Automatic protein pretreatment detection kit based on magnetic beads and application thereof

Abstract

The invention discloses an automatic protein pretreatment detection kit based on magnetic beads, the kit is used for all pretreatment steps of protein extraction, reductive alkylation, proteolysis, peptide fragment desalination, peptide fragment redissolution and the like, and the treated peptide fragment can be directly loaded on a mass spectrum. After the kit is matched with automatic equipment, enzymolysis and desalting of 96 samples can be achieved within 4 h, the pretreatment speed is greatly increased, the labor cost is reduced, and meanwhile compared with manual operation, the kit has higher precision, higher stability, higher efficiency and the like. The pretreatment detection kit is suitable for all sample types, has no preference, such as cells, blood, tissues, plants, fungi and the like, and is also suitable for most of protein extraction reagents in the market, such as SDS, urea, SDC, RIPA, NP-40 and the like.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to an automated protein pre-treatment detection kit based on magnetic beads and an application thereof in proteomics based on mass spectrometry. Background Art

[0002] Mass spectrometry-based proteomics is a large-scale qualitative or quantitative study of all proteins in biological systems using liquid chromatography-tandem mass spectrometry (LC-MS / MS) technology. At present, mass spectrometry-based proteomics is mainly divided into three modules: module one is biological sample pretreatment: including protein extraction, enzymatic hydrolysis, desalting, classification and other sample preparation steps; module two is liquid chromatography-tandem mass spectrometry detection and the development of its methods; module three is the search and analysis of mass spectrometry data and bioinformatics statistical analysis. In recent years, with the continuous updating and iteration of mass spectrometry instruments, from the initial Q-Exacive series of mass spectrometers to open a new stage of proteomics research, to the 4D-based tims-TOF Pro series of mass spectrometers, and now the advent of higher-throughput mass spectrometers such as Astral and tims-TOF HT, it is easy to detect 180 samples a day, greatly improving the throughput of mass spectrometry detection. On the other hand, more and more downstream bioinformation retrieval software can also realize real-time library search functions, and greatly improve the speed of proteomics data analysis through the upgrade of servers and computer hardware. On the other hand, at present, the pre-treatment technology of the entire proteomics still relies more on high-quality professional and technical personnel, and the pre-treatment of most proteomics research institutions is different, and most of them are manual operations. Furthermore, the currently commonly used pre-treatment process is cumbersome, and the enzymatic hydrolysis step requires overnight enzymatic hydrolysis. Therefore, protein pre-treatment has become a key rate-limiting step in the entire proteomics process, which cannot match the mass spectrometry detection flux, greatly limiting the entire proteomics detection flux. Summary of the invention

[0003] In order to solve the deficiencies in the above-mentioned prior art, the present invention proposes an automated protein pre-treatment detection kit based on magnetic beads, which is used for all sample pre-treatment steps such as protein extraction, reduction alkylation, proteolysis, peptide desalting, peptide redissolution, etc., and the treated peptides can be directly put on the mass spectrometer. The kit of the present invention is used to carry out a proteomics sample pre-treatment experiment based on mass spectrometry, and the entire operation process only takes 5 hours, the operation is simple and easy to use, and the throughput is high. After the kit of the present invention is adapted to the automated equipment, the enzymatic hydrolysis and desalting of 96 samples can be achieved in 4 hours, which greatly improves the pre-treatment speed and reduces the labor cost. At the same time, compared with manual operation, it has higher precision, higher stability, higher efficiency, etc. The pretreatment detection kit of the present invention is applicable to all sample types without preference, such as cells, blood, tissues, plants, fungi, etc., and can also be adapted to most protein extraction reagents on the market, such as sodium dodecyl sulfate (SDS), urea (urea), sodium deoxycholate (SDC), radioimmunoprecipitation assay lysis buffer (RIPA), ethylphenyl polyethylene glycol (NP-40), etc.

[0004] The present invention provides an automated protein pretreatment detection kit based on magnetic beads. The kit comprises an extractant, a magnetic bead solution, a replacement solution, a cleaning solution 1, a cleaning solution 2, a reaction solution 1, a reaction solution 2, an activation solution, a balancing solution, an eluent, and a desalting plate.

[0005] The extractant in the kit of the present invention is used for protein extraction, the magnetic bead solution, the replacement solution, the cleaning solution 1 and the cleaning solution 2 are used for enriching the protein on the magnetic beads and washing away the extraction reagent, the reaction solution 1 is used for the reduction alkylation of the protein, the reaction solution 2 is used for the enzymatic hydrolysis of the protein, and the activation solution, the balancing solution, the eluent and the desalting plate are used for the desalting and re-dissolving of the peptide segments.

[0006] In the present invention, the extractant is composed of a mixed aqueous solution of SDS, tris(hydroxymethylaminomethane) (Tris), sodium pyrophosphate, β-glycerophosphate, sodium orthovanadate, sodium fluoride, ethylene diaminetetraacetic acid (EDTA), and leupeptin; wherein the mass volume ratio concentration of SDS is 1% to 4%, the concentration of Tris is 50mM, the concentration of sodium pyrophosphate is 2.5mM, the concentration of β-glycerophosphate is 1mM, the concentration of sodium orthovanadate is 1mM, the concentration of sodium fluoride is 10mM, the concentration of ethylenediaminetetraacetic acid is 1mM, and the concentration of leupeptin is 0.5μg / μL; preferably, the mass volume ratio concentration of SDS is 4%.

[0007] In the present invention, the volume of the extractant is 30 mL.

[0008] In the present invention, the magnetic beads are Sera-Mag magnetic beads with a diameter of 1 μm and surface modified with carboxyl groups.

[0009] In the present invention, the concentration of the magnetic bead solution is 50 μg / μL; the volume of the magnetic bead solution is 1 mL.

[0010] In the present invention, the replacement fluid is acetonitrile; the volume of the replacement fluid is 20 mL.

[0011] Wherein, the concentration of acetonitrile is 100%.

[0012] In the present invention, the cleaning solution 1 is composed of ethanol and water; wherein the volume ratio of the ethanol:water is 80:20.

[0013] In the present invention, the volume of the cleaning solution 1 is 20 mL.

[0014] In the present invention, the cleaning solution 2 is composed of acetonitrile and water; wherein the volume ratio of acetonitrile:water is 80:20.

[0015] In the present invention, the volume of the cleaning solution 2 is 20 mL.

[0016] In the present invention, the composition of the reaction solution 1 is ammonium bicarbonate (NH 4 HCO 3 ), tris(2-carboxyethyl)phosphine (TCEP), chloroacetamide (CAA), or Tris-HCl, TCEP, CAA; wherein the NH 4 HCO 3The concentration is 25mM to 50mM, the concentration of TCEP is 5mM to 10mM, and the concentration of CAA is 20mM to 40mM. Preferably, the NH 4 HCO 3 The concentration is 50 mM; or the concentration of Tris-HCl is 50 mM, the concentration of TCEP is 10 mM, and the concentration of CAA is 40 mM.

[0017] In the present invention, the volume of the reaction solution 1 is 5 mL.

[0018] In the present invention, the reaction solution 2 is composed of trypsin and lysine protease Lys-C; wherein the concentration of Trypsin is 0.04 μg / μL, and the concentration of lysine protease Lys-C is 0.02 μg / μL.

[0019] In the present invention, the volume of the reaction solution 2 is 5 mL.

[0020] In the present invention, the activation solution is one or more of methanol, acetonitrile, etc.; preferably, it is methanol.

[0021] In the present invention, the volume of the activation solution is 20 mL.

[0022] In the present invention, the balance solution is composed of formic acid and water; wherein the volume ratio of formic acid to water is 0.1%:99.9%.

[0023] In the present invention, the volume of the equilibrium solution is 50 mL.

[0024] In the present invention, the eluent is composed of formic acid, acetonitrile and water; wherein the volume ratio of formic acid:acetonitrile:water is 0.1%:40%-60%:39.9%-59.9%; preferably, the volume ratio of formic acid:acetonitrile:water is 0.1%:40%:59.9%.

[0025] In the present invention, the volume of the eluent is 20 mL.

[0026] In the present invention, the desalting plate is a 96-well HLB single-well detachable solid phase extraction plate, with 2 mg of filler in each well.

[0027] In the present invention, the number of desalination plates is 1 to 2; preferably, there are 2.

[0028] In a specific embodiment, the specific components and specifications of the kit of the present invention are shown in Table 1 below:

[0029] Table 1

[0030]

[0031]

[0032] At present, most of the protein pretreatment kits available on the market are based on in-solution enzymatic hydrolysis and filter-aided sample preparation (Filter-Aided Sample Preparation, abbreviated as FASP), in which in-solution enzymatic hydrolysis has restrictions on the composition of the protein solution. Common strong lysates such as SDS and NP-40 will affect the activity of the protease and thus the enzymatic hydrolysis effect. Therefore, the lysates used are compatible with enzymatic hydrolysis and mass spectrometry, such as Urea, SDC, etc., but such lysates have poor protein solubility compared to SDS and NP-40, thus affecting the number of protein identifications; and FASP enzymatic hydrolysis steps are cumbersome, time-consuming, and large sample loss, which is not conducive to small samples. Therefore, the kit of the present invention is easy to use for workers with certain proteomics operation experience, and can be operated by reading the instructions.

[0033] The present invention also proposes the application of the kit in all pre-treatment steps of protein extraction, reduction alkylation, proteolysis, peptide desalting and peptide redissolution.

[0034] The method for sample extraction, sample reduction alkylation, sample enzymatic hydrolysis, and sample desalting of the kit of the present invention comprises the following steps:

[0035] Step 1: Add the extractant to the sample, followed by ultrasonic extraction;

[0036] Step 2: After the ultrasound is finished, centrifuge at low temperature to obtain the supernatant and perform BCA quantification;

[0037] Step 3: Take a certain amount of protein solution according to the BCA quantitative results, and add magnetic beads to the sample at a mass ratio of protein: magnetic beads = 1:10;

[0038] Step 4: Immediately add a certain volume of replacement solution and incubate at room temperature (25°C) for 20 minutes with shaking at 1000 rpm / min.

[0039] Step 5: After the shaking incubation is completed, the sample is separated and placed on a magnetic rack for two minutes, and then the supernatant is removed.

[0040] Step 6: Immediately add a certain volume of cleaning solution 1 and incubate at room temperature (25°C) for 5 minutes with shaking at 1000 rpm / min.

[0041] Step 7: After the shaking incubation is completed, the sample is separated and placed on a magnetic rack for two minutes, and then the supernatant is removed.

[0042] Step 8: Immediately add a certain volume of cleaning solution 2 and incubate at room temperature (25°C) for 5 minutes with shaking at 1000 rpm / min.

[0043] Step 9: After the shaking incubation is completed, the sample is separated and placed on a magnetic rack for two minutes, then the supernatant is removed. After removing the supernatant, the sample is placed at room temperature for 5-10 minutes.

[0044] Step 10: Add a certain volume of reaction solution 1 and incubate at 55°C and 1000 rpm for 30 minutes.

[0045] Step 11: The sample was immediately separated and returned to room temperature. A certain volume of reaction solution 2 was added and incubated at 37°C and 1000 rpm for 1-2 hours.

[0046] Step 12: After the incubation reaction is completed, the sample is separated and placed on a magnetic rack for two minutes, then the supernatant is aspirated and placed in a new EP tube for later use.

[0047] The formulation of the reaction solution 1 in step (10) of the present invention is of great importance. In the prior art, dithiothreitol (DTT) is often used to reduce the sample, and then iodoacetamide (IAM) is used for alkylation. The reaction rate of DTT is slow, and it takes at least 30 minutes. IAA also needs to be protected from light for reaction, which is not suitable for automation and the two cannot be mixed. Therefore, the composition of TCEP and CAA is used in this step. TCEP has a fast reaction speed and can complete the reduction in a few minutes, while CAA does not need to be protected from light. Moreover, TCEP and CAA can be mixed and used together, which greatly improves the efficiency. On the one hand, the steps of traditional reduction and alkylation are combined and completed in one step, and on the other hand, the efficiency of sample reduction and alkylation is improved.

[0048] The formulation of reaction solution 2 in step (11) of the present invention is also crucial. The prior art generally uses lysine protease Lys-C for 4 hours of enzymatic hydrolysis, and then adds Trypsin for 16 hours of enzymatic hydrolysis. In the present invention, lysine protease Lys-C and Trypsin are combined together, and the ratio of Lys-C to trypsin is optimized. Only one enzymatic hydrolysis is required. The design of reaction solution 2 greatly improves the enzymatic hydrolysis efficiency, and increases the traditional overnight enzymatic hydrolysis time to only 1-2 hours without reducing the enzymatic hydrolysis efficiency. This greatly improves the sample processing throughput and solves the key rate-limiting step of proteomics.

[0049] Step 13: Sample desalting step

[0050] a. Take out the desalination plate and place it on the negative pressure or positive pressure adapter.

[0051] b. Add a certain volume of activation solution to the desalting plate, 0.05MPa, and pass through the column for 1min;

[0052] c. Add a certain volume of equilibrium solution to the desalting plate, 0.1MPa, and pass through the column for 1min;

[0053] d. Add the supernatant in step 12 to the desalting plate, 0.1 MPa, and pass through the column for 1 min;

[0054] e. Add a certain volume of equilibrium solution to the desalting plate, 0.1MPa, and pass through the column for 1min;

[0055] f. Add a certain volume of eluent to the desalting plate, 0.05MPa, and pass through the column for 1 minute.

[0056] Step 14: The desalted sample is freeze-dried or evaporated and analyzed by liquid chromatography-mass spectrometry.

[0057] In a specific embodiment scheme, in the present invention, the automated pre-treatment kit based on magnetic beads is used for sample pre-treatment of rat kidney samples, Hela cell samples, and rice leaf samples. Since magnetic beads are used for protein enrichment, organic reagents can be used to wash away all extraction reagents in the magnetic bead cleaning stage, which greatly avoids various surfactants from entering the mass spectrometer and causing contamination. In addition, the magnetic beads are processed based on magnetism, so they are easy to automate, and the magnetic absorption process of steps such as centrifugation and precipitation in traditional proteolysis is convenient for automation. On the other hand, the ratio of reaction solution 1 and reaction solution 2 in the kit greatly improves the efficiency of the entire experiment.

[0058] The beneficial effects of the present invention include but are not limited to: the present invention proposes an automated protein pre-treatment detection kit based on magnetic beads, the kit is used for all pre-treatment steps such as protein extraction, reduction alkylation, proteolysis, peptide desalting, peptide redissolution, etc., and the treated peptides can be directly put on the mass spectrometer. The entire operation process of the kit of the present invention takes 5 hours, the operation is simple and easy to use, and the throughput is high. After the kit of the present invention is adapted to the automated equipment, the enzymatic hydrolysis and desalting of 96 samples can be achieved in 4 hours, which greatly improves the pre-treatment speed and reduces the labor cost. At the same time, compared with manual operation, it has higher precision, higher stability, higher efficiency, etc. The pre-treatment kit of the present invention is suitable for all sample types without preference, such as cells, blood, tissues, plants, fungi, etc., and is also suitable for most protein extraction reagents on the market, such as SDS, urea, SDC, RIPA, NP-40, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without paying any creative work.

[0060] Figure 1 This is a statistical chart of the protein peptide identification results of rat kidney tissue.

[0061] Figure 2 Venn diagram of proteins from three replicate samples of rat kidney tissue.

[0062] Figure 3 This is a box plot of the relative standard deviation (RSD) of rat kidney tissue protein quantification.

[0063] Figure 4 Statistical chart of protein peptide identification results after automated processing in Hela cells and rice leaves.

[0064] Figure 5 A Venn diagram of proteins processed automatically for three replicate samples of HeLa cells and rice leaves.

[0065] Figure 6 Box plots of relative standard deviation (RSD) of automated protein quantification for Hela cells and rice leaves. DETAILED DESCRIPTION

[0066] The invention is further described in detail with reference to the following specific examples. The process, conditions, experimental methods, etc. for implementing the present invention, except for the contents specifically mentioned below, are all common knowledge and common common sense in the art and are not particularly limited by the present invention.

[0067] Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. The following description of at least one exemplary embodiment is actually only illustrative and is by no means intended to limit the present invention and its application or use. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0068] In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limiting. Therefore, other examples of the exemplary embodiments may have different values.

[0069] The present invention discloses an automated protein pre-treatment detection kit based on magnetic beads, and the kit is used for all pre-treatment steps such as protein extraction, reduction alkylation, proteolysis, peptide desalting, peptide redissolution, etc., and the treated peptides can be directly put on a mass spectrometer. After the kit of the present invention is adapted to the automated equipment, the enzymatic hydrolysis and desalting of 96 samples can be achieved in 4 hours, which greatly improves the pre-treatment speed and reduces the labor cost. At the same time, compared with manual operation, it has higher accuracy, higher stability, higher efficiency, etc. The pre-treatment detection kit of the present invention is applicable to all sample types without preference, such as cells, blood, tissues, plants, fungi, etc., and is also adapted to most protein extraction reagents on the market, such as SDS, urea, SDC, RIPA, NP-40, etc.

[0070] Unless otherwise specified, the test materials used in the examples are all conventional biochemical reagents.

[0071] In the examples of the present invention, the compositions and contents of the reagents used are shown in the following table:

[0072]

[0073]

[0074] Example 1

[0075] This example describes the application of the kit of the present invention in rat kidney samples. The specific steps are as follows:

[0076] Step 1: Sample extraction and enzymatic hydrolysis

[0077] Required reagents: extraction agent, magnetic bead solution, replacement solution, cleaning solution 1, cleaning solution 2, reaction solution 1, reaction solution 2.

[0078] 1. Cut the rat kidney tissue into pieces with scissors, weigh three rat kidney samples of the same mass (10 mg) into a 1.5 mL low-absorption centrifuge tube, and add 300 μL of extractant to cover the tissue sample.

[0079] 2. The samples were ultrasonically disrupted on ice using contact ultrasound with a power of 80 W, 1.0 s on, 1.0 s off, for a total of 2 min.

[0080] 3. After the ultrasound is finished, centrifuge for 10 minutes (4°C, 12000 rpm) and take the supernatant into a clean EP tube.

[0081] 4. Use a commercial BCA quantification kit to quantify the sample and obtain the protein concentration.

[0082] 5. According to the BCA quantitative results, take 50 μg of protein solution into a new EP tube and use the extraction agent to supplement the volume of each well to 50 μL.

[0083] 6. Pipette 10 μL of magnetic bead solution and add it to each sample.

[0084] 7. Pipette 200 μL of replacement solution and add it to each sample. Incubate in an oscillator for 20 minutes (25°C, 1000 rpm). After the reaction is completed, separate the EP tube and place it on a magnetic stand for 2 minutes. Remove the supernatant with a pipette and retain the magnetic beads.

[0085] 8. Pipette 200 μL of cleaning solution 1 and add it to each sample. Incubate in an oscillator for 5 minutes (25°C, 1000 rpm). After the reaction is completed, separate the EP tube and place it on a magnetic stand for 2 minutes. Remove the supernatant with a pipette and retain the magnetic beads.

[0086] 9. Pipette 200 μL of cleaning solution 2 and add it to each sample. Incubate in an oscillator for 5 minutes (25°C, 1000 rpm). After the reaction is completed, separate the EP tube and place it on a magnetic stand for 2 minutes. Remove the supernatant with a pipette and retain the magnetic beads.

[0087] 10. After removing cleaning solution 2, leave it at room temperature for 5-10 minutes to allow the solution to evaporate.

[0088] 11. Add 50 μL of reaction solution 1 and shake in an oscillator for 30 min (55°C, 1000 rpm).

[0089] 12. After the reaction, the sample was immediately centrifuged to return to room temperature, 50 μL of reaction solution 2 was added, and the mixture was shaken in an oscillator for 1-2 h (37°C, 1000 rpm).

[0090] 13. After the reaction is completed, the sample is immediately placed on a magnetic rack for 2 minutes, and the supernatant is transferred to a new EP tube for later use.

[0091] Step 2: Sample extraction and enzymatic hydrolysis

[0092] Required reagents: activation solution, equilibration solution, elution solution, desalting plate.

[0093] 1. Take out the desalination plate and place it on the negative pressure adapter;

[0094] 2. Add 200 μL of activation solution to the desalting plate, 0.05 MPa, and pass through the column for 1 min;

[0095] 3. Add 200 μL of equilibrium solution to the desalting plate, 0.1 MPa, and pass through the column for 1 min;

[0096] 4. Add the supernatant from the extraction and enzymatic hydrolysis step 13 to the desalting plate, 0.1 MPa, and pass through the column for 1 min;

[0097] 5. Add 200 μL of equilibrium solution to the desalting plate, 0.1 MPa, and pass through the column for 1 min;

[0098] 6. Add 200 μL of eluent to the desalting plate, 0.05 MPa, and pass through the column for 1 min.

[0099] 7. The collected eluate was placed in a evaporator for liquid chromatography-mass spectrometry analysis.

[0100] Step 3: LC-MSMS mass spectrometry analysis and mass spectrometry data interpretation

[0101] Required reagents: Balance solution

[0102] In this example, a liquid chromatography Nanoelute (Bruker) and mass spectrometer TimsTOF Pro2 (Bruker) were used to evaluate the effect of the kit.

[0103] The evaporated peptides were re-dissolved with the equilibrium solution, and 200 ng of the peptide solution was chromatographed with the nanoliter liquid phase Nanoelute, and the Ionpticks chromatographic analysis column (15 cm*75 μm, C18, 1.6 μm). Mobile phase A (0.1% formic acid-water solution), mobile phase B (0.1% formic acid-acetonitrile). The liquid phase separation gradient was as follows: 0min-20min, the linear gradient of phase B was from 5% to 22%; 20min-24min, the linear gradient of phase B was from 22% to 37%; 24min-27min, the linear gradient of phase B was from 37% to 80%; 27min-30min, the gradient of phase B was maintained at 80%. The liquid phase flow rate was 400 nL / min.

[0104] The mass spectrometry system uses the TimsTOF Pro2 mass spectrometry system of Bruker, and the mass spectrometry parameters are as follows:

[0105] PASER-DIA mode was used for data acquisition, with the capillary voltage set to 1500 V, the primary and secondary mass spectrometer scanning ranges of 100-1700 m / z, and the ion mobility window range (1 / K0) of 0.65-1.35.

[0106] The data was searched and identified using Biognosys Spectronaut software (version 18.4) and analyzed qualitatively and quantitatively. The rat kidney samples were subjected to three repeated experiments according to the above example. The original mass spectrometry data of the three repeated experiments were analyzed and identified by the software. The results are as follows: Figure 1 As shown in the figure, the number of proteins identified in the three repeated experiments remained above 7,000, and the number of peptides remained above 80,000, indicating that the kit has good extraction, enzymatic hydrolysis and desalting effects. A Venn diagram was made using the proteins identified in the three repeated experiments. The results are shown in the figure. Figure 2 As shown, the proteins identified in all three experiments were as high as 99%, indicating that the repeatability of the kit of the present invention can meet the needs of proteomics qualitative analysis. In addition, the stability of the kit of the present invention for three re-readings of the sample was investigated as follows: Figure 3 As shown, the median RSD of the protein quantitative values ​​identified by three sets of repeated experiments was below 20%, indicating that the quantitative repeatability of the kit of the present invention can meet the requirements of proteomics quantitative analysis.

[0107] Example 2

[0108] This example describes the application of the kit of the present invention in combination with an automated instrument in Hela cell samples and rice leaf samples. The specific steps are as follows:

[0109] Step 1: Sample extraction, enzymatic hydrolysis and desalting

[0110] Required reagents: extraction agent, magnetic bead solution, replacement solution, cleaning solution 1, cleaning solution 2, reaction solution 1, reaction solution 2, activation solution, balancing solution, elution solution, desalting plate.

[0111] 1. Hela cell samples and rice leaf samples were manually extracted, and commercial BCA quantification kit was used to quantify the samples and obtain protein concentration.

[0112] 2. According to the BCA quantitative results, take 50 μg of protein solution into a new 96-well reaction plate and add the extraction agent to make up the volume of each well to 50 μL.

[0113] 3. Pipette the magnetic bead solution, replacement solution, cleaning solution 1, cleaning solution 2, reaction solution 1, reaction solution 2, activation solution, balancing solution, and elution solution into another corresponding 96-well plate for later use.

[0114] 4. Accordingly, the desalting plates, new 96-well plates, gun tips and other consumables are placed in the automated instrument stack for use.

[0115] 5. The above steps are manual, and the remaining steps are all automated instrument operations.

[0116] 6. Use the liquid workstation to aspirate 10 μL of magnetic bead solution and add it to the 96-well reaction plate.

[0117] 7. The liquid workstation draws 200 μL of replacement solution and adds it to the 96-well reaction plate. The mechanical gripper transfers the 96-well reaction plate to the shaker and incubates for 20 minutes (25°C, 1000 rpm). After the reaction is completed, the mechanical gripper transfers the 96-well reaction plate to the magnetic suction module and leaves it for 2 minutes. The liquid workstation removes the supernatant and retains the magnetic beads.

[0118] 8. The liquid workstation draws 200 μL of cleaning solution 1 and adds it to the 96-well reaction plate. The mechanical gripper transfers the 96-well reaction plate to the shaker and incubates for 5 minutes (25°C, 1000 rpm). After the reaction is completed, the mechanical gripper transfers the 96-well reaction plate to the magnetic suction module and leaves it for 2 minutes. The liquid workstation removes the supernatant and retains the magnetic beads.

[0119] 9. The liquid workstation draws 200 μL of cleaning solution 2 and adds it to the 96-well reaction plate. The mechanical gripper transfers the 96-well reaction plate to the shaker and incubates for 5 minutes (25°C, 1000 rpm). After the reaction is completed, the mechanical gripper transfers the 96-well reaction plate to the magnetic suction module and leaves it for 2 minutes. The liquid workstation removes the supernatant and retains the magnetic beads.

[0120] 10. After removing cleaning solution 2, leave it at room temperature for 5-10 minutes to allow the solution to evaporate.

[0121] 11. The liquid workstation aspirates 50 μL of reaction solution 1 and adds it to the 96-well reaction plate. The mechanical gripper transfers the 96-well reaction plate to the shaker for shaking reaction for 30 minutes (55°C, 1000 rpm).

[0122] 12. After the reaction is completed, let the sample stand for 10 minutes to return to room temperature. Use the liquid workstation to aspirate 50 μL of reaction solution 2 and add it to the 96-well reaction plate. Oscillate the reaction on an oscillator for 1-2 hours (37°C, 1000 rpm).

[0123] 13. After the reaction is completed, the mechanical gripper transfers the 96-well reaction plate to the magnetic rack and leaves it for later use.

[0124] 14. The mechanical gripper takes out the desalination plate from the stack and places it on the positive pressure adapter;

[0125] 15. The liquid workstation draws 200 μL of activation solution into the desalting plate, 0.05 MPa, and passes through the column for 1 min; 16. The liquid workstation draws 200 μL of equilibrium solution into the desalting plate, 0.1 MPa, and passes through the column for 1 min; 17. The liquid workstation draws the supernatant from the 96-well reaction plate in step 13 into the desalting plate, 0.1 MPa, and passes through the column for 1 min;

[0126] 18. Use the liquid workstation to draw 200 μL of the equilibrium solution and add it to the desalting plate, 0.1 MPa, and pass through the column for 1 minute;

[0127] 19. The liquid workstation draws 200 μL of eluent and adds it to the desalting plate. The mechanical gripper transfers the new 96-well plate to the collection area, 0.05 MPa, and passes through the column for 1 minute.

[0128] 20. The collected eluate is placed in a evaporator for liquid chromatography-mass spectrometry analysis.

[0129] Step 2: LC-MSMS mass spectrometry analysis and mass spectrometry data interpretation

[0130] Required reagents: Balance solution

[0131] In this example, a liquid chromatography Nanoelute (Bruker) and mass spectrometer TimsTOF Pro2 (Bruker) were used to evaluate the effect of the kit.

[0132] The evaporated peptides were re-dissolved with the equilibrium solution, and 200 ng of the peptide solution was chromatographed with the nanoliter liquid phase Nanoelute, and the Ionpticks chromatographic analysis column (15 cm*75 μm, C18, 1.6 μm). Mobile phase A (0.1% formic acid-water solution), mobile phase B (0.1% formic acid-acetonitrile). The liquid phase separation gradient was as follows: 0min-20min, the linear gradient of phase B was from 5% to 22%; 20min-24min, the linear gradient of phase B was from 22% to 37%; 24min-27min, the linear gradient of phase B was from 37% to 80%; 27min-30min, the gradient of phase B was maintained at 80%. The liquid phase flow rate was 400 nL / min.

[0133] The mass spectrometry system uses the TimsTOF Pro2 mass spectrometry system of Bruker, and the mass spectrometry parameters are as follows:

[0134] PASER-DIA mode was used for data acquisition. The capillary voltage was set to 1500 V, the primary and secondary scan ranges of the mass spectrometer were 100-1700 m / z, and the ion mobility window range (1 / K0) was 0.65-1.35.

[0135] The data was searched and identified by Biognosys Spectronaut software (version 18.4) and analyzed qualitatively and quantitatively. The Hela cell sample and rice leaf sample in the above embodiment were subjected to three repeated experiments. The original mass spectrometry data of the three repeated experiments were analyzed and identified by the software. The results are as follows: Figure 4 As shown in the figure, the number of proteins identified in the three repeated experiments of Hela cells remained above 7500, and the number of peptides remained above 110,000. The number of proteins identified in the three repeated experiments of rice leaves remained above 7400, and the number of peptides remained above 65,000. This shows that the kit has good extraction, enzymatic hydrolysis and desalting effects when used with automated instruments. A Venn diagram was made using the proteins identified in the three repeated experiments. The results are shown in the figure. Figure 5 As shown in the figure, the proteins identified in the three experiments of Hela cells and rice leaves were as high as 99.5% and 99.1%, respectively, indicating that the repeatability of the kit of the present invention when used with an automated instrument can meet the needs of proteomics qualitative analysis. In addition, the stability of the three repeated quantitative tests of the kit of the present invention on the samples when used with an automated instrument is shown in the figure. Figure 6 As shown, the medians of the RSD of the protein quantitative values ​​identified by the three sets of repeated experiments were 10% and 20% lower, respectively, indicating that the quantitative repeatability of the kit of the present invention can meet the requirements of proteomics quantitative analysis. It can be seen from this example that the kit of the present invention can meet the qualitative and quantitative requirements of proteomics when used with an automated pretreatment instrument, and the 4h throughput can be as high as 96 samples.

[0136] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0137] As used in the present invention, the terms "include" and "comprising" are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0138] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0139] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the attached claims.

Claims

1. An automated protein pre-treatment detection kit based on magnetic beads, characterized in that: The kit comprises an extractant, a magnetic bead solution, a replacement solution, a cleaning solution 1, a cleaning solution 2, a reaction solution 1, a reaction solution 2, an activation solution, a balancing solution, an eluent, and a desalting plate.

2. The kit according to claim 1, characterized in that The extractant is composed of a mixed aqueous solution of SDS with a mass volume ratio of 1% to 4%, 50mM tris(hydroxymethyl)aminomethane, 2.5mM sodium pyrophosphate, 1mM β-glycerophosphate, 1mM sodium orthovanadate, 10mM sodium fluoride, 1mM ethylenediaminetetraacetic acid, and 0.5μg / μL leupeptin; and / or the concentration of the magnetic bead solution is 50μg / μL; and / or the replacement fluid is acetonitrile.

3. The kit according to claim 1, characterized in that The magnetic beads are Sera-Mag magnetic beads with a diameter of 1 μm and surface modified with carboxyl groups.

4. The kit according to claim 1, characterized in that The cleaning solution 1 is composed of ethanol and water; and / or the cleaning solution 2 is composed of acetonitrile and water.

5. The kit according to claim 4, characterized in that The volume ratio of the ethanol:water is 80:20; and / or the volume ratio of the acetonitrile:water is 80:

20.

6. The kit according to claim 1, characterized in that The composition of the reaction solution 1 is 25mM to 50mM ammonium bicarbonate, 10mM tris(2-carboxyethyl)phosphine, and 40mM chloroacetamide, or 50mM tris(2-carboxyethyl)phosphine, 10mM tris(2-carboxyethyl)phosphine, and 40mM chloroacetamide; and / or the composition of the reaction solution 2 is 0.04μg / μL trypsin and 0.02μg / μL lysine protease Lys-C.

7. The kit according to claim 1, characterized in that The activation solution is one or two of methanol and acetonitrile; and / or the balance solution is composed of formic acid and water, and the volume ratio of formic acid: water is 0.1%:99.9%; and / or the eluent is composed of formic acid, acetonitrile and water, and the volume ratio of formic acid:acetonitrile: water is 0.1%:60%-40%:39.9%-59.9%.

8. The kit according to claim 1, wherein The desalting plate is a 96-well HLB single-well detachable solid phase extraction plate, with 2 mg of filler in each well.

9. The kit according to claim 1, characterized in that The volume of the extractant is 30 mL, the volume of the magnetic bead solution is 1 mL, the volume of the replacement solution is 20 mL, the volume of the cleaning solution 1 is 20 mL, the volume of the cleaning solution 2 is 20 mL, the volume of the reaction solution 1 is 5 mL, the volume of the reaction solution 2 is 5 mL, the volume of the activation solution is 20 mL, the volume of the balancing solution is 50 mL, the volume of the eluent is 20 mL, and there are 1 to 2 desalting plates.

10. Use of the kit according to any one of claims 1 to 9 in protein extraction, reductive alkylation, proteolysis, peptide desalting and peptide redissolution.

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