to the sample inlet and outlet by the sample moving stage;

[0056] S6. The data acquisition and imaging system performs imaging based on the mass spectrometry data detected by the ion flight detection module.

[0057] Specifically:

[0058] The vacuum operation in the vacuum drying operation in step S3 of this embodiment includes the steps:

[0059] S3.1. The first vacuum degree detection module detects the first vacuum degree value in the matrix spraying chamber;

[0060] S3.2. The second vacuum degree detection module detects the second vacuum degree value in the ion flight detection area;

[0061] S3.3. The vacuum system performs a vacuum operation on the matrix spraying chamber according to the first vacuum degree value and the second vacuum degree value. Specifically, when the first vacuum degree value and the second vacuum degree value reach consistency, the vacuum extraction of the matrix spraying chamber is stopped.

[0062] It should be noted that an MALDI-TOF mass spectrometer integrating matrix coating function provided in this embodiment is similar to that in Embodiment 1, and will not be described in detail here.

[0063] The invention described above only describes the preferred embodiments of the present invention, and does not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope of the present invention.

Claims

1. An MALDI-TOF mass spectrometer integrating matrix coating function, comprising a working chamber, an optical system, a vacuum system, and a data acquisition and imaging system. The working chamber includes an ion flight detection area and a sample inlet / outlet area. A sample moving stage is provided at the bottom of the working chamber. It is characterized in that It further includes a matrix spraying area provided between the ion flight detection area and the sample inlet / outlet area. An ion flight detection component is provided in the ion flight detection area. A sample inlet / outlet is provided at the sample inlet / outlet area where the working chamber is located. A matrix spraying chamber is provided in the matrix spraying area. A first spraying chamber inlet / outlet is provided on the side of the matrix spraying chamber close to the sample inlet / outlet area. A second spraying chamber inlet / outlet is provided on the side of the matrix spraying chamber close to the ion flight detection area. The matrix spraying chamber is connected to the vacuum system. The data acquisition and imaging system is electrically connected to the ion flight detection component. The optical system is used to provide detection laser for the ion flight detection component. A first vacuum degree detection module is provided in the matrix spraying chamber, and the first vacuum degree detection module is connected to the vacuum system. A second vacuum degree detection module is provided in the ion flight detection area, and the second vacuum degree detection module is connected to the vacuum system.

2. The MALDI-TOF mass spectrometer integrating matrix coating function according to claim 1, characterized in that A spraying component for spraying matrix on the sample is provided in the matrix spraying chamber.

3. The MALDI-TOF mass spectrometer integrating matrix coating function according to claim 2, characterized in that The spraying component includes an ultrasonic nozzle.

4. The MALDI-TOF mass spectrometer integrating matrix coating function according to claim 3, characterized in that The spraying component further includes a driving clamping part for moving the ultrasonic nozzle.

5. The MALDI-TOF mass spectrometer integrating matrix coating function according to claim 1, characterized in that A heating and drying component is further provided in the matrix spraying chamber. The heating and drying component includes a heating rod and a heating probe. The heating rod is used to heat the sample, and the heating probe is used to control the temperature in the matrix spraying chamber.

6. The MALDI-TOF mass spectrometer integrating matrix coating function according to claim 1, characterized in that The ion flight detection component includes an ion source, an ion flight tube, and an ion flight detection module. The ion flight detection module is used to detect the mass spectrometry data of different ions in the ion flight tube.

7. A method for using an MALDI-TOF mass spectrometer integrating matrix coating function, based on the MALDI-TOF mass spectrometer integrating matrix coating function according to any one of claims 1 to 6, characterized in that It includes the following steps: S1. The sample enters the sample inlet / outlet area through the sample inlet / outlet and is moved into the matrix spraying chamber through the sample moving stage; S2. The first spraying chamber inlet / outlet is closed, and the ultrasonic nozzle coats the sample with matrix; S3. After the coating is completed, the sample is vacuum-dried through the vacuum system and the heating and drying component; S4. After the vacuum drying is completed, the second spraying chamber inlet / outlet is opened, the sample is transferred to the ion flight detection area, and the detection starts through the ion flight detection component; S5. After the detection is completed, the sample is transferred back to the matrix spraying chamber and the second spraying chamber inlet / outlet is closed. The matrix spraying chamber is evacuated through the vacuum system. After the normal pressure is restored in the matrix spraying chamber, the first spraying chamber inlet / outlet is opened, and the sample is moved back to the sample inlet / outlet; S6. The data acquisition and imaging system performs imaging based on the mass spectrometry data detected by the ion flight detection module.

8. The method for using an MALDI-TOF mass spectrometer integrating matrix coating function according to claim 7, characterized in that The vacuum operation in the vacuum drying operation in step S3 specifically includes the steps: S3.

1. The first vacuum degree detection module detects the first vacuum degree value in the matrix spraying chamber; S3.

2. The second vacuum degree detection module detects the second vacuum degree value in the ion flight detection area; S3.

3. The vacuum system performs vacuum operation on the matrix spraying chamber according to the first vacuum degree value and the second vacuum degree value.

Images