CN115327016B - Extraction and detection method of tobacco protoplast proteome - Google Patents
Extraction and detection method of tobacco protoplast proteome Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
- G01N2030/8831—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving peptides or proteins
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Abstract
The application belongs to the technical field of plant physiology, and particularly relates to a method for extracting and detecting proteomes in tobacco protoplasts. The method takes tobacco protoplast as an extraction object, and specifically comprises the following steps: preparing tobacco protoplast, cell splitting, obtaining whole protein, detecting and the like. In the application, based on the research requirement of the tobacco proteome, the inventor optimizes the extraction method and the detection method of the tobacco proteome, and combines the stress treatment of related abiotic factors to perform preliminary research on the change condition of the tobacco protein components. The result shows that the extraction method and the detection method provided by the application have higher sensitivity, and can effectively support the research and application of metabolic proteome, thereby laying a good technical foundation for the research of related molecular regulation pathways and the research of gene functions, and presenting better practical value.
Description
Technical Field
The application belongs to the technical field of plant physiology, and particularly relates to a method for extracting and detecting proteomes in tobacco protoplasts.
Background
In the plant physiology research of the cell level, the whole component extraction and analysis detection are often required to be carried out on the plant cell content, thereby laying a technical foundation for the research of related molecular regulation mechanism. Tobacco (Nicotiana tabacum) is often a better representative of tobacco cell content studies as one of the model plants in botanicals studies.
In plant cell structures, plant cells can generally be divided into two parts, a cell wall and a protoplast, i.e., protoplasts refer to cells in which the cell wall is absent, because the cell wall structure often has independence. For plant content extraction and analysis, protoplasts are often used as analysis study objects, so that the accuracy of the study results is ensured.
In proteomics research, it is often necessary to extract specific whole protein components from the cell content. In practical research, proteomics research often determines related regulation paths by comparing and analyzing protein components or abundance changes caused by certain environmental condition changes (such as stress treatment under abiotic factors), so as to lay a mechanism foundation for researching molecular regulation paths. Therefore, the integrity, accuracy, and associated detection analysis accuracy of proteome extraction often have a substantial impact on the actual research results.
Disclosure of Invention
The application aims to provide an extraction and detection method of proteomes in tobacco protoplasts, thereby laying a certain technical foundation for relevant tobacco proteomics research.
The technical scheme adopted by the application is briefly described below.
The method for extracting and detecting the proteome in the tobacco protoplast takes the tobacco protoplast as an extraction object and specifically comprises the following steps:
firstly, preparing materials
According to the prior art, taking tobacco mesophyll cells as raw materials, and conventionally preparing tobacco protoplasts for later use;
(II) cell lysis
Dissolving a protoplast sample in a Sodium Dodecyl Sulfate (SDS) buffer solution, and performing ultrasonic disruption; crushing, adding pre-cooled acetone, and precipitating at-20deg.C for more than 4 hr; the specific operation references are as follows:
protoplast samples (1 ml sample, cell amount in the sample is not less than 10) 6 Dissolving in 1-5 mL Sodium Dodecyl Sulfate (SDS) buffer solution, transferring into a 10 mL-specification centrifuge tube after dissolving, and placing on ice;
ultrasonic treatment is carried out for 30-60 seconds (ultrasonic treatment is carried out for 2s and stopping is carried out for 3s, and total time is 30-60 s) with power of 60 percent;
after ultrasonic crushing, adding 4-6 volumes of pre-cooled acetone, and precipitating for more than 4 hours at the temperature of minus 20 ℃;
(III) obtaining holoprotein
Centrifuging the mixed solution after the precipitation treatment in the step (II), discarding the supernatant, washing with precooled acetone, re-suspending with heavy suspension (under the condition of the dosage of 1-3 mL), performing ultrasonic treatment again, and centrifuging and collecting to obtain whole protein;
the resuspension: the heavy suspension contains 6-8M urea and 50 mM ammonium bicarbonate;
the specific operation references are as follows:
centrifuging the mixed solution after the precipitation treatment in the step (II) for 5-20 minutes at the temperature of 4 ℃ and under the condition of 13000-20000 and g, and reserving precipitation;
after resuspension washing the pellet with 1ml of pre-chilled acetone, it is centrifuged at 4℃and 13000-20000, g for 5-20 minutes (this operation is repeated twice to ensure the washing effect); a precipitate is left;
resuspension the pellet with 1-3 mL resuspension and sonication (60% power, total sonication for 5min, period of sonication for 2s, stop for 3 s);
centrifuging at 4deg.C and 13000-20000 and g for 5-20 min after ultrasonic treatment, and collecting to obtain whole protein;
for collection of whole proteins, further protein concentration can be determined using the (BCA) protein assay kit;
(IV) detection
Taking the whole protein sample extracted in the step (III), and carrying out the following pretreatment:
(1) Uniformly mixing the whole protein with Dithiothreitol (DTT) with a final concentration of 5 mM, and incubating for 30 min at 56 ℃; the protein was then alkylated with 40 mM Iodoacetamide (IAA) under dark conditions for 30 min; adding DTT to the final concentration of 10mM, and continuously treating for 10min under dark conditions;
(2) Diluting the protein alkylated sample with 50 mM ammonium bicarbonate, adding trypsin, and performing enzymolysis digestion for 12-24 hours at 37 ℃ on a shaking table;
protein: trypsin= (25-50): 1;
(3) Desalting the digested sample in step (2) by using an HLB solid phase extraction column (SPE), and washing the obtained polypeptide sample with 1% trifluoroacetic acid (TFA) (typically 3 times);
finally, eluting the sample from the HLB solid phase extraction column (SPE) by using 1-3 mL of 80% Acetonitrile (ACN) and 0.1% TFA, and preserving at-80 ℃ for standby detection after drying;
in the final detection, the method can be divided into: data dependent acquisition (data-dependent acquisition, DDA) methods and data independent acquisition methods (data-independent acquisition, DIA); specifically:
data Dependent Acquisition (DDA) method:
mobile phase a:0.1% formic acid-MS water;
mobile phase B:0.1% formic acid-MS CAN;
linear gradient:
in the analysis process, 30-50 ions with the largest response value are selected for secondary acquisition;
the mass range of TOF-MS is 350-1250 m/z, the high resolution mode (> 2.5 ten thousand), and the acquisition time is 250 MS;
the spray voltage of the heated capillary tube is set to 2.5 kV, and the temperature is set to 150 ℃;
the data file was processed using the ProteinPilot software (AB SCIEX, version 5.0);
using the K326 database, the reference parameters are as follows: the enzyme is set as trypsin, and the Cys alkylation reaction is the solidifying reaction of iodoacetamide; when the minimum peptide length is set to 7, the False Discovery Rate (FDR) is less than 1.0%;
data Independent Acquisition (DIA) method:
LC parameter conditions are the same as above;
80 dynamic windows are constructed, the mass range is 350-1500 Da, and the ion accumulation time is 20-50 ms;
SWATH quantification by Peakview software (version 2.1.10, AB SCIEX) matched to ion library (. Wiff and. Wiffscan file); derived from software ProteinPilot (AB SCIEX, version 5.0);
setting peptide extraction window parameters: 5 peptide fragments of each protein, 4 fragment ions of each peptide fragment, the confidence score of the peptide is more than or equal to 99%, the FDR is less than or equal to 1%, the extraction window time of extraction ion chromatography (XIC) is 10min, and the XIC window is 0.01 and Da;
protein and peptide quantitative information was derived. mrkv file, and Marker View software (version 1.2.1.1, AB SCIEX) was imported for area and normalization and t-test.
In the application, based on the research requirement of the tobacco proteome, the inventor optimizes the extraction method and the detection method of the tobacco proteome, and combines the stress treatment of related abiotic factors to perform preliminary research on the change condition of the tobacco protein components. The result shows that the extraction method and the detection method provided by the application have higher sensitivity, and can effectively support the research and application of metabolic proteome, thereby laying a good technical foundation for the research of related molecular regulation pathways and the research of gene functions, and presenting better practical value.
Drawings
FIG. 1 DDA mass spectrometry chromatogram of tobacco mesophyll cell protoplasts;
FIG. 2 DIA mass spectrometry chromatogram of tobacco mesophyll cell protoplasts;
fig. 3 OPLS-plots of control and treatment groups;
FIG. 4S-plot of control and treatment groups;
FIG. 5 differential protein heat maps of control and treatment groups.
Detailed Description
The present application is further illustrated below with reference to examples. Before describing the specific embodiments, the following description will briefly explain some experimental contexts in the following embodiments.
Biological material:
tobacco (tobacco)Nicotiana tabacum) K326, a common tobacco variety for cultivation and research; applicant has long stored this study material as a professional tobacco research institution;
main experimental facilities:
triple TOF 5600+ mass spectrometer, ekspertTM nano LC 415 liquid phase system (used in conjunction with mass spectrometer), trap column (ChromaXP C18-CL, 3 μm, 120A, 350 μm X0.5 mm), chromatographic column (C18-CL-120, 3 μm, 120A, 75 μm X15 cm), all were AB SCIEX (USA) company products.
Example 1
Taking proteome extraction and detection in K326 tobacco mesophyll cells as an example, related technical schemes are described in detail below.
Firstly, preparing materials
According to the prior art, taking tobacco mesophyll cells (30-35 days old) as raw materials, conventionally preparing tobacco protoplasts, and finally, centrifuging the prepared tobacco mesophyll protoplasts at 400-600 rpm, discarding the supernatant, and reserving the sediment (namely the protoplasts).
For protoplast preparation, reference may be made specifically to the following procedure:
taking 1 leaf of about 30 days old seedling, cutting into filaments with width of about 10mm, placing into a centrifuge tube, adding 8 mL mixed enzyme solution (1% cellulase, 0.5% segregation enzyme and 9% CPW9 solution), and slightly oscillating to thoroughly mix tobacco filaments with enzyme solution; placing the mixture on a constant temperature shaking table, and performing enzymolysis at 28 ℃ for 2.5 h;
filtering (removing blade tissues which are not subjected to enzymolysis) after enzymolysis is finished;
centrifuging the filtrate for 5min to remove supernatant, and precipitating to obtain protoplast cells.
(II) cell lysis
Dissolving a protoplast sample in a Sodium Dodecyl Sulfate (SDS) buffer solution, and performing ultrasonic disruption; crushing, adding pre-cooled acetone, and precipitating at-20deg.C for more than 4 hr; the specific operation references are as follows:
protoplast samples (protoplast cell quantity 10) 6 About two) are dissolved in 1mL Sodium Dodecyl Sulfate (SDS) buffer solution, and the solution is transferred into a centrifuge tube with the specification of 10 mL after being dissolved, and the solution is placed on ice;
ultrasonic for 60 seconds (ultrasonic for 2s, stop for 3s, total time of 60 s) at 60% power;
after ultrasonication, 4 volumes of pre-chilled acetone were added and precipitated at-20℃for 4 hours.
(III) obtaining holoprotein
Centrifuging the mixed solution after the precipitation treatment in the step (II), discarding the supernatant, washing with precooled acetone, re-suspending with heavy suspension, performing ultrasonic treatment again, and centrifuging and collecting to obtain whole protein;
the resuspension (aqueous solution): contains 8M urea and 50 mM ammonium bicarbonate;
the specific operation references are as follows:
centrifuging the mixed solution after the precipitation treatment in the step (two) for 10 minutes at the temperature of 4 ℃ and at the temperature of 15000 and g, and reserving precipitation;
after resuspension washing the pellet with 1ml of pre-chilled acetone, it was centrifuged at 15000 g for 10min at 4 ℃ (this operation was repeated twice to ensure the washing effect); a precipitate is left;
the pellet was resuspended with 2 ml resuspension and sonicated (60% power, total sonication for 5min, period of 2s, 3s stopped);
centrifuging at 4deg.C and 15000 g for 10min after ultrasonic treatment, and collecting to obtain total protein;
for collection of whole proteins, the concentration of the whole protein obtained was measured using a bicinconiic acid (BCA) protein assay kit (product of Thermo Scientific company) using Bovine Serum Albumin (BSA) (2 mg/mL) as a standard. The measurement results are as follows: 0.16 mg/ml.
(IV) detection
Taking the whole protein sample extracted in the step (III), and carrying out the following pretreatment:
(1) After mixing whole protein (100. Mu.g) with Dithiothreitol (DTT) at a final concentration of 5 mM, incubating for 30 min at 56 ℃; the protein was then alkylated with 40 mM Iodoacetamide (IAA) under dark conditions for 30 min; adding DTT to the final concentration of 10mM, and continuously treating for 10min under dark conditions;
(2) Diluting the protein alkylated sample with 50 mM ammonium bicarbonate, adding trypsin, and performing enzymolysis and digestion on a shaking table at 37 ℃ for 16 hours;
protein: trypsin = 50:1;
(3) Desalting the digested sample in the step (2) by using an HLB solid phase extraction column (SPE), and washing the obtained polypeptide sample with 1% trifluoroacetic acid (TFA) (3 times);
finally, eluting the sample from the HLB solid phase extraction column (SPE) with 3 mL of 80% Acetonitrile (ACN) and 0.1% TFA, drying, and preserving at-80 ℃ for standby detection;
in the final detection, the method can be divided into: data dependent acquisition (data-dependent acquisition, DDA) methods and data independent acquisition methods (data-independent acquisition, DIA).
Data Dependent Acquisition (DDA) method:
the liquid chromatography-mass spectrometry analysis comprises the following steps:
mobile phase a:0.1% formic acid-MS water;
mobile phase B:0.1% formic acid-MS CAN;
linear gradient:
in the analysis process, carrying out secondary acquisition on 30-50 ions with maximum response values;
for TOF-MS (time of flight mass spectrometry) analysis, the analysis parameters were set as: the mass range is 350-1250 m/z, the high resolution mode (> 2.5 ten thousand), and the acquisition time is 250 ms;
the spray voltage of the heated capillary tube was set to 2.5 kV and the temperature was set to 150 ℃.
The data file was processed using the ProteinPilot software (AB SCIEX, version 5.0).
Meanwhile, the K326 database constructed and owned by the applicant is combined for comparison analysis, and the reference parameters are as follows: the enzyme is set as trypsin, and the Cys alkylation reaction is the solidifying reaction of iodoacetamide; when the minimum peptide length was set to 7, the False Discovery Rate (FDR) was less than 1.0%.
Data Independent Acquisition (DIA) method:
LC (liquid chromatography) parameter conditions are the same as above;
in the analysis process, 80 dynamic windows are constructed, the mass range is 350-1500 Da, and the ion accumulation time is 20-50 ms;
SWATH quantification by Peakview software (version 2.1.10, AB SCIEX) matched to ion library (wiff and Wiffscan file); derived from software ProteinPilot (AB SCIEX, version 5.0);
setting peptide extraction window parameters: 5 peptide fragments of each protein, 4 fragment ions of each peptide fragment, the confidence score of the peptide is more than or equal to 99%, the FDR is less than or equal to 1%, the extraction window time of extraction ion chromatography (XIC) is 10min, and the XIC window is 0.01 and Da;
protein and peptide quantitative information was exported as mrkv files, and Marker View software (version 1.2.1.1, AB SCIEX) was imported for area and normalization and t-test.
Analysis of the results obtained from protoplasts obtained under normal conditions but obtained from different collection methods (fig. 1 and 2) shows that the relevant protein detection gradient is more suitable, the elution is more uniform, and the secondary spectrum collection is more reasonable, so that the method can be used for practical detection analysis application.
In order to further examine and evaluate the sensitivity of the proteome detection method, the inventor carries out Cd treatment (10 mu M treatment) on the prepared tobacco mesophyll cell protoplast, and further carries out detection analysis on the change condition of the proteome. The results show that: in the control group, more than 3000 proteins were identified in total, while 453 differential proteins were obtained by statistical analysis of the proteomes of the treated and control samples (two groups of results were analyzed with T-test (p < 0.05) and VIP values of SIMCA software (VIP > 1), respectively). Specifically:
by analyzing the OPLS-graph of FIG. 3, it can be demonstrated that the samples are obviously separated after treatment and before treatment, and there is a significant difference between groups, which lays a foundation for further protein differentiation and identification;
the S-plot of FIG. 4 gives the distribution of the variable contributing to the difference, namely the difference protein, which lays a foundation for further distinguishing the proteins;
FIG. 5 is a thermal diagram of a differential protein, which is a visual treatment of the differential protein, thereby facilitating more visual screening of the target protein; and the related data result shows that: more than half of the processed differential proteins show a descending trend, and the small half of the processed differential proteins show an ascending trend, so that further guidance and reference can be provided for further screening and defining which proteins possibly participate in the Cd-resistant function of tobacco.
Claims (3)
1. The method for extracting and detecting the proteome in the tobacco protoplast is characterized by taking the tobacco protoplast as an extraction object and comprises the following steps:
firstly, preparing materials
According to the prior art, taking tobacco mesophyll cells as raw materials, and preparing tobacco protoplasts for later use;
(II) cell lysis
Dissolving a protoplast sample in a sodium dodecyl sulfate buffer solution, and performing ultrasonic crushing; crushing, adding pre-cooled acetone, and precipitating at-20deg.C for more than 4 hr;
(III) obtaining holoprotein
Centrifuging the mixed solution after the precipitation treatment in the step (II), discarding the supernatant, washing with precooled acetone, re-suspending with heavy suspension, performing ultrasonic treatment again, and centrifuging and collecting to obtain whole protein;
the resuspension: the heavy suspension contains 6-8M urea and 50 mM ammonium bicarbonate; the specific operation is as follows:
centrifuging the mixed solution after the precipitation treatment in the step (II) for 5-20 minutes at the temperature of 4 ℃ and under the condition of 13000-20000 and g, and reserving precipitation;
re-suspending and cleaning the precipitate with 1ml of pre-cooled acetone, and centrifuging at 4 ℃ for 5-20 minutes with 13000-20000 and g; a precipitate is left;
re-suspending the precipitate with 1-3 mL heavy suspension, and performing ultrasonic treatment;
centrifuging at 4deg.C and 13000-20000 and g for 5-20 min after ultrasonic treatment, and collecting to obtain whole protein;
(IV) detection
Taking the whole protein sample extracted in the step (III), carrying out trypsin digestion and enzymolysis after alkylation treatment, and purifying peptide fragments after digestion and enzymolysis by utilizing an HLB solid phase extraction column; the specific processing operation is as follows:
(1) Uniformly mixing the whole protein with dithiothreitol DTT with the final concentration of 5 mM, and incubating for 30 min at 56 ℃; then alkylating the protein with 40 mM iodoacetamide IAA under dark conditions for 30 min; adding DTT to the final concentration of 10mM, and continuously treating for 10min under dark conditions;
(2) Diluting the protein alkylated sample with 50 mM ammonium bicarbonate, adding trypsin, and performing enzymolysis digestion on a shaker at 37 ℃ for 12-24 hours;
protein: trypsin = 25-50:1;
(3) Desalting the digested sample in the step (2) by using an HLB solid phase extraction column, and washing the obtained polypeptide sample by using 1% trifluoroacetic acid TFA;
finally, eluting the sample from the HLB solid phase extraction column with 1-3 mL of 80% acetonitrile ACN and 0.1% TFA, and drying;
during detection, the method can be divided into: a data dependent acquisition DDA method and a data independent acquisition DIA method; specifically:
data dependent acquisition DDA method:
mobile phase a:0.1% formic acid-MS water;
mobile phase B:0.1% formic acid-MS CAN;
linear gradient:
in the analysis process, 30-50 ions with the largest response value are selected for secondary acquisition;
the mass range of TOF-MS is 350-1250 m/z, the high resolution mode of > 2.5 ten thousand, and the acquisition time is 250 MS; the spray voltage of the heated capillary tube is set to 2.5 kV, and the temperature is set to 150 ℃;
data independent acquisition DIA method:
LC parameter conditions are the same as above;
80 dynamic windows are constructed, the mass range is 350-1500 Da, and the ion accumulation time is 20-50 ms.
2. The method for the extraction and detection of proteomes in tobacco protoplasts according to claim 1, wherein in the step (one), the tobacco protoplasts are Cd-treated protoplasts.
3. The method for extracting and detecting proteomes from tobacco protoplasts according to claim 1, wherein in the step (two), the ultrasonic disruption is: crushing is carried out by ultrasonic waves with the power of 60 percent for 30-60 seconds.
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