High-capacity automatic crop proteome sample pretreatment method

Abstract

The invention belongs to the field of proteomics analysis, and particularly discloses a high-capacity automatic crop proteome sample pretreatment method which comprises the following steps: putting a sample into a centrifugal tube filled with nano magnetic beads; carrying out operations such as protein solid-phase adsorption by the nano magnetic beads, cleaning of non-specific binding protein, protein enzymolysis, peptide fragment elution and the like in an automatic liquid workstation; and finally, moving the sample out of the automatic liquid workstation, and carrying out low-temperature drying and mass spectrometric detection on the sample. According to the method, the step of desalting by using an SDB column and a C18 column in a traditional method is omitted, a centrifugal tube does not need to be replaced in the preparation process of the sample, time and labor are saved, the consumption of consumables is reduced, and convenient and rapid high-throughput treatment of the sample is realized.

Description

Technical Field

[0001] The invention relates to the field of proteomics analysis, and in particular to a high-capacity automated crop proteomics sample pre-treatment method. Background Art

[0002] Proteomics is a technology that performs high-throughput analysis of proteins across the entire genome. Its research goal is to reveal the relationship between protein function and the laws of cellular life activities from the perspective of systems biology. Similar to other bio-omics technologies, the analytical capabilities of proteomics are also developing rapidly, especially with the advancement of mass spectrometry analysis technology. The sample throughput has been reduced from 90-180 minutes for a single sample mass spectrometry acquisition to less than 10 minutes.

[0003] Crops are the most important source of food and nutrients for humans. Humans have accumulated a large number of crop germplasm resources and have a large crop planting area. In order to explore the important regulatory genes in germplasm resources and understand the growth of large-scale crops, it is often necessary to collect hundreds of samples for genome, transcriptome, proteome and metabolome detection and analysis.

[0004] At present, the method of extracting the proteome of crop samples requires each sample to be operated separately, which has the problems of cumbersome operation, long time consumption, low throughput, poor stability between samples, etc. For example, using filter-aided sample preparation (FASP) to process samples, the operation time for every 8 samples is about 8 hours. Crop tissue samples are different from human disease samples, cells and other samples. They are often easy to sample and have a large sample volume, but the protein content is not high. Therefore, the crop tissue samples used to extract protein are generally more than 0.5 grams. The existing sample processing system has a small capacity and is not suitable for the processing of crop tissue samples.

[0005] As mentioned above, it is of special value to conduct high-capacity, high-throughput, automated sample pretreatment research for crop tissue samples. Summary of the invention

[0006] The purpose of the present invention is to provide a high-capacity automated crop proteome sample pretreatment method to address the above problems, thereby achieving high-throughput and rapid detection of crop proteins.

[0007] To achieve the above-mentioned purpose, the high-capacity automated crop proteome sample pretreatment method provided by the present invention comprises the following steps: step S200, placing the sample in a centrifuge tube filled with nanomagnetic beads, and performing solid-phase adsorption of proteins on the nanomagnetic beads, cleaning of non-specific binding proteins, protein enzymatic hydrolysis, and peptide elution in an automated liquid workstation; step S300, removing the sample from the automated liquid workstation, and performing low-temperature drying and mass spectrometry detection on the sample.

[0008] Furthermore, the sample is selected from protein purifications, proteomes, peptide purifications or peptide groups from different crops, parts and purification methods.

[0009] Furthermore, the acquisition of the sample used in step S200 includes step S100, crop tissue protein extraction and quantitative determination, and step S100 specifically includes: step S101, taking the crop tissue material and grinding it into powder in a cryo-grinder pre-cooled with liquid nitrogen; step S102, taking the ground powder and adding it to a lysis solution for denaturation and reductive alkylation, and after completion, centrifuging to obtain the supernatant, and determining the protein content in the supernatant.

[0010] Furthermore, the step S200 includes the following specific steps: step S201, in an automated liquid workstation, placing the sample in multiple centrifuge tubes containing nanomagnetic beads, adding anhydrous ethanol, incubating in the automated liquid workstation, using magnetic force to adsorb the precipitate, retaining precipitate A, and removing the supernatant; step S202, on the automated liquid workstation, adding 80% ethanol to the centrifuge tube retaining precipitate A, blowing and resuspending precipitate A, using magnetic force to adsorb the precipitate, retaining precipitate B, and removing the supernatant, repeating this step no less than 6 times; step S203, on the automated liquid workstation, adding NH4HCO3 to the centrifuge tube retaining precipitate B, and then adding trypsin and lysine protease for enzymatic hydrolysis; step S204, after the enzymatic hydrolysis is completed, using a solvent to desorb the peptide segment on the automated liquid workstation.

[0011] Furthermore, in step S101, the weight of the crop tissue material is greater than 0.5 g, and each batch of samples processed by automated grinding is no less than 12.

[0012] Furthermore, in step S102, the time of the reductive alkylation does not exceed 20 minutes.

[0013] Furthermore, in step S201, the volume of the centrifuge tube is not less than 1.5 ml, the number of the centrifuge tubes is 1-24, and there is oscillation during the incubation process, and the frequency is 1000 rpm.

[0014] Furthermore, in step S202, the volume of 80% ethanol added each time is greater than 500 ul, and the addition of 80% ethanol is repeated 6-8 times, with oscillation during the resuspension process at a frequency of 1000 rpm.

[0015] Furthermore, in step S203, the trypsin and lysine protease are added at one time, the enzymatic hydrolysis time does not exceed 4 hours, and there is oscillation during the enzymatic hydrolysis process with a frequency of 700 rpm.

[0016] Furthermore, in step S300, the low-temperature drying is carried out at a temperature of -10 to 0°C, and is assisted by high-speed centrifugation, with a centrifugal speed of not less than 1000 rpm.

[0017] In summary, the beneficial effects of the present invention are:

[0018] (1) The present invention simplifies the peptide desalting step, removes salt impurities in the sample through multiple washings, and utilizes the volatility of ammonium bicarbonate in an acidic environment, eliminating the traditional desalting step of using SDB columns and C18 columns.

[0019] (2) During the sample preparation process, there is no need to replace the centrifuge tube. All processes can be completed on the same multi-porous screen plate in the automated liquid workstation, saving time and effort, reducing the consumption of consumables, and achieving convenient, rapid and high-throughput sample processing.

[0020] (3) The automated liquid workstation used in the present invention has a sieve plate with many holes, so that a single sieve plate can carry a large number of samples. In addition, the sieve plate has a large aperture, which is convenient for accommodating centrifuge tubes of different specifications, so that they can be flexibly combined according to different sample quantities. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a flow chart of an embodiment of the high-capacity automated crop proteome sample pre-processing method of the present invention;

[0022] Figure 2 It is a structural schematic diagram of the automated liquid workstation and its component modules in the present invention.

[0023] In the figure: 100 - automated liquid workstation, 101 - magnetic attraction, oscillation, heating module, 102 - pipette module, 103 - cooling module, 104 - solution tank module. DETAILED DESCRIPTION

[0024] The present invention aims to provide a high-capacity automated crop proteome sample pretreatment method to solve the problems in the prior art that the crop tissue sample volume is large but the protein content is not high, and the existing sample processing system has a small capacity and is not suitable for crop tissue sample processing, thereby achieving high-throughput and rapid detection of crop proteins.

[0025] like Figure 1 As shown, the high-capacity automated crop proteome sample pre-treatment method of the present invention mainly comprises the following steps:

[0026] Step S100: Extraction and quantitative determination of crop tissue protein.

[0027] Step S100 specifically includes: step S101, taking crop tissue materials weighing more than 0.5 g, grinding them into powder in a cryo-grinder pre-cooled with liquid nitrogen, and automatically grinding each batch of samples to process no less than 12 portions; step S102, taking the ground powder and adding it into a lysis solution for denaturation and reductive alkylation, the reductive alkylation time shall not exceed 20 minutes, and after completion, centrifuging to obtain the supernatant, and determining the protein content in the supernatant.

[0028] Step S200, placing the sample in a centrifuge tube containing nanomagnetic beads, and performing solid phase adsorption of proteins on the nanomagnetic beads, cleaning of non-specifically bound proteins, protein enzymatic hydrolysis, and peptide elution in an automated liquid workstation.

[0029] Step S200 specifically includes: step S201, in an automated liquid workstation, placing the sample in a plurality of centrifuge tubes containing nanomagnetic beads, the volume of the centrifuge tubes being not less than 1.5 ml, the number of the centrifuge tubes being 1-24, adding anhydrous ethanol, and incubating in an automated liquid workstation, with oscillation during the incubation process, and the frequency being 1000 rpm, after incubation, using magnetic force to adsorb the precipitate, retaining the precipitate A, and removing the supernatant; step S202, on the automated liquid workstation, adding 80% ethanol to the centrifuge tube retaining the precipitate A, blowing and resuspending the precipitate A, using magnetic force to adsorb the precipitate, retaining the precipitate B, and removing the supernatant, repeating this step for not less than 6 times, in which the volume of 80% ethanol added each time is greater than 500 ul, there is oscillation during the resuspension process, and the frequency is 1000rpm; step S203, on the automated liquid workstation, add NH4HCO3 to the centrifuge tube retaining the precipitate B, and then add trypsin and lysine protease for enzymatic hydrolysis. In this step, trypsin and lysine protease are added once, and the enzymatic hydrolysis time does not exceed 4 hours. There is oscillation during the enzymatic hydrolysis process, and the frequency is 700rpm; step S204, after the enzymatic hydrolysis is completed, use a solvent to desorb the peptides on the automated liquid workstation.

[0030] Step S300, remove the sample from the automated liquid workstation, perform low-temperature drying and mass spectrometry on the sample. In this step, the low-temperature drying temperature is -10 to 0°C, and is assisted by high-speed centrifugation, with a centrifugal speed of not less than 1000 rpm.

[0031] Reference Figure 2 The automated liquid workstation in the present invention mainly includes magnetic attraction, oscillation, heating modules, a pipette module, a refrigeration module, and a solution tank module.

[0032] The samples in the present invention can be selected from protein purifications, proteomes, peptide purifications or peptideomes from different crops, parts and purification methods, such as roots, stems, leaves, flowers, fruits, seeds, cell culture fluids and the like.

[0033] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with the embodiments (taking wheat grains and wheat rootlets as examples). The following embodiments are only used to illustrate the present invention and should not be construed as limiting the present invention. The specific techniques or conditions not specified in the embodiments are all operated according to the documents in the art or the conventional technical means in the art.

[0034] Example 1: Automated sample pre-processing of 24 wheat grain proteomes

[0035] The automated liquid workstation used in this embodiment is RayKol Vitae 100 automated liquid transfer workstation (including a robotic arm), with 24 channels, manufactured by Xiamen Ruike Group. The automated liquid workstation mainly has functions such as accurate automated liquid transfer. The present invention mainly uses the magnetic suction, liquid transfer, oscillation and other functions of the automated liquid workstation.

[0036] 1. Fresh wheat grains 15 days after flowering were collected and quickly transferred to liquid nitrogen, ground into powder in a cryo-grinder pre-cooled with liquid nitrogen, and the powder was transferred to a 50 mL centrifuge tube. 2 mL of lysis buffer was added, and the lysis buffer included 2-4% sodium dodecyl sulfate (SDS), 100 mM Tris-HCl pH=7.5, 5-20 mM tris(2-formylethyl) phosphine hydrochloride (TCEP), and 20-60 mM chloroacetamide (CAA). The mixture was shaken and mixed to obtain a sample.

[0037] 2. Incubate the sample in a 95°C water bath for 5 minutes, place the centrifuge tube containing the sample in ice water for ultrasonic disruption, and set the ultrasonic disruption parameters to 10 seconds of working time, 10 seconds of rest, and a total ultrasonic time of 3 minutes. Incubate again at 95°C for 5 minutes.

[0038] 3. Centrifuge the sample at 10000G for 10 minutes, transfer the supernatant to a new 1.5 mL centrifuge tube, centrifuge again at 10000G for 5 minutes, transfer the supernatant to a new 1.5 mL centrifuge tube. Take 10 μL of the sample and dilute it 20 times, and determine the protein concentration using the BCA protein quantification kit.

[0039] 4. On the work platform of the automated liquid handling workstation, place 200 μL of cleaned nanomagnetic beads in each of 24 centrifuge tubes, adjust the sample protein concentration to 1 μG / μL, take 200 μG protein and add it to the centrifuge tube containing nanomagnetic beads, then add 400 μL of anhydrous ethanol, shake and mix for 10 minutes, and the shaking frequency is 600 RPM. The multi-porous magnetic stand is raised and left for 5 minutes, the liquid is discarded, and the magnetic pole is lowered.

[0040] 5. Add 500 μL of 80% ethanol to each tube, mix by pipetting, and oscillate for 10 minutes at a frequency of 1000 RPM. Let the porous magnetic stand stand for 5 minutes with the pole raised, discard the liquid, and lower the pole.

[0041] 6. Repeat step 5 six times (the specific number of repetitions can be increased or decreased depending on the color depth of the sample), making sure to fully disperse and wash the magnetic beads in each step, and be gentle.

[0042] 7. Add 50 mM ammonium bicarbonate to each of the 24 centrifuge tubes to provide an alkaline environment for proteolysis.

[0043] 8. Protease hydrolysis. The mass ratio of protease to protein in each tube is 1:50. The reaction temperature of the porous magnetic rack is 37°C, the oscillation frequency is 700 rpm, and the enzymatic reaction time is 4 hours.

[0044] 9. After the enzymatic hydrolysis is completed, the multi-porous magnetic stand is raised and the magnetic pole is allowed to stand for 5 minutes, and the supernatant is transferred to the corresponding new tube. 150 μL of ammonium bicarbonate is added to each tube, and the mixture is blown and shaken for 10 minutes at a frequency of 1000 rpm. After standing for 5 minutes, the supernatant is transferred to the corresponding new tube. After the enzymatic hydrolysis reaction is terminated, the final concentration of formic acid is 0.1%. The peptide concentration is determined by BCA, and the peptide is freeze-dried in a frozen centrifugal concentrator and stored at -80 ℃ for loading.

[0045] The samples were re-dissolved in 10.2% acetonitrile (ACN) (containing 0.2% formic acid (FA)) solution and subjected to high-resolution mass spectrometry analysis (LC-MS / MS) and subsequent proteomic data analysis.

[0046] The experimental results are as follows:

[0047] The sample results obtained by this method are shown in Table 1. The results of high-resolution mass spectrometry analysis of protein samples extracted by the method described in Example 1 showed that they could meet the detection requirements, and the number of detected proteins was more than 20,000. The enzymatic hydrolysis efficiency of the 24 samples was also maintained at about 50%, demonstrating the ultra-high stability of automated processing.

[0048] Table 1 Comparison of enzymatic hydrolysis efficiency, peptide fragments and protein identification numbers in wheat leaves

[0049] Sample No. Enzymatic efficiency Number of identified peptides Number of identified proteins 1 62.4% 109627 21997 2 62.4% 116494 22718 3 51.6% 112238 22186 4 55.2% 105866 21651 5 58.8% 119870 22941 6 51.6% 115361 22638 7 56.2% 111490 22064 8 59.3% 111656 22024 9 55.2% 100107 21689 10 53.2% 113252 22361 11 59.3% 115419 22468 12 55.2% 112901 22177 13 69.5% 114884 22254 14 55.2% 114370 22231 15 62.4% 115122 22289 16 66.0% 113165 22103 17 62.4% 108369 22218 18 58.8% 113443 22305 19 58.8% 112007 22182 20 55.2% 113119 22182 21 51.6% 115199 22195 22 58.8% 115189 22201 23 62.4% 116063 22224 24 58.8% 110625 21731

[0050] Example 2: Automated sample pre-processing of 24 wheat root proteomes

[0051] The automated liquid workstation used in this embodiment is RayKol Vitae 100 automated liquid transfer workstation (including a robotic arm), with 24 channels, manufactured by Xiamen Ruike Group. The automated liquid workstation mainly has functions such as accurate automated liquid transfer. The present invention mainly uses the magnetic suction, liquid transfer, oscillation and other functions of the automated liquid workstation.

[0052] 1. Collect fresh wheat rootlets that have germinated for 5 days and quickly transfer them to liquid nitrogen. Grind them into powder in a cryo-grinder pre-cooled with liquid nitrogen. Transfer the powder to a 50 mL centrifuge tube and add 2 mL of lysis buffer (2-4% sodium dodecyl sulfate (SDS), 100 mM Tris-HCl pH=7.5, 5-20 mM tris(2-formylethyl) phosphate hydrochloride (TCEP), 20-60 mM chloroacetamide (CAA)). Mix by oscillation.

[0053] 2. Incubate the sample in a 95 ℃ water bath for 5 minutes, place the centrifuge tube in ice water for ultrasonic disruption, with a working time of 10 seconds, a rest of 10 seconds, and a total ultrasonic time of 3 minutes, and incubate again at 95 ℃ for 5 minutes.

[0054] 3. Centrifuge at 10,000 g for 10 minutes, transfer the supernatant to a new 1.5 mL centrifuge tube, centrifuge at 10,000 g for 5 minutes again, transfer the supernatant to a new 1.5 mL centrifuge tube. Take 10 uL of the sample and dilute it 20 times, and measure the protein concentration using the BCA protein quantification kit.

[0055] 4. On the automated platform of the automated liquid handling workstation, place 200 μL of cleaned nanomagnetic beads in each of 24 centrifuge tubes, adjust the sample protein concentration to 1 μg / μL, take 200 μg of protein and add it to the centrifuge tube containing nanomagnetic beads, then add 400 μL of anhydrous ethanol, shake and mix for 10 minutes, and the shaking frequency is 600 rpm. The porous magnetic stand is raised and left for 5 minutes, the liquid is discarded, and the magnetic pole is lowered.

[0056] 5. Add 500 μL of 80% ethanol to each tube, mix by pipetting and oscillating for 10 minutes at a frequency of 1000 rpm. Let the porous magnetic stand stand for 5 minutes with the pole raised, discard the liquid, and lower the pole.

[0057] 6. Repeat step 5 six times (the specific number of repetitions can be increased or decreased depending on the color depth of the sample), making sure to fully disperse and wash the magnetic beads in each step, and be gentle.

[0058] 7. Add 50 mM ammonium bicarbonate to each of the 24 centrifuge tubes to provide an alkaline environment for proteolysis.

[0059] 8. Protease hydrolysis. The mass ratio of protease to protein in each tube is 1:50. The reaction temperature of the porous magnetic rack is 37°C, the oscillation frequency is 700 rpm, and the enzymatic reaction time is 4 hours.

[0060] 9. After the enzymatic hydrolysis is completed, the multi-porous magnetic stand is raised and the magnetic pole is allowed to stand for 5 minutes, and the supernatant is transferred to the corresponding new tube. 150μL of ammonium bicarbonate is added to each tube, and the mixture is blown and shaken for 10 minutes at a frequency of 1000rpm. After standing for 5 minutes, the supernatant is transferred to the corresponding new tube. After the enzymatic hydrolysis reaction is terminated, the final concentration of formic acid is 0.1%. The peptide concentration is determined by BCA, and the peptide is freeze-dried in a frozen centrifugal concentrator and stored at -80℃ for loading.

[0061] The samples were re-dissolved in 10.2% ACN (containing 0.2% FA) solution and subjected to high-resolution mass spectrometry analysis (LC-MS / MS) and subsequent proteomic data analysis.

[0062] The experimental results are as follows:

[0063] The sample results obtained by this method are shown in Table 2. The results of high-resolution mass spectrometry analysis of the protein samples extracted by the method described in Example 2 showed that they could meet the detection requirements, and the number of detected proteins was more than 20,000. The enzymatic hydrolysis efficiency of the 24 samples was also maintained at about 50%, demonstrating the ultra-high stability of the automated processing.

[0064] Table 2 Comparison of enzymatic hydrolysis efficiency, peptide fragments and protein identification numbers of wheat seed proteins

[0065] Sample No. Enzymatic efficiency Number of identified peptides Number of identified proteins 1 61% 158706 32344 2 52.4% 162922 33194 3 59.25% 167099 33097 4 50.7% 161492 32522 5 48.95% 161122 32638 6 35.25% 162134 32444 7 47.25% 168669 33199 8 35.25% 163842 32647 9 45.55% 164424 32813 10 43.85% 173322 33739 11 40.4% 171987 33630 12 42.1% 167566 33380 13 47.25% 157949 32191 14 54.1% 164648 32774 15 47.25% 165837 33065 16 47.25% 169484 33366 17 43.85% 167830 33473 18 43.85% 173221 33830 19 43.85% 159401 32150 20 45.55% 160253 32394 21 45.55% 163809 32873 22 45.55% 156538 32130 23 54.1% 161029 32716 24 48.95% 166006 33062

[0066] Compared with the prior art, the high-capacity automated crop proteome sample pretreatment method provided by the present invention has the following advantages and effects:

[0067] (1) Compared with the previous protein enrichment process, this process significantly optimized the peptide desalting step in order to realize the automated process more simply and quickly. The previous desalting process required the use of SDB columns and C18 columns for adsorption, washing and desorption. We used multiple washing methods to remove salt impurities in the sample in advance. The subsequent addition of ammonium bicarbonate, taking advantage of its volatility in an acidic environment, eliminated the desalting step.

[0068] (2) During the entire sample preparation process, there is no need to replace the centrifuge tube. All processes can be completed on the same porous sieve plate, which saves time and effort, reduces the consumption of consumables, and realizes convenient, rapid and high-throughput sample processing.

[0069] (3) If the sieve plate used has 24 holes, a single sieve plate can hold 24 samples, and can be flexibly adjusted according to the number of samples, such as 8 holes, 12 holes, and 24 holes. Since the sieve plate has a large aperture, centrifuge tubes of specifications such as 1.5ml and 2ml can be placed, and can be flexibly matched according to the sample quantity.

Claims

1. A high-capacity automated crop proteome sample pre-treatment method, characterized in that: The method comprises the following steps: Step S200, placing the sample in a centrifuge tube containing nanomagnetic beads, and performing solid phase adsorption of proteins on the nanomagnetic beads, cleaning of non-specific binding proteins, protein enzymatic hydrolysis, and peptide elution in an automated liquid workstation; Step S300, removing the sample from the automated liquid workstation, and performing low-temperature drying and mass spectrometry detection on the sample.

2. The high-capacity automated crop proteome sample pre-processing method according to claim 1, characterized in that: The sample is selected from protein purifications, proteomes, peptide purifications or peptideomes from different crops, parts and purification methods.

3. The high-capacity automated crop proteome sample pre-processing method according to claim 1, characterized in that: The acquisition of the sample used in step S200 includes step S100, crop tissue protein extraction and quantitative determination, and step S100 specifically includes: Step S101, taking crop tissue material and grinding it into powder in a cryo-grinder pre-cooled with liquid nitrogen; Step S102, taking the ground powder and adding it into the lysis solution for denaturation and reductive alkylation, after completion, centrifuging and taking the supernatant, and measuring the protein content in the supernatant.

4. The high-capacity automated crop proteome sample pre-processing method according to claim 1, characterized in that: The step S200 includes the following specific steps: Step S201, in an automated liquid workstation, placing the sample in a plurality of centrifuge tubes containing nanomagnetic beads, adding anhydrous ethanol, incubating in the automated liquid workstation, adsorbing the precipitate using magnetic force, retaining the precipitate A, and removing the supernatant; Step S202, on an automated liquid handling station, add 80% ethanol to the centrifuge tube retaining precipitate A, resuspend precipitate A by pipetting, adsorb the precipitate by magnetic force, retain precipitate B, and remove the supernatant, and repeat this step for no less than 6 times; Step S203, on an automated liquid handling station, adding NH4HCO3 to the centrifuge tube retaining the precipitate B, and then adding trypsin and lysine protease for enzymatic hydrolysis; Step S204, after the enzymatic hydrolysis is completed, the peptides are desorbed using a solvent on an automated liquid handling station.

5. The high-capacity automated crop proteome sample pre-processing method according to claim 3, characterized in that: In step S101, the weight of the crop tissue material is greater than 0.5 g, and each batch of samples processed by automated grinding is no less than 12 portions.

6. The high-capacity automated crop proteome sample pre-processing method according to claim 3, characterized in that: In the step S102, the time of the reductive alkylation is no more than 20 minutes.

7. The high-capacity automated crop proteome sample pre-processing method according to claim 4, characterized in that: In step S201, the volume of the centrifuge tube is not less than 1.5 ml, the number of the centrifuge tubes is 1-24, and there is oscillation during the incubation process, and the frequency is 1000 rpm.

8. The high-capacity automated crop proteome sample pre-processing method according to claim 4, characterized in that: In the step S202, the volume of the 80% ethanol added each time is greater than 500 ul, and the addition of 80% ethanol is repeated 6-8 times. There is oscillation during the resuspension process, and the frequency is 1000 rpm.

9. The high-capacity automated crop proteome sample pre-processing method according to claim 4, characterized in that: In the step S203, the trypsin and lysine protease are added at one time, the enzymatic hydrolysis time does not exceed 4 hours, and there is oscillation during the enzymatic hydrolysis process, and the frequency is 700 rpm.

10. The high-capacity automated crop proteome sample pre-processing method according to claim 1, characterized in that: In the step S300, the low-temperature drying is performed at a temperature of -10 to 0°C, and is assisted by high-speed centrifugation, with the centrifugal speed being not less than 1000 rpm.

Images