Method for detecting a compound of interest in a sample based on filter cartridge filtration and MALDI-TOF MS

Abstract

The application discloses a method for detecting a compound of interest in a sample based on filter core filtering and MALDI-TOF MS, which comprises the following steps: S1, selecting a target plate with filtering function; S2, uniformly mixing a sample with medium particles with adsorption groups to form a suspension, and the adsorption groups on the medium particles are combined with the compound of interest in the sample during the mixing process; S3, transferring the suspension to the target plate with filtering function to perform filtering, so that the medium particles and the compound of interest combined with the medium particles are intercepted on the target plate with filtering function; S4, covering a matrix on the medium particles and the compound of interest combined with the medium particles; and S5, placing the target plate with filtering function covered with the matrix into a MALDI-TOF MS system to perform detection. The application adopts the medium particles to adsorb the compound of interest in the sample, and combines the target plate with filtering function to intercept the medium particles and the compound of interest, so that high-sensitivity detection of the compound of interest can be realized.

Description

Technical Field

[0001] This application relates to the field of MALDI-TOF sample detection technology, and in particular to a method for detecting compounds of interest in samples based on cartridge filtration and MALDI-TOFMS. Background Technology

[0002] Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) has high sensitivity and a high-quality detection range, capable of detecting relative molecular masses up to hundreds of thousands. As a type of "soft ionization" mass spectrometry, it has the advantages of samples that are not easily broken and strong molecular ion peaks. It solves the problem of desorption and ionization of non-volatile and thermally unstable biomolecules, and has powerful analytical capabilities, and has been widely used in the field of medical diagnostics.

[0003] Each disease produces a series of specific proteins and other compounds during its occurrence and development. MALDI-binding target plates are currently a commonly used technique for detecting these compounds. The detection method is as follows: First, the target plate is chemically modified to attach adsorption groups to the target sites. Then, the sample solution to be tested is spotted onto the target sites with adsorption groups. The proteins and other compounds in the sample solution bind to the adsorption groups. Because the target site diameter is 1-2 mm, which is small and only aggregates on the target site surface, the adsorption groups cannot fully contact the proteins and other compounds. Therefore, the target plate needs to be shaken on a shaker for a long time. For example, for serum samples from COVID-19 infected individuals, it needs to be shaken for 6 hours to allow the proteins and other compounds of interest to fully contact the adsorption groups and bind well. After several tens of minutes of shaking to remove interfering substances, the target plate is removed, and the matrix is ​​covered on the target sites. After drying, matrix-assisted laser desorption / ionization time-of-flight mass spectrometry is used to acquire and process the spectrum for analysis of the sample. However, the above detection method requires chemical modification of the target plate, which is a relatively complex manufacturing process and has a high cost. Moreover, in order to make the protein and other compounds bind well with the adsorption groups, the target plate needs to be shaken on a shaker for several hours and then rinsed for more than ten minutes, resulting in a long detection time.

[0004] Currently, solid-phase extraction (SPE) columns are also used to avoid chemical modification of the target plate and shaking. After adsorbing proteins and other compounds using SPE columns, elution is required, and the eluted proteins and other compounds are then spotted onto the target sites on the target plate. This method requires a large sample volume, and the necessary elution step results in a large liquid volume, necessitating concentration, which is cumbersome. During elution, due to the presence of strongly adsorbed compounds, some compounds of interest may not be eluted, leading to the loss of important information and affecting the accuracy of the detection results. Summary of the Invention

[0005] To address at least one of the aforementioned technical problems and to develop a detection method that is short in detection time, low in cost, and relatively simple, this application provides a method for detecting compounds of interest in samples based on filter cartridge filtration and MALDI-TOF MS.

[0006] The method for detecting compounds of interest in samples based on cartridge filtration and MALDI-TOF MS provided in this application includes the following steps: S1. Select a target plate with filtering function and set it aside; S2. The sample is mixed evenly with medium particles having adsorption groups to prepare a suspension. During the mixing process, the adsorption groups on the medium particles combine with the compound of interest in the sample. S3. The suspension is transferred to the target plate with filtration function for filtration, so that the media particles and the compound of interest combined with the media particles are trapped on the target plate with filtration function. S4. Cover the medium particles and the compound of interest that is combined with the medium particles with the matrix to obtain a sample loading target plate; S5. Place the sample target plate into the MALDI-TOF MS system for detection.

[0007] By adopting the above technical solution, this application abandons the relatively complex processes of chemically modifying the target plate and eluting the compound of interest required by existing detection methods. Instead, it uses medium particles to adsorb the compound of interest in the sample, and combines a target plate with a filtering function to retain the medium particles and the compound of interest bound to the medium particles. This enables highly sensitive detection of the compound of interest, and the detection method is relatively simple, requires less time, and has lower cost.

[0008] Optionally, in step S1, the target plate with filtration function includes a target plate body and a filter element. The target plate body is provided with perforations, and the filter element is installed in the perforations.

[0009] By adopting the above technical solution, the structure is relatively simple, and the filter element can trap media particles in the mixture as well as the compounds of interest that are bound to the media particles.

[0010] Optionally, the filter element has filter pores, the pore size of which is smaller than the size of the medium particles with adsorption groups.

[0011] By adopting the above technical solution, the filter pores can effectively filter out the filtrate and retain the media particles in the suspension as well as the compounds of interest that are bound to the adsorption groups of the media particles.

[0012] Optionally, the filter element is a PP cotton filter element.

[0013] Optionally, the medium particles may be activated before step S2.

[0014] By employing the above technical solution to activate the medium particles, the adsorption groups on the medium particles can be better bound to the protein molecules in the sample.

[0015] Optionally, the activation process can be performed by immersing the media particles in methanol, filtering them after thorough immersion, and rinsing the media particles with a buffer solution.

[0016] Optionally, the buffer solution is a sodium acetate buffer solution, the pH of the buffer solution is 4 to 6, and the molar concentration of the buffer solution is 90 to 110 mmol / L.

[0017] Optionally, in step S2, the medium particles are one of weak cation exchange resin, weak anion exchange resin, strong cation exchange resin, strong anion exchange resin, and carbon nanotubes.

[0018] Optionally, before step S2, the sample may be pretreated, and the sample may be a serum sample, a blood sample, or a urine sample. The pretreatment method is to centrifuge the sample at 800-1000 r / min and 4-6℃ for 3-5 min, and then take the supernatant.

[0019] Optionally, in step S2, the sample and the medium particles are mixed evenly in a vortex mixer.

[0020] By adopting the above technical solution, the sample and medium particles can be mixed more thoroughly, thereby allowing the adsorption groups in the medium particles to bind more fully with the compounds of interest in the sample.

[0021] In summary, the present invention has at least one of the following beneficial technical effects: 1. This application abandons the complex processes of existing detection methods, which require chemical modification of the target plate and elution of the compound of interest. Instead, it uses medium particles to adsorb the compound of interest in the sample, and combines a target plate with a filtering function to retain the medium particles and the compound of interest that binds to the adsorption groups of the medium particles. This enables highly sensitive detection of the compound of interest, and the detection method is relatively simple, requires less time, and has a lower cost.

[0022] 2. The sample and media particles are mixed in a vortex mixer, which allows the adsorption groups in the media particles to bind more fully with the compounds of interest in the sample.

[0023] 3. The target plate with filtration function has a relatively simple structure. The filter element installed on the target plate can effectively trap media particles in the mixture and the compounds of interest that are bound to the media particles. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the target plate with filtering function provided in this application; Figure 2 This is a schematic diagram of the structure of the matrix covering the compound of interest provided in this application; Figure 3 This is a comparison chart of the detection results obtained by testing serum samples from patients with thrombotic thrombocytopenic purpura using existing detection methods and the detection method provided in Example 1. Figure 4 This application provides protein fingerprints of COVID-19 infected patients and healthy individuals. Explanation of reference numerals in the attached figures: 1. Target plate body; 11. Perforation; 2. Filter element; 3. Media particles; 4. Compound of interest; 5. Matrix. Detailed Implementation

[0025] The present application will be further described in detail below with reference to the accompanying drawings and embodiments.

[0026] This application designs a method for detecting compounds of interest in samples based on cartridge filtration and MALDI-TOF MS, including the following steps: S1. Select a target plate with filtering function and set it aside; S2. The sample is mixed evenly with medium particles having adsorption groups to prepare a suspension. During the mixing process, the adsorption groups on the medium particles combine with the compound of interest in the sample. S3. The suspension is transferred to the target plate with filtration function for filtration, so that the media particles and the compound of interest combined with the media particles are trapped on the target plate with filtration function. S4. Cover the medium particles and the compound of interest that is combined with the medium particles with the matrix to obtain a sample loading target plate; S5. Place the sample target plate into the MALDI-TOF MS system for detection.

[0027] This application abandons the complex processes required by existing detection methods, such as chemical modification of the target plate and elution of the compound of interest. Instead, it uses medium particles to adsorb the compound of interest in the sample, and combines a target plate with a filtering function to retain the medium particles and the compound of interest bound to the medium particles. This enables highly sensitive detection of the compound of interest, and the detection method is simple, requires less time, and is less expensive.

[0028] Example 1 The method for detecting compounds of interest in samples based on cartridge filtration and MALDI-TOF MS includes the following steps: S1. Select a target plate with filtering function. like Figure 1 As shown, the target plate with filtration function includes a target plate body 1 and a filter element 2. The target plate body 1 is made of stainless steel and has 48 perforations 11 with a diameter of 2mm. The filter element 2 is a PP cotton filter element that has undergone hydrophilic treatment to give it hydrophilic properties. The radial height of the filter element 2 is 2mm, and the axial length of the filter element 2 is 3mm. The axial length of the filter element 2 is slightly... The filter element 2 has a filter hole with a diameter of 0.5 μm, which is larger than the aperture of the perforation 11. During installation, the filter element 2 is horizontally inserted into the upper part of the perforation 11, and the radial end face of the filter element 2 is tightly attached to the inner sidewall of the perforation 11. The axial length of the filter element 2 is slightly larger than the aperture of the perforation 11. The filter element 2 undergoes slight deformation without affecting the filtration effect, so that the filter element 2 is stably installed in the perforation 11. It is easy to install and remove. One filter element 2 is installed in each perforation 11.

[0029] S2, Activation treatment of media particles The medium particles are weak cation exchange resins, such as carboxymethyl cation exchange resins. The medium particles are spherical in shape, with an average particle size of 2.5 μm, which is larger than the pore size of the filter. During activation, 0.1 mg of medium particles are immersed in 99.9% pure methanol for 5 seconds, then filtered. The medium particles are then rinsed with buffer solution for 5 seconds to obtain activated medium particles. After activation, the adsorption groups on the medium particles can bind well to the compound of interest. The buffer solution is sodium acetate buffer solution with a pH of 5 and a molar concentration of 90 mmol / L.

[0030] S3. Sample preprocessing Centrifuge the serum sample at 800 r / min and 6℃ for 5 min, and then take 5 mL of the supernatant.

[0031] S4, Mix All activated media particles from step S2 and the supernatant from step S3 are added to containers respectively. The containers are placed in a vortex mixer and shaken for 3 minutes to ensure that the supernatant and activated media particles are fully mixed to obtain a mixture. The shaking treatment allows the adsorption groups on the media particles to fully combine with the compounds of interest in the supernatant.

[0032] S5, Filter Use a pipette to remove all the mixture prepared in step S4, and then transfer the mixture from the pipette to the 6 filter cartridges 2 in step S1. The amount of mixture transferred to each filter cartridge 2 is the same. After the mixture is filtered by the filter cartridge 2, the media particles and the compounds of interest that are bound to the adsorption groups on the media particles are retained on the filter cartridge 2.

[0033] S6, Add matrix Using a pipette, matrix 5 was applied to compounds of interest 4 that were bonded to adsorption groups on media particles 3, such as... Figure 2 As shown, the matrix 5 is used to cover the medium particles 3 and the compound of interest 4 which is bound to the adsorption groups on the medium particles 3, and then dried at room temperature.

[0034] S7, Mass Spectrometry Detection The target plate covered with the matrix in step S6 was placed into the Ebio Reader 3700 time-of-flight mass spectrometry system for spectral acquisition, processing, and analysis.

[0035] Example 2 The difference between Example 2 and Example 1 is that: In step S1, the diameter of the perforation 11 is 5mm, the number of perforations 11 is 24, the axial length of the filter element 2 is 7mm, and the diameter of the filter holes of the filter element 2 is 2.5um. In step S2, the average particle size of the medium particles is 5 μm. During the activation treatment, 0.3 mg of medium particles are immersed in methanol, and the medium particles are rinsed with buffer solution for 6 seconds. The pH of the buffer solution is 4.

[0036] In step S3, the serum sample is centrifuged at 1000 r / min and 4℃ for 5 min, and then 25 mL of supernatant is taken.

[0037] In step S4, the container is oscillated in a vortex mixer for 5 minutes.

[0038] In step S5, the mixture in the pipette is transferred to 12 filter cartridges, and the amount of mixture transferred to each filter cartridge 2 is the same.

[0039] Example 3 The difference between Example 3 and Example 1 is that: In step S1, the diameter of the perforation 11 is 3mm, the number of perforations 11 is 12, the radial height of the filter element 2 is 2.2mm, the axial length of the filter element 2 is 4.5mm, and the diameter of the filter holes of the filter element 2 is 6um. In step S2, the average particle size of the medium particles is 9 μm. During the activation treatment, 0.7 mg of medium particles are immersed in methanol for 6 seconds. The medium particles are rinsed with buffer solution for 4 seconds. The pH of the buffer solution is 6 and the molar concentration of the buffer solution is 110 mmol / L.

[0040] In step S3, the serum sample is centrifuged at 900 r / min and 6℃ for 3 min, and then 10 mL of supernatant is taken.

[0041] In step S4, the container is oscillated in a vortex mixer for 4 minutes.

[0042] In step S5, the mixture in the pipette is transferred to eight filter cartridges, and the amount of mixture transferred to each filter cartridge 2 is the same.

[0043] Example 4 The difference between Example 4 and Example 1 is that: In step S1, the diameter of the perforation 11 is 8mm, the number of perforations 11 is 8, the radial height of the filter element 2 is 2.5mm, the axial length of the filter element 2 is 10mm, and the diameter of the filter holes of the filter element 2 is 8um. In step S2, the medium particles are strong cation exchange resins with an average particle size of 12 μm. During the activation treatment, 1 mg of medium particles are immersed in methanol for 4 seconds, and the molar concentration of the buffer solution is 100 mmol / L.

[0044] In step S3, the serum sample is centrifuged at 850 r / min and 5℃ for 4 min, and then 20 mL of supernatant is taken.

[0045] In step S4, the container is oscillated in a vortex mixer for 4 minutes.

[0046] In step S5, the mixture in the pipette is transferred to eight filter cartridges, and the amount of mixture transferred to each filter cartridge 2 is the same.

[0047] In another embodiment, the difference from Embodiment 1 is that, in step S2, the medium particles can also be selected as weak anion exchange resin, strong cation exchange resin, strong anion exchange resin or carbon nanotubes as needed. This is prior art and will not be described in detail here.

[0048] In other embodiments, the difference from Embodiment 1 is that, in step S3, the sample may be a plasma sample or a urine sample.

[0049] Precision testing The serum samples were from patients with thrombotic thrombocytopenic purpura. The serum samples were divided into two portions, both originating from the same patient. One serum sample was analyzed using an existing detection method, namely the chemically modified target plate method mentioned in the background section. The other serum sample was analyzed using the method described in Example 1. The operating conditions of the mass spectrometry system were as follows: Ebio Reader 3700 time-of-flight mass spectrometry system, linear positive ion mode, laser intensity 68%, ion acceleration voltage 20 kV, and ion delay extraction time 80 ns. The detection results are as follows: Figure 3 As shown, from Figure 3 It is evident that the detection method provided in this application has high accuracy.

[0050] Application testing Serum samples P1 to P6 were detected using the detection method described in Example 1 above. Serum samples P1 and P2 were obtained from serum of different healthy individuals, serving as a control group. Serum sample P3 was obtained from serum of a patient nearing recovery from COVID-19 infection. Serum samples P4 to P6 were obtained from serum samples of different COVID-19 patients. In step S7, the parameters of the Ebio Reader 3700 time-of-flight mass spectrometry system were set as follows: linear positive ion mode, laser intensity 68%, ion acceleration voltage 20 kV, and ion delay extraction time 80 ns. The obtained protein fingerprint spectrum is shown below. Figure 4 As shown, from Figure 4 It can be seen that the four protein peaks of 5905 Da, 6781 Da, 7885 Da, and 8115 Da can be used to distinguish between patients infected with the novel coronavirus, patients who are close to recovery from the novel coronavirus infection, and healthy people.

[0051] The above are all preferred embodiments of this application, and are not intended to limit the scope of protection of this application. Therefore, all equivalent changes made in accordance with the structure, shape and principle of this application should be covered within the scope of protection of this application.

Claims

1. A method for detecting compounds of interest in samples based on cartridge filtration and MALDI-TOF MS, characterized in that, Includes the following steps: S1. Select a target plate with filtering function and set it aside; The target plate with filtration function includes a target plate body and a filter element. The target plate body is made of stainless steel and has perforations. The filter element is a PP cotton filter element, which has undergone hydrophilic treatment to give it hydrophilic properties. The filter element is laterally inserted into the upper part of the perforation, and the radial end face of the filter element is in close contact with the inner sidewall of the perforation. The filter element has filter pores, and the pore size of the filter pores is smaller than the size of the medium particles with adsorption groups. S2. The sample is mixed evenly with medium particles having adsorption groups to prepare a suspension. During the mixing process, the adsorption groups on the medium particles combine with the compound of interest in the sample. The media particles with adsorption groups are carboxymethyl cation exchange resin. The process of mixing the sample with the media particles with adsorption groups includes: immersing 0.1 mg of the media particles with adsorption groups in 99.9% pure methanol for 5 seconds, filtering, and rinsing the media particles with adsorption groups with a buffer solution (90 mmol / L sodium acetate buffer, pH 5) for 5 seconds to obtain activated media particles; the sample is a serum sample, centrifuged at 800 rpm for 5 minutes at 6°C, and 5 mL of supernatant is collected; the activated media particles and supernatant are added to a container and vortexed for 3 minutes. S3. The suspension is transferred to the target plate with filtration function for filtration, so that the media particles and the compound of interest combined with the media particles are trapped on the target plate with filtration function. S4. Cover the medium particles and the compound of interest that is combined with the medium particles with the matrix to obtain a sample loading target plate; S5. Place the sample target plate into the MALDI-TOF MS system for detection.

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