Crop proteome sample pretreatment method based on solid phase enrichment technology

Abstract

The invention belongs to the field of proteomics analysis, and particularly discloses a crop proteome sample pretreatment method based on a solid phase enrichment technology, and the method comprises the following steps: carrying out pyrolysis denaturation and reductive alkylation on protein in a crop tissue sample; carrying out solid-phase enrichment on crop protein by utilizing nano magnetic beads; carrying out protein enzymolysis, and carrying out elution and low-temperature drying on peptide fragments; and performing mass spectrometry on the peptide fragment sample. According to the method, various secondary metabolites such as chlorophyll in the plant tissues can be effectively removed, proteins are effectively purified, interference of impurities on subsequent mass spectrometry is reduced, the identification number and data quality stability of proteomes in the plant tissues rich in pigments are effectively improved, the sample treatment steps are simplified, and the detection efficiency is improved. The method does not need an additional reductive alkylation step and a peptide fragment desalting step, is efficient and rapid, reduces experimental operation errors, and is suitable for an automatic sample pretreatment process.

Description

Technical Field

[0001] The invention relates to the field of proteomics analysis, and in particular to a crop proteomics sample pre-treatment method based on solid phase enrichment technology. Background Art

[0002] As an important branch of multi-omics research in the field of life sciences, the progress of proteomics has always attracted much attention. Proteomics studies all proteins expressed by a cell, tissue, organ or organism at a specific period, analyzes the dynamic changes in protein composition, expression level and modification state in the cell from a holistic perspective, explores the interactions between different proteins and their roles in cells, tissues or organisms, and thus reveals the laws of protein function and cell life activities. Therefore, proteomics is a technology for high-throughput analysis of proteins in the whole genome. In recent years, with the rapid development of analytical technology, the way of proteomics data collection has also rapidly developed from the early two-dimensional gel electrophoresis separation combined with mass spectrometry analysis to a high-throughput liquid chromatography-mass spectrometry joint analysis mode. From the original qualitative identification, it has developed to the current efficient quantification that can quickly complete the precise quantification of tens of thousands of peptides. Because proteins exist in biological cells, current analytical technology cannot directly read their content information. Before proteomics analysis, proteins need to be released from cells and go through dozens of time-consuming and laborious steps of proteomics extraction preparation. Pretreatment is crucial to ensuring sample quality and is also a key step in determining the quality of subsequent proteomic data collection.

[0003] Compared with other biological samples, plants are usually more difficult to prepare for proteomics because plant tissues are rich in high levels of proteases, secondary metabolites, cell walls and chlorophyll, which can seriously interfere with protein extraction, separation and identification. Commonly used sample pretreatment methods: in-solution enzymatic preparation and filter-aided sample preparation (FASP), etc., are not the most ideal preparation schemes. Among them, in-solution enzymatic cleavage has restrictions on the composition of protein solutions, and common denaturants (such as urea and guanidine hydrochloride) will affect the activity of trypsin and thus the enzymatic effect; while FASP enzymatic hydrolysis steps are cumbersome, time-consuming, and cause large sample losses, which is not conducive to trace samples. Therefore, it is particularly urgent to develop sample preparation pretreatment methods suitable for crop samples with simple pretreatment steps. Summary of the invention

[0004] The purpose of the present invention is to provide a crop proteome sample pretreatment method based on protein solid phase enrichment technology to shorten the sample processing time and achieve convenient, rapid and high-throughput sample processing.

[0005] To achieve the above-mentioned purpose, the crop proteome sample pretreatment method based on solid phase enrichment technology provided in the present invention comprises the following steps: step S100, cleavage denaturation and reductive alkylation of proteins in crop tissue samples; step S200, solid phase enrichment of crop proteins using nanomagnetic beads; step S300, protein enzymatic hydrolysis, elution and low-temperature drying of peptides; step S400, mass spectrometry analysis of peptide samples.

[0006] Further, the step S100 includes: taking a crop tissue sample and adding a lysis solution for denaturation and reduction alkylation, centrifuging to obtain a supernatant after completion, and quantitatively detecting the protein in the supernatant. Step S200 includes: taking the protein sample obtained in step S100, adding magnetic beads to the sample at a mass ratio of protein: magnetic beads = 1:5-10, adding anhydrous ethanol to immerse the sample, shaking the sample to mix and react, adsorbing the magnetic beads with a magnetic stand and removing the supernatant. Step S300 includes: rinsing the solid obtained in step S200, adding NH4HCO3, and then adding trypsin or / and lysine protease to the solid for enzymatic hydrolysis, and eluting the peptides and drying at low temperature after enzymatic hydrolysis. Step S400 includes: taking the peptides obtained in step S300 for high-resolution mass spectrometry analysis (LC-MS / MS) and performing spectral comparison and identification on the sample.

[0007] Furthermore, before step S100, the method further includes the step of crushing the sample to be tested under liquid nitrogen conditions.

[0008] Furthermore, the lysate in step S100 includes 2%-4% SDS, 5-20 mM TCEP, 20-60 mM CAA reagent, and is assisted by ultrasonic oscillation and incubation at above 90°C.

[0009] Furthermore, in step S200, the particle size of the nanomagnetic beads is 200-1000 nm, and the surface of the magnetic beads is modified with functional groups such as carboxyl groups.

[0010] Furthermore, the step S200 also includes the following operation: washing with 80% ethanol for 6 times.

[0011] Furthermore, in the step S300, the concentration of NH4HCO3 is 50-100 mM, the enzymatic hydrolysis is a single enzymatic hydrolysis, the added mass ratio of protease is sample protein: protease = 50-100:1, and the enzymatic hydrolysis conditions are a constant temperature of 37°C and 1000 rpm for 4 hours.

[0012] Furthermore, the peptide elution and low-temperature drying steps in step S300 include the following operations: high-speed centrifugation after enzymatic hydrolysis, taking the supernatant, adding formic acid solution for acidification, and freeze-drying the supernatant.

[0013] Furthermore, the high-resolution mass spectrometry detection and analysis in step S400 includes: using liquid chromatography-tandem mass spectrometry for detection, using software to extract the obtained data, and obtaining qualitative and quantitative data of proteins and peptides.

[0014] Furthermore, in the high-resolution mass spectrometry analysis of step S400, the chromatographic conditions adopt a reverse phase chromatography separation method of a C18 chromatographic column; the mass spectrometry analysis conditions adopt a positive ion scanning mode, and the analysis time is 8-30 minutes.

[0015] In summary, the beneficial effects of the present invention are as follows: the present method can not only effectively remove various secondary metabolites such as chlorophyll in plant tissues, effectively purify proteins, reduce the interference of impurities on subsequent mass spectrometry analysis, and effectively improve the identification number and data quality stability of proteomes in pigment-rich plant tissues, but also simplify the sample processing steps, no longer require additional reduction alkylation steps and peptide desalting steps, and is efficient and fast while reducing experimental operation errors, and is suitable for automated sample pretreatment processes. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a flow chart of an embodiment of the method for pre-processing crop proteome samples based on solid phase enrichment technology in the present invention; Figure 2 It is the gene function annotation of the identified protein, including the protein cellular localization information (the annotation comes from the agriGO database); Figure 3 It is the gene function annotation for identifying proteins, including protein biological function information (annotation comes from the agriGO database). DETAILED DESCRIPTION

[0017] The present invention aims to provide a crop proteome sample pretreatment method based on protein solid phase enrichment technology to shorten the sample processing time and achieve convenient, rapid and high-throughput sample processing.

[0018] Reference Figure 1The technical scheme of the present invention is as follows: 1. The protein in the crop tissue sample is lysed, denatured and reduced by alkylation: take an appropriate amount of crop tissue and put it into an appropriate container and grind it under liquid nitrogen conditions, add lysis solution to the ground tissue cells for denaturation and reduction by alkylation (the lysis solution composition is: 2%-4% SDS, 100 mM Tris-HCl PH=7.5, 5-20 mM TCEP, 20-60 mM CAA), mix well, ultrasonically crush and then lyse in a water bath at high temperature. 2. Use nanomagnetic beads to solid phase enrich crop proteins: add nanomagnetic beads to the crude extract and replace the lysis solution, and perform multiple washings on the magnetic rack to remove the denaturant and non-protein impurities that affect the activity of trypsin. 3. Perform protein enzymatic hydrolysis, elute and low-temperature dry the peptides: add trypsin and other enzymes for hydrolysis, and the peptides obtained by enzymatic hydrolysis are eluted in situ and freeze-dried to complete the treatment. 4. Perform mass spectrometry analysis on the peptide samples: the freeze-dried samples are subjected to liquid chromatography-tandem mass spectrometry analysis.

[0019] This method can not only effectively remove chlorophyll and other secondary metabolites in plant tissues, effectively purify proteins, reduce the interference of impurities on subsequent mass spectrometry analysis, and effectively improve the identification number and data quality stability of proteomes in pigment-rich plant tissues, but also simplify the sample processing steps, no longer requiring additional reduction alkylation steps and peptide desalting steps. It is efficient and fast, reduces experimental operation errors, and is suitable for automated sample pretreatment processes.

[0020] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments (taking young wheat leaves as an example). The following embodiments are only used to illustrate the present invention and should not be regarded 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. Example 1

[0021] 1. Collect 5-day-old wheat leaves and quickly transfer them to liquid nitrogen. Grind them into powder in a cryo-grinder. Transfer the powder to a 50-mL centrifuge tube, add 2 mL of lysis buffer (4% sodium dodecyl sulfate (SDS), 100 mMTris-HCl PH=7.5, 10 mM tris(2-formylethyl)phosphine hydrochloride (TCEP), 40 mM chloroacetamide (CAA), and shake to mix.

[0022] 2. Incubate the sample in a 95℃ water bath for 5 minutes, place the sample tube in ice water for ultrasonic disruption, with a working time of 10 seconds, a rest period of 10 seconds, and a total ultrasonic time of 3 minutes, and incubate again at 95℃ for 5 minutes.

[0023] 3. Centrifuge at 10000g for 10 minutes, transfer the supernatant to a new 1.5 mL centrifuge tube, centrifuge at 10000g 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.

[0024] 4. Add 100ul of pure water to the washed nanomagnetic beads, mix by pipetting, then add 100ug of protein, and then add an equal volume of anhydrous ethanol to ensure that the volume ratio of ethanol is 50%. Mix by gently pipetting, oscillate at 600 rpm for 10 minutes, let stand on the magnetic stand for 5 minutes, and discard the liquid.

[0025] 5. Add 1 mL of 80% ethanol to each tube and mix by pipetting. Place the tube on a magnetic rack for 5 minutes and discard the liquid.

[0026] 6. Repeat step 5 five more times (the number of repetitions can be increased or decreased depending on the color depth of the sample). Pay attention to fully break up and clean the magnetic beads in each step, and be gentle.

[0027] After washing the beads, 50 mM ammonium bicarbonate was used to provide an alkaline environment for proteolysis.

[0028] 8. Protease enzymolysis: the mass ratio of protease to protein in each tube is 1:50, the reaction temperature is 37°C, the turntable is rotated, and the enzymolysis reaction time is 4 hours.

[0029] 9. After the enzymatic hydrolysis is completed, let it stand on the magnetic stand for 5 minutes and transfer the supernatant to a new tube. Then add 150μL of ammonium bicarbonate to the tube containing the nanomagnetic beads, mix and shake for 5 minutes, let it stand for 5 minutes, transfer the supernatant to the corresponding new tube, add formic acid to the new tube to a concentration of 0.1%, and terminate the enzymatic hydrolysis reaction. Determine the peptide concentration by BCA, freeze-dry in a frozen centrifugal concentrator, and freeze at -80℃ for loading.

[0030] 10. Redissolve the sample in 2% acetonitrile (containing 0.2% formic acid) solution for high-resolution mass spectrometry analysis (LC-MS / MS) and subsequent proteomic data analysis.

[0031] The experimental results are as follows: The results of high-resolution mass spectrometry analysis of protein samples extracted by the method described in Example 1 showed that it could fully meet the detection requirements. The number of peptide segments identified was large and stable, with three replicates of 106320, 112451, and 114800 respectively; the corresponding protein entries all exceeded 20,000, namely 21740, 22478, and 22759 respectively. Further analysis results can be found in Figure 2 , Figure 3According to the display in the figure, this method has good universality and has no preference in the protein enrichment and enzymatic hydrolysis process. It identifies that proteins come from multiple components of the cell, and the functions of the proteins are also widely distributed.

[0032] Compared with the prior art, this method has the following advantages and effects: The present invention provides a crop proteome sample pretreatment method based on protein solid phase enrichment technology. The method of the present invention can quickly complete reduction alkylation and use nanomagnetic beads as solid phase to efficiently adsorb crop proteins, can reduce sample pretreatment steps, effectively shorten sample processing time, and is conducive to convenient, fast and high-throughput sample processing. The protein sample extracted by the method described in Example 1 was subjected to high-resolution mass spectrometry analysis, and the results showed that it can fully meet the detection needs, and the number of peptide segments identified is large and stable, and the corresponding protein entries are all more than 20,000. Further analysis results show that the method has good universality and has no preference in protein enrichment and enzymatic hydrolysis. This method can not only effectively remove various secondary metabolites such as chlorophyll in plant tissues, effectively purify proteins, reduce the interference of impurities on subsequent mass spectrometry analysis, and effectively improve the identification number and data quality stability of proteomes in pigment-rich plant tissues, but also simplify the sample processing steps, no longer need additional reduction alkylation steps and peptide desalting steps, and is efficient and fast while reducing experimental operation errors, and is suitable for automated sample pretreatment processes.

Claims

1. A method for pre-treatment of crop proteome samples based on solid phase enrichment technology, characterized in that: The method comprises the following steps: Step S100, performing cleavage denaturation and reductive alkylation on proteins in the crop tissue sample; Step S200, using nanomagnetic beads to perform solid phase enrichment of crop proteins: taking the protein sample obtained in step S100, adding magnetic beads to the sample at a mass ratio of protein: magnetic beads = 1:5-10, and adding anhydrous ethanol to immerse the sample, shaking the sample to mix and react, adsorbing the magnetic beads with a magnetic stand, removing the supernatant, and washing with ethanol solution for multiple times; Step S300, performing protein enzymatic hydrolysis, eluting peptides and low-temperature drying: the solid obtained in step S200 is rinsed clean, NH4HCO3 is added, and then trypsin and / or lysine protease are added to the solid for enzymatic hydrolysis, and after enzymatic hydrolysis, the peptides are eluted and low-temperature dried; Step S400: performing mass spectrometry analysis on the peptide samples.

2. The method for pre-treatment of crop proteome samples based on solid phase enrichment technology according to claim 1, characterized in that: The step S100 comprises: taking a crop tissue sample and adding a lysis solution to perform denaturation and reductive alkylation, centrifuging to obtain a supernatant after completion, and quantitatively detecting the protein in the supernatant; The step S400 includes: performing high-resolution mass spectrometry detection and analysis on the peptide segments obtained in step S300 and performing spectrum comparison and identification on the samples.

3. The method for pre-processing crop proteome samples based on solid phase enrichment technology according to claim 2, characterized in that: The step S100 also includes the step of crushing the sample to be tested under liquid nitrogen conditions.

4. The method for pre-processing crop proteome samples based on solid phase enrichment technology according to claim 2, characterized in that: The lysate in step S100 includes 2%-4% SDS, 5-20 mM TCEP, 20-60 mM CAA reagent, and is assisted by ultrasonic oscillation and incubation at above 90°C.

5. The crop proteome sample pretreatment method based on solid phase enrichment technology according to claim 2, characterized in that: In the step S200, the particle size of the nanomagnetic beads is 200-1000 nm, and the surface of the magnetic beads is modified with functional groups such as carboxyl groups.

6. The method for pre-processing crop proteome samples based on solid phase enrichment technology according to claim 2, characterized in that: The step S200 specifically includes the following operations: washing with 80% ethanol for 6 times.

7. The method for pre-processing crop proteome samples based on solid phase enrichment technology according to claim 2, characterized in that: In the step S300, the concentration of NH4HCO3 is 50-100 mM, the enzymatic hydrolysis is a single enzymatic hydrolysis, and the added mass ratio of protease is sample protein: protease = 50-100:

1.

8. The method for pre-processing crop proteome samples based on solid phase enrichment technology according to claim 2, characterized in that: The peptide elution and low-temperature drying steps in step S300 include the following operations: high-speed centrifugation after enzymatic hydrolysis, taking the supernatant, adding formic acid solution for acidification, and freeze-drying the supernatant.

9. The method for pre-processing crop proteome samples based on solid phase enrichment technology according to claim 2, characterized in that: The high-resolution mass spectrometry detection and analysis in step S400 includes: using liquid chromatography-tandem mass spectrometry for detection, using software to extract the obtained data, and obtaining qualitative and quantitative data of proteins and peptides.

10. The method for pre-processing crop proteome samples based on solid phase enrichment technology according to claim 9, characterized in that: In the high-resolution mass spectrometry analysis of step S400, the chromatographic conditions adopt the reverse phase chromatographic separation method of the C18 chromatographic column; the mass spectrometry analysis conditions adopt the positive ion scanning mode, and the analysis time is 8-30 minutes.

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