CN108828126B - Method for qualitatively identifying strawberry stigma protein based on two-dimensional liquid chromatography tandem mass spectrometry - Google Patents
Method for qualitatively identifying strawberry stigma protein based on two-dimensional liquid chromatography tandem mass spectrometry 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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- 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
Abstract
The invention discloses a method for qualitatively identifying strawberry stigma protein based on a two-dimensional liquid chromatography tandem mass spectrometry platform, which comprises the following steps: 1) extracting and purifying the strawberry stigma to obtain protein powder; 2) protein quantification and reductive alkylation; 3) performing proteolysis; 4) separating the peptide fragment mixture by a first dimension SCX strong cation exchange liquid chromatogram; 5) second dimension reversed C18 liquid phase separation tandem Q active mass spectrometry; 6) and data analysis: and identifying the original file obtained by the mass spectrometer by a Maxquant library so as to obtain a protein and peptide fragment information list. The method can be used for qualitatively identifying the red-cheek strawberry stigma protein and provides a foundation for constructing a red-cheek strawberry stigma protein information base.
Description
Technical Field
The invention relates to the field of plant proteomics, and provides a method for identifying strawberry (especially red-cheek strawberry) stigma protein by using strong cation exchange chromatography and reverse polarity chromatography two-dimensional separation and connecting a Q exact mass spectrometer in series.
Background
The strawberry belongs to Rosaceae (Rosaceae) strawberry (Fragaria), is popular among people due to sweet taste and rich nutrient elements of the fruit, and the market demand for new varieties is gradually expanded along with the increase of the consumption level of people. The red-cheek strawberry is a high-quality big fruit type variety (introduced by Hangzhou city agricultural science research institute in 2006) bred in Japan, and becomes one of the most popular varieties in the market at present due to the big and regular fruit shape, proper sour and sweet taste and high fruit hardness, but the variety has weak resistance to anthracnose, gray mold and the like. The method for hybridizing the red cheeks with the disease-resistant variety is an idea for cultivating a new strawberry variety, and the research on stigmatiselity has important significance on hybridization breeding. The most common method for detecting stigma receptivity at present is the benzidine-hydrogen peroxide method, which can intuitively and effectively detect stigma receptivity, but the current mechanism research on receptivity change is still few.
Proteins are important components of living organisms and are direct executives of life activities, and proteins are involved in all important physiological phenomena of living organisms such as development, metabolism and stress. Proteomics reveals the essential mechanism of life activities from the level of whole proteins by analyzing the expression level of proteins, the interaction relationship among proteins, and the like. In recent years, with the improvement of resolution and scanning speed of mass spectrometry instruments, proteomics analysis technology based on a mass spectrometry platform is also rapidly developed. At present, the protein identification method by using the mass spectrometry technology mainly comprises 3 methods: bottom-up, Middle-down, Top-down. Among them, the Bottom-up method, also called as the shotgun method, is the most mainstream method at present, and the method is to finally obtain protein information by separating peptide fragments formed by proteolysis through liquid chromatography, identifying a mass spectrometer and assembling a bioinformatics tool. Due to the limited detection capability of mass spectrometry in unit time, the pre-separation quality of peptide fragments is often the key factor for determining the identification amount of protein. The currently used peptide fragment separation methods comprise 3 conventional reverse C18 column one-dimensional liquid phase separation, SCX strong cation exchange column and conventional reverse C18 column two-dimensional liquid phase separation, high pH reverse C18 column and conventional reverse C18 column two-dimensional liquid phase separation.
However, the peptide fragment obtained by only using the C18 one-dimensional liquid phase has high complexity, insufficient separation degree and limited protein amount identified by mass spectrometry, and usually the Q active single needle protein identification amount is only about 2000-3500; the separation effect of the two-dimensional liquid phase of the SCX-C18 serial connection and the high-pH reverse C18-conventional reverse C18 serial connection is greatly influenced by the liquid phase gradient, the separation effect of the common gradient reported in the literature on the red-cheek strawberry stigma protein peptide fragment is poor, and the separation gradient needs to be optimized in order to improve the identification amount.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for qualitatively identifying strawberry (especially red-cheek strawberry) stigma protein by using high performance liquid chromatography tandem mass spectrometry, and providing a foundation for constructing a strawberry stigma protein information base.
In order to solve the technical problem, the invention provides a method for qualitatively identifying strawberry stigma protein based on a two-dimensional liquid chromatography tandem mass spectrometry platform, which sequentially comprises the following steps:
1) preparing a sample, extracting and purifying;
mixing strawberry stigma in a small bud stage, a medium bud stage and a large bud stage according to the quantity of 1:1:1, and then taking the mixture as a sample, and extracting and purifying to obtain protein powder;
2) protein quantification and reductive alkylation;
dissolving protein powder in lysate to obtain protein solution (generally, dissolving 0.25 + -0.02 g of lysate in 400 + -40 μ L to obtain protein powder);
a protein solution containing 200. mu.g of protein was added to 100mM NH4HCO3Diluting the solution (pH7.8) to 100 μ L, adding 11 μ L100 mM DTT (DL-Dithiothreitol, Dithiothreitol) solution, mixing, and reacting at 37 + -1 deg.C (metal bath) for 1 + -0.1 h; adding 12 μ L500 mM IAM (Iodoacetamide ) solution, and reacting at room temperature for 30 + -2 min in dark place;
remarking: the above steps can be performed in a 1.5mL centrifuge tube, and the protein quantification is performed by using a Bradford protein concentration determination kit (petunia), and the steps are described in the specification;
3) performing proteolysis;
ultrafiltering the reaction liquid obtained in the step 2) to remove impurities, and then adding 100mM NH4HCO3(pH8.0) to the total volume of 100 mu L, adding 4 mu g of Trypsin (Promega), and reacting for 12-16 h (overnight) at 37 +/-1 ℃ (metal bath), thereby realizing enzymolysis;
centrifuging after the above enzymolysis is completed, and adding 100mM NH into the precipitate obtained by centrifuging4HCO3Repeatedly centrifuging once after 100 mu L, and combining the liquid obtained by twice centrifugation to obtain a peptide fragment solution; the centrifugation is carried out for 15 plus or minus 2min at 12000 plus or minus 2000rpm at 4 plus or minus 1 ℃;
adding 5% (V/V) TFA aqueous solution into the peptide fragment solution until the final concentration of TFA (Trifluoroacetic acid ) is 0.1-1% (V/V), desalting, and freeze-drying (freeze-drying at-70-20 ℃ to constant weight) to obtain freeze-dried powder;
4) separating the peptide fragment mixture by a first dimension SCX strong cation exchange liquid chromatogram;
SCX Buffer A：10mMKH2PO425% acetonitrile, pH 2.9;
SCX Buffer B：10mM KH2PO4400mM KCl, 25% acetonitrile, pH 2.9;
the preparation method of the SCX Buffer A comprises the following steps: 10mmol of KH are added to 1L of 25% strength by volume aqueous acetonitrile2PO4Adjusting to pH 2.9;
the preparation method of the SCX Buffer B comprises the following steps: 10mmol of KH are added to 1L of 25% strength by volume aqueous acetonitrile2PO4400mmol of KCl, and adjusting the pH value to 2.9;
redissolving all the freeze-dried powder obtained in the step 3) by using SCX Buffer A (100 mu l), and performing gradient separation by using a Biobasic SCX column (Thermofeisher); the separation gradient was:
0～5min，0％SCX Buffer B；
5～8min,0％～3％SCX Buffer B；
8～50min,3％～40％SCX Buffer B；
50～60min,40～100％SCX Buffer B；
60～65min,100％SCX Buffer B；
65-70min,100～0％SCX buffer B；
the balance of the mobile phase in each time period is SCX Buffer A; in each time period, the concentration of SCX Buffer B is uniformly changed; the elution flow rate is 1 ml/min; collecting the elution component containing the peptide fragment (about 10-50min of elution component, wherein the time period is the time period when the peptide fragment flows out); freeze-drying (freeze-drying to constant weight at-70-20 ℃) after desalting; obtaining peptide fragment powder;
5) second dimension reversed C18 liquid phase separation tandem Q active mass spectrometry;
c18Buffer A0.1% formic acid solution;
c18Buffer B0.1% formic acid acetonitrile solution;
performing the following mass spectrometry on the peptide fragment powder obtained in the step 4):
redissolving with C18Buffer A, AcclaimTMPepMapTMPerforming gradient on-line separation on a 100C 18 liquid chromatographic column (Thermofoisher), and then analyzing by a Q exact mass spectrometer (Thermofoisher); separation gradient: 0-3 min, 4-7% of C18Buffer B; 3-103 min, 7-18% of C18Buffer B; 103-113 min, 18-35% of C18Buffer B; 113-117 min, 35-75% of C18Buffer B, 117-120 min and 75% of C18Buffer B;
the balance of the mobile phase in each time period is C18Buffer A; the concentration of C18Buffer B was changed uniformly in each time period; the elution flow rate is 1 ml/min;
6) and data analysis:
the raw file from the mass spectrometer was identified by a Maxquant search (protein library downloaded from Strawberry Garden, with reference to Maxquant instructions for the search parameters) to obtain a list of protein and peptide fragment information.
The method for qualitatively identifying the strawberry stigma protein based on the two-dimensional liquid chromatography tandem mass spectrometry platform is improved as follows:
in the step 4), one tube is collected every 10-18min, one tube is collected every 4 min at 18-50min (namely, one tube is collected every minute, and every 4 tubes are combined into 1 tube), and 16 tubes are counted; freeze-drying (freeze-drying to constant weight at-70-20 ℃) after desalting each tube; thus obtaining 16 peptide fragment powders;
in the step 5), performing mass spectrometry on the 16 kinds of peptide fragment powder obtained in the step 4) respectively; each peptide fragment powder was reconstituted with 20. mu. L C18Buffer A.
As a further improvement of the qualitative identification method of strawberry stigma protein based on the two-dimensional liquid chromatography tandem mass spectrometry platform, the step 1) is as follows:
mixing the strawberry stigmas in the small bud stage, the medium bud stage and the large bud stage according to the number of 1:1:1 to obtain a sample (for example, randomly mixing 50 of the strawberry stigmas in the small bud stage, the strawberry stigmas in the medium bud stage and the strawberry stigmas in the large bud stage), uniformly grinding by using liquid nitrogen, taking 0.25 +/-0.02 g of the strawberry stigmas ground substance (which can be transferred to a 2mL centrifuge tube), adding 1.8 +/-0.02 mL of an extracting agent, shaking and uniformly mixing on ice for 30 +/-5 min, and then carrying out primary centrifugation at 4 +/-1 ℃; adding 4.5-5.5 volume times of-20 +/-2 ℃ precooled 0.1 +/-0.02M ammonium acetate methanol solution into the obtained supernatant, standing for 12-16 hours at-20 +/-2 ℃, and centrifuging for the second time at 4 +/-1 ℃ (discarding the supernatant); washing the precipitate obtained by secondary centrifugation with precooled methanol at-20 +/-2 ℃, and freeze-drying (freeze-drying at 70 to-20 ℃ to constant weight) to obtain protein powder;
the extractant is prepared by mixing pre-cooled extracting solution at 4 +/-1 ℃ and Tris saturated phenol according to the volume ratio of 1: 1;
the extracting solution is as follows: 0.7M sucrose, 0.1M KCl, 0.5M Tris-HCl, 2% mercaptoethanol, 50mM EDTA, adjusted to pH 8.0;
the preparation method of the extracting solution comprises the following steps: adding 0.7mol of sucrose, 0.1mol of KCl, 20mL of mercaptoethanol and 50mmol of EDTA into 1L of 0.5mol/L Tris-HCl buffer solution, and adjusting the pH value to 8.0;
the Tris saturated phenol is: a solution of phenol in Tris-HCl buffer (pH8.0) fully saturated.
As a further improvement of the method for qualitatively identifying strawberry stigma protein based on the two-dimensional liquid chromatography tandem mass spectrometry platform, in the step 1), a filter cake obtained by primary centrifugation replaces 0.25g of strawberry stigma ground substance, and the steps of adding the extracting agent on ice, vibrating and centrifuging are repeated; the repetition times are 1-3 times; and combining the supernatants obtained by centrifugation, and then carrying out the subsequent steps.
As a further improvement of the qualitative identification method of strawberry stigma protein based on the two-dimensional liquid chromatography tandem mass spectrometry platform, in the step 1), the primary centrifugation and the secondary centrifugation are both: centrifuging at 5000 + -100 g at 4 + -1 deg.C for 30 + -5 min;
as a further improvement of the qualitative identification method of strawberry stigma protein based on the two-dimensional liquid chromatography-tandem mass spectrometry platform, the ultrafiltration impurity removal of the step 3) is to remove impurities from the reaction liquid obtained in the step 2) by using an Amicon-Ultra-15 ultrafiltration tube (Millipore).
The method specifically comprises the following steps: transferring the reaction solution obtained in the step 2) into an Amicon-Ultra-15 ultrafiltration tube (Millipore), and centrifuging at 12000 +/-2000 rpm at 4 +/-1 ℃ for 20 +/-2 min; discard the waste stream from centrifugation and add 150. mu.L of 100mM NH to the ultrafiltration tube4HCO3The solution was centrifuged at 12000rpm for 20min at 4 deg.C (100 mM NH added as described above was repeated)4HCO3Solution, step 1 of centrifugation).
As a further improvement of the qualitative identification method of strawberry stigma protein based on the two-dimensional liquid chromatography tandem mass spectrometry platform, the desalting in the step 3) and the step 4) adopts PierceTMC18 tips (thermolfisher) removes salt. Desalting procedure strictly as PierceTMC18 tips (thermolfisher) instructions.
As a further improvement of the qualitative identification method of strawberry stigma protein based on the two-dimensional liquid chromatography tandem mass spectrometry platform, the preparation method of the lysate in the step 2) comprises the following steps: 48g of urea and 0.017g of PMSF are dissolved in 0.1mol/L of Tris-HCl buffer solution, the volume is determined to be 100mL, and the pH value is adjusted to 8.0 (adjusted by using 1M hydrochloric acid).
The lysis solution is: 8M Urea, 0.1M Tris-HCl, 1mM PMSF (Phenylmethanesulfonylfluoride, phenylmethylsulfonyl fluoride), adjusted to pH 8.0.
As a further improvement of the qualitative identification method of strawberry stigma protein based on the two-dimensional liquid chromatography tandem mass spectrometry platform, the strawberry is red-buccal strawberry.
According to the method, the total identification quantity of the strawberry stigma protein in the small bud period, the medium bud period and the large bud period is more than 8000, the identification quantity of the peptide segment is 39000, and a solid foundation is provided for constructing a strawberry stigma protein information base. The small bud, medium bud and large bud period of stigma of red-cheek strawberry basically covers the process of weakening stigma receptivity from weak to strong and then weakening in the development process. If only one bud stage is taken, the protein information related to stigma granting sex in a certain development period is lost.
Mass spectrometers have limited detection capabilities per unit time, and total protein samples are often very complex, far exceeding the detection capabilities of current mass spectrometers. The peptide fragments of the proteolysis products are pre-separated in the step 4), so that the peptide fragments entering the mass spectrum in unit time are kept consistent as much as possible, and the detection result can be greatly improved. Amino acids can be classified into polar amino acids (12 species) and non-polar amino acids (8 species) according to the structure of the side chain group R. The peptide segments obtained by protein enzymolysis also show differences in overall polarity due to differences in the composition and content of internal amino acids, and the peptide segments with multiple polar amino acid residues are strong in hydrophilicity, and conversely, are strong in hydrophobicity. In the separation process of the reverse C18 chromatographic column, the hydrophilic peptide fragment flows out firstly, and the hydrophobic peptide fragment flows out later, so that the separation purpose is achieved; the invention therefore provides for the second dimension of step 5) to be reversed in the C18 liquid phase. The result of a preliminary experiment shows that the proportion of the hydrophilic peptide segment in the red-cheek strawberry stigma proteolysis product is far greater than that of the hydrophobic peptide segment, so that the final protein identification amount is influenced if the conventional separation gradient separation effect is poor. The invention equally divides the total protein of the red-cheek strawberry stigma into 16 components (namely, 16 large tubes corresponding to the step 4) by optimizing the SCX chromatographic separation gradient and the component combination method); and by optimizing the chromatographic separation gradient of C18, the organic phase proportion is reduced, the flow time of the low organic phase is prolonged, and a liquid phase peak diagram and a mass spectrum identification result with better separation effect are obtained.
According to the invention, the mechanism of the permissive change in the red-cheek strawberry stigma development process is researched on the protein level, and the qualitative identification and the information base construction of the red-cheek strawberry stigma protein are firstly carried out. The invention qualitatively identifies the red-cheek strawberry stigma protein by two-dimensional liquid phase separation of an SCX strong cation exchange column and a conventional reverse C18 column and connecting a Q-exact high-resolution mass spectrometer in series, and provides a foundation for constructing a red-cheek strawberry stigma protein information base.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 is a comparison of the amount of protein identified in example 1 and comparative examples 1 to 3;
FIG. 2 is a graph of protein wien identified in example 1 and comparative examples 1-3;
FIG. 3 is a graph comparing the separation effect of C18 liquid chromatography in example 1 (upper panel) and comparative example 1 (lower panel);
FIG. 4 is a graph comparing the separation effect of SCX liquid chromatography in example 1 (upper panel) and comparative example 4 (lower panel).
Detailed Description
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
the strawberries in the following cases are all referred to as red-buccal strawberries.
Example 1: a method for qualitatively identifying strawberry stigma protein based on two-dimensional liquid chromatography tandem mass spectrometry sequentially comprises the following steps:
1) preparing a sample, extracting and purifying;
randomly taking 50 strawberry stigmas in a small bud period, a medium bud period and a large bud period respectively, uniformly grinding by using liquid nitrogen, transferring 0.25g of strawberry stigmas ground substance to a 2mL centrifuge tube, adding an extracting agent consisting of 900 mu L of 4 ℃ precooled extracting solution and 900 mu L of Tris saturated phenol, uniformly mixing by shaking on ice for 30min, centrifuging at 4 ℃ by 5000g for 30min, and transferring supernatant to a clean 10mL tube. And (3) replacing 0.25g of strawberry stigma ground substance with the precipitate (filter cake) obtained by centrifugation, repeating the extraction step for 2 times (namely, repeating the steps of adding the extracting agent, shaking on ice and centrifuging for 2 times), and combining the supernatants for 3 times to obtain combined supernatants.
The extracting solution is as follows: 0.7M sucrose, 0.1M KCl, 0.5M Tris-HCl, 2% mercaptoethanol, 50mM EDTA, adjusted to pH 8.0;
the preparation method of the extracting solution comprises the following steps: adding 0.7mol of sucrose, 0.1mol of KCl, 20mL of mercaptoethanol and 50mmol of EDTA into 1L of 0.5mol/L Tris-HCl buffer solution, and adjusting to pH8.0 (by using 1M hydrochloric acid);
the Tris saturated phenol is: a solution of phenol in Tris-HCl buffer (pH8.0) fully saturated;
adding a pre-cooled 0.1M ammonium acetate methanol solution with the temperature of-20 ℃ and the volume 5 times that of the combined supernatant into the combined supernatant, precipitating at the temperature of-20 +/-2 ℃ overnight (standing for 12-16 hours), centrifuging for 30min at 5000g, and discarding the supernatant; after washing the precipitate with precooled methanol at-20 ℃ for three times (filter cake obtained by centrifugation), freeze-drying (freeze-drying to constant weight at-70-20 ℃) to obtain protein powder (about 800 mu g).
2) Protein quantification and reductive alkylation;
dissolving the protein powder obtained in the step 1) into a protein solution by 400 mu L of lysate, taking the protein solution containing 200 mu g of protein after quantification, and adding 100mM NH4HCO3Diluting the solution (pH7.8) to 100 μ L, adding 11 μ L of 100mM DTT (DL-Dithiothreitol, Dithiothreitol) solution, mixing, and reacting in 37 deg.C metal bath for 1 h; adding 12 μ L500 mM IAM (Iodoacetamide ) solution, and reacting at room temperature in dark place for 30 min;
the preparation method of the lysate comprises the following steps: dissolving 48g of urea and 0.017g of PMSF (phenylmethylmethanesulphonylfluoride) in 0.1mol/L of Tris-HCl buffer solution, diluting to 100mL, and adjusting the pH to 8.0 (by using 1M hydrochloric acid);
remarking: the steps can be carried out in a 1.5mL centrifuge tube;
protein quantification was performed using the Bradford protein concentration assay kit (bi yun days), with the procedures described in the instructions;
3) performing proteolysis;
transferring the reaction solution obtained in the step 2) to an Amicon-Ultra-15 ultrafiltration tube (Millipore), and removing impurities according to the instructions of the ultrafiltration tube; the method specifically comprises the following steps: centrifuge at 12000rpm for 20min at 4 ℃. Discard the waste liquid in the collecting tube, and add 150. mu.L 100mM NH into the ultrafiltration tube4HCO3The solution was centrifuged at 12000rpm at 4 ℃ for 20min and the addition of 100mM NH repeated as described above4HCO3Solution, centrifugation step 1 time. Replacing the collecting pipe with a new one, and adding 100mM NH into the ultrafiltration pipe4HCO3Adding 4 mu g of Trypsin (Promega) until the total volume is 100 mu L, and reacting for 12-16 h (overnight) on a metal bath at 37 ℃; thereby realizing enzymolysis.
Centrifuging at 12000rpm at 4 deg.C for 15min, and adding 100mM NH into the ultrafilter tube (to obtain precipitate4HCO3(pH8.0) repeatedly centrifuging (4 deg.C and 12000rpm for 15min) once after 100 μ L, and mixing the two centrifugations to obtain peptide fragment solution.
Adding 5% (V/V) TFA aqueous solution to the peptide fragment solutionTFA (Trifluoroacetic acid ) was used as a peptide sample (about 200. mu.L) at a final concentration of 1% (V/V) using PierceTMC18 tips (thermolfisher) desalting, the procedure of which is described in the specification.
The desalting is specifically (this is a conventional technique): the C18 tip was immobilized tightly on a 100. mu.L pipette, pipetted out after aspirating 100. mu.L of 50% ACN, and the procedure was repeated once. Sucking and blowing 100 mu L of 0.1% FA (Formic acid ) for 2 times, sucking and blowing the peptide fragment sample for 10 times, and sucking and blowing 100 mu L of 0.1% FA for 3 times to clean and remove salt. 50 μ L of 0.1% FAin 50%, 60%, 70%, 80% ACN (Acetonitrile ) was slowly pipetted in sequence to elute the peptide fragment and collect the eluate into a new 1.5mL centrifuge tube. A final volume of about 200. mu.L of sample was obtained.
Mixing the PierceTMC18 tips (thermoldissher) peptide fragment sample after desalting is lyophilized (to constant weight at-70-20 ℃) to obtain lyophilized powder.
4) Separating the peptide fragment mixture by a first dimension SCX strong cation exchange liquid chromatogram;
SCX Buffer A：10mMKH2PO425% acetonitrile, pH 2.9;
SCX Buffer B：10mM KH2PO4400mM KCl, 25% acetonitrile, pH 2.9;
the preparation method of the SCX Buffer A comprises the following steps: 10mmol of KH are added to 1L of 25% strength by volume aqueous acetonitrile2PO4Adjusting to pH 2.9;
the preparation method of the SCX Buffer B comprises the following steps: 10mmol of KH are added to 1L of 25% strength by volume aqueous acetonitrile2PO4400mmol of KCl, and adjusting the pH value to 2.9;
redissolving the dry powder (all the freeze-dried powder) obtained in the step 3) by using 100 mu L of SCX Buffer A, and performing gradient separation by using a Biobasic SCX column (Thermofeisher); the separation gradient was:
0～5min，0％SCX Buffer B；
5～8min,0％～3％SCX Buffer B；
8～50min,3％～40％SCX Buffer B；
50～60min,40～100％SCX Buffer B；
60～65min,100％SCX Buffer B；
65-70min,100～0％SCX buffer B；
the balance of the mobile phase in each time period is SCX Buffer A; in each time period, the concentration of SCX Buffer B is uniformly changed; the elution flow rate was 1 mL/min.
Collecting the eluent for 10-50min, which comprises the following steps: collecting one tube every 10-18min, and collecting one tube every 4 min for 18-50min (i.e. collecting one tube every 4 tubes, and combining into 1 tube every 4 tubes), for 16 tubes in total; freeze-drying (freeze-drying to constant weight at-70-20 ℃) after desalting each tube; thus obtaining 16 peptide fragment powders;
desalting with PierceTMC18 tips (thermolfisher), according to the instructions; similar to step 3 above).
5) Second dimension reversed C18 liquid phase separation tandem Q active mass spectrometry;
c18Buffer A0.1% formic acid solution;
c18Buffer B0.1% formic acid in acetonitrile.
Each of the 16 peptide fragment powders obtained in the step 4) is subjected to the following operations:
the peptide fragment powder was reconstituted with 20. mu. L C18Buffer A and processed through AcclaimTMPepMapTMQ exact mass spectrometer (Thermofeisher) analysis was performed after on-line separation of 100C 18 liquid chromatography column (Thermofeisher) gradient; separation gradient: 0-3 min, 4-7% of C18Buffer B; 3-103 min, 7-18% of C18Buffer B; 103-113 min, 18-35% of C18Buffer B; 113-117 min, 35-75% of C18Buffer B, 117-120 min and 75% of C18Buffer B;
the balance of the mobile phase in each time period is C18Buffer A; the concentration of C18Buffer B was changed uniformly in each time period; the elution flow rate was 1 mL/min.
Remarking: the components separated by the second-dimension C18 chromatographic column directly enter a mass spectrometer for analysis in real time, the liquid phase carrying the C18 chromatographic column is in matched online connection with the mass spectrum, and the components eluted from the liquid phase at a certain time can enter the mass spectrum within a few seconds to obtain a fragmentation ion spectrogram.
6) And analyzing data.
Identifying an original file obtained by mass spectrometry by searching a Maxquant, downloading a protein library from Strawberry Garden, and referring to Maxquant use instructions for searching the library parameters; thereby obtaining a list of protein and peptide fragment information.
Remarking: the original file is in a raw file format which records mass spectrum information such as mass-to-charge ratio, peak height, retention time and the like of parent ions and daughter ions of the peptide fragments, and can be read into protein and peptide fragment information after database searching is carried out by database searching software.
According to the method, 16 kinds of freeze-dried powder obtained in the step 4) are obtained; the total identification quantity of red-cheek strawberry stigma protein in the small bud period, the medium bud period and the large bud period reaches more than 8000, the identification quantity of peptide segments reaches 39000, and a solid foundation is provided for constructing a red-cheek strawberry stigma protein information base.
Comparative example 1: only the second-dimensional C18 liquid chromatography separation gradient of step 5) was modified relative to example 1, the gradient being set to: 0-3 min, 4% -7% of C18Buffer B; 3-95 min, 7% -25% of C18Buffer B; 95-113 min, 25-35% of C18Buffer B; 113-117 min, 35-75% of C18Buffer B, 117-120 min and 75% of C18Buffer B. The rest is equivalent to embodiment 1.
Compared with example 1, the chromatogram of C18 obtained by the method of comparative example 1 has uneven distribution and poor separation effect, the amount of protein identified by mass spectrometry is reduced by 9.5% and the amount of peptide fragment is reduced by 10.4% compared with example 1, as shown in FIGS. 1, 2 and 3.
Comparative example 2: compared with the embodiment 1, the step 4) is omitted, namely the freeze-dried powder obtained by 3) proteolysis is directly subjected to reverse C18 liquid phase separation and Q active mass spectrometry analysis in the step 5); the rest is equivalent to embodiment 1.
The amount of protein identified by the method of comparative example 2 was 55.0% and the amount of peptide fragments was 55.3% as compared to example 1, as shown in fig. 1 and 2.
Comparative example 3: compared with the embodiment 1, the step 4) is omitted, namely the freeze-dried powder obtained by 3) proteolysis is directly subjected to reverse C18 liquid phase separation and Q active mass spectrometry analysis in the step 5); and the C18 liquid chromatography separation gradient conditions were the same as in comparative example 1; the rest is equivalent to embodiment 1.
Compared with the method of example 1, the amount of the protein identified by the method of comparative example 3 is reduced by 60.2 percent, and the amount of the peptide fragment is reduced by 58.5 percent; as shown in fig. 1 and 2.
The comparison of the identification results of example 1, comparative example 1 to comparative example 3 is shown in table 1 below.
TABLE 1 comparison of the results of the different test treatments
| Examples of the invention | Identification of Total protein | Peptide fragment identification |
| Example 1 | 8189 | 39042 |
| Comparative example 1 | 7414 | 34995 |
| Comparative example 2 | 3687 | 17458 |
| Comparative example 3 | 3262 | 16197 |
Comparative example 4: steps 1) to 3) are identical with respect to example 1; the first dimension SCX liquid chromatography separation gradient of the step 4) is changed into:
the gradient is set as: 0-5 min, 0% SCX Buffer B; 5-50 min, 0% -60% of SCX Buffer B; 60-100% SCX Buffer B for 50-60 min; 60-65 min, 100% SCX Buffer B; 65-70 min, 100-0% SCX Buffer B.
And, SCX Buffer B: 10mM KH2PO4500mM KCl, 25% acetonitrile, pH 2.9;
SCX Buffer A is as in example 1.
Compared with example 1, the peak area of the SCX chromatographic separation chart obtained by the method of comparative example 4 is reduced by 24.3%, and the peptide fragment separation effect is poor, as shown in FIG. 4.
Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Claims (9)
1. The method for qualitatively identifying the strawberry stigma protein based on the two-dimensional liquid chromatography tandem mass spectrometry is characterized by sequentially comprising the following steps of:
1) preparing a sample, extracting and purifying;
mixing strawberry stigma in a small bud stage, a medium bud stage and a large bud stage according to the quantity of 1:1:1, and then taking the mixture as a sample, and extracting and purifying to obtain protein powder;
2) protein quantification and reductive alkylation;
dissolving the protein powder in the lysate to obtain a protein solution;
a protein solution containing 200. mu.g of protein was added to 100mM NH4HCO3The solution is added to 100 mu L with constant volume, 11 mu L of 100mM DTT solution is added, and the mixture is evenly mixed and placed at 37 +/-1 ℃ for reaction for 1 +/-0.1 h; adding 12 mu L500 mM IAM solution, and reacting for 30 +/-2 min at room temperature in a dark place;
3) performing proteolysis;
ultrafiltering the reaction liquid obtained in the step 2) to remove impurities, and then adding 100mM NH4HCO3Adding 4 mu g of Trypsin until the total volume is 100 mu L, and reacting at 37 +/-1 ℃ for 12-16 h to realize enzymolysis;
centrifuging after the enzymolysis is finishedAdding 100mM NH to the obtained precipitate4HCO3Repeatedly centrifuging once after 100 mu L, and combining the liquid obtained by twice centrifugation to obtain a peptide fragment solution; the centrifugation is carried out for 15 plus or minus 2min at 12000 plus or minus 2000rpm at 4 plus or minus 1 ℃;
adding a 5% TFA aqueous solution into the peptide fragment solution until the final concentration of TFA is 0.1-1%, desalting, and freeze-drying to obtain freeze-dried powder; the% is volume%;
4) separating the peptide fragment mixture by a first dimension SCX strong cation exchange liquid chromatogram;
SCX Buffer A：10mMKH2PO425% acetonitrile, pH 2.9;
SCX Buffer B：10mM KH2PO4400mM KCl, 25% acetonitrile, pH 2.9;
redissolving all the freeze-dried powder obtained in the step 3) by using SCX Buffer A, and performing gradient separation by using a Biobasic SCX column; the separation gradient was:
0～5min，0％SCX Buffer B；
5～8min,0％～3％SCX Buffer B；
8～50min,3％～40％SCX Buffer B；
50～60min,40～100％SCX Buffer B；
60～65min,100％SCX Buffer B；
65-70min,100～0％SCX buffer B；
the balance of the mobile phase in each time period is SCX Buffer A; in each time period, the concentration of SCX Buffer B is uniformly changed; the elution flow rate is 1 ml/min; collecting the eluted fraction containing the peptide fragments; desalting and freeze-drying to obtain peptide fragment powder;
5) second dimension reversed C18 liquid phase separation tandem Q active mass spectrometry;
c18Buffer A0.1% formic acid solution;
c18Buffer B0.1% formic acid acetonitrile solution;
performing the following mass spectrometry on the peptide fragment powder obtained in the step 4):
redissolving with C18Buffer A, AcclaimTMPepMapTMPerforming gradient online separation on a 100C 18 liquid chromatographic column, and then analyzing by a QOxctive mass spectrometer; separation gradient: 0-3 min, 4-7% of C18Buffer B; 3～103min,7～18％C18Buffer B；103～113min,18～35％C18 Buffer B；113～117min,35～75％C18 Buffer B,117～120min,75％C18 Buffer B；
The balance of the mobile phase in each time period is C18Buffer A; the concentration of C18Buffer B was changed uniformly in each time period; the elution flow rate is 1 ml/min;
6) and data analysis:
and identifying the original file obtained by the mass spectrometer by a Maxquant library so as to obtain a protein and peptide fragment information list.
2. The method for qualitatively identifying strawberry stigma protein based on two-dimensional liquid chromatography tandem mass spectrometry as claimed in claim 1, wherein:
in the step 4), one tube is collected every 10-18min, and one tube is collected every 4 min at 18-50min, and 16 tubes are counted; freeze-drying after desalting each tube, thus obtaining 16 peptide fragment powders;
in the step 5), performing mass spectrometry on the 16 kinds of peptide fragment powder obtained in the step 4) respectively; each peptide fragment powder was reconstituted with 20. mu. L C18Buffer A.
3. The method for qualitatively identifying strawberry stigma protein based on two-dimensional liquid chromatography tandem mass spectrometry according to claim 1 or 2, characterized in that the step 1) is as follows:
mixing the strawberry stigma in the small bud stage, the medium bud stage and the large bud stage according to the quantity of 1:1:1 to obtain a sample, uniformly grinding the sample by using liquid nitrogen, adding 1.8 +/-0.02 mL of an extracting agent into 0.25 +/-0.02 g of the ground strawberry stigma, uniformly shaking the mixture on ice for 30 +/-5 min, and then centrifuging the mixture for the first time at the temperature of 4 +/-1 ℃; adding 4.5-5.5 volume times of-20 +/-2 ℃ precooled 0.1 +/-0.02M ammonium acetate methanol solution into the obtained supernatant, standing for 12-16 hours at-20 +/-2 ℃, and carrying out secondary centrifugation at 4 +/-1 ℃; washing the precipitate obtained by secondary centrifugation with precooled methanol at-20 +/-2 ℃, and freeze-drying to obtain protein powder;
the extractant is prepared by mixing pre-cooled extracting solution at 4 +/-1 ℃ and Tris saturated phenol according to the volume ratio of 1: 1;
the extracting solution is as follows: 0.7M sucrose, 0.1M KCl, 0.5M Tris-HCl, 2% mercaptoethanol, 50mM EDTA, adjusted to pH 8.0;
the Tris saturated phenol is: phenol in Tris-HCl buffer fully saturated solution.
4. The method for qualitatively identifying strawberry stigma protein based on two-dimensional liquid chromatography tandem mass spectrometry as claimed in claim 3, wherein: in the step 1), replacing 0.25g of strawberry stigma grinding material with the filter cake obtained by primary centrifugation, and repeating the steps of adding the extracting agent, vibrating on ice and centrifuging; the repetition times are 1-3 times; and combining the supernatants obtained by centrifugation, and then carrying out the subsequent steps.
5. The method for qualitatively identifying strawberry stigma protein based on two-dimensional liquid chromatography tandem mass spectrometry as claimed in claim 4, wherein:
in the step 1), the first centrifugation and the second centrifugation are both: centrifuging at 5000 + -100 g for 30 + -5 min at 4 + -1 deg.C.
6. The method for qualitatively identifying strawberry stigma protein based on two-dimensional liquid chromatography tandem mass spectrometry as claimed in claim 3, wherein: the ultrafiltration impurity removal of the step 3) is to remove impurities from the reaction solution obtained in the step 2) by using an Amicon-Ultra-15 ultrafiltration tube.
7. The method for qualitatively identifying strawberry stigma protein based on two-dimensional liquid chromatography tandem mass spectrometry as claimed in claim 6, wherein: the desalting in the step 3) and the step 4) adopts PierceTMDesalting with C18 Tips.
8. The method for qualitatively identifying strawberry stigma protein based on two-dimensional liquid chromatography tandem mass spectrometry as claimed in claim 3, wherein:
the preparation method of the lysate in the step 2) comprises the following steps: 48g of urea and 0.017g of PMSF are dissolved in 0.1mol/L Tris-HCl buffer solution, the volume is determined to be 100mL, and the pH value is adjusted to be 8.0.
9. The method for qualitatively identifying strawberry stigma protein based on two-dimensional liquid chromatography tandem mass spectrometry as claimed in claim 1, wherein: the strawberry is red cheek strawberry.
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