CN114591392A - Sample preparation method for high-salt dilute soy sauce microbial macroproteomics analysis - Google Patents
Sample preparation method for high-salt dilute soy sauce microbial macroproteomics analysis Download PDFInfo
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- 235000013555 soy sauce Nutrition 0.000 title claims abstract description 30
- 238000004458 analytical method Methods 0.000 title claims abstract description 14
- 230000000813 microbial effect Effects 0.000 title claims abstract description 11
- 238000005464 sample preparation method Methods 0.000 title claims abstract description 7
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 30
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 28
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 27
- 238000001514 detection method Methods 0.000 claims abstract description 8
- 238000001914 filtration Methods 0.000 claims abstract description 8
- 238000000034 method Methods 0.000 claims abstract description 7
- 230000009471 action Effects 0.000 claims abstract description 6
- 238000001976 enzyme digestion Methods 0.000 claims abstract description 6
- 239000012535 impurity Substances 0.000 claims abstract description 6
- 229920001184 polypeptide Polymers 0.000 claims abstract description 6
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 6
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 6
- 230000029936 alkylation Effects 0.000 claims abstract description 4
- 238000005804 alkylation reaction Methods 0.000 claims abstract description 4
- 239000002808 molecular sieve Substances 0.000 claims abstract description 4
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 claims abstract description 4
- 238000011033 desalting Methods 0.000 claims abstract description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 27
- 239000000706 filtrate Substances 0.000 claims description 24
- 239000000243 solution Substances 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- 238000010009 beating Methods 0.000 claims description 5
- 102000004142 Trypsin Human genes 0.000 claims description 3
- 108090000631 Trypsin Proteins 0.000 claims description 3
- 230000001580 bacterial effect Effects 0.000 claims description 3
- 239000011324 bead Substances 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 3
- 238000000227 grinding Methods 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 239000002244 precipitate Substances 0.000 claims description 3
- 239000012460 protein solution Substances 0.000 claims description 3
- 239000008223 sterile water Substances 0.000 claims description 3
- 239000012588 trypsin Substances 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 230000010355 oscillation Effects 0.000 claims 2
- 238000005119 centrifugation Methods 0.000 claims 1
- 238000004140 cleaning Methods 0.000 claims 1
- 239000012716 precipitator Substances 0.000 claims 1
- 238000009210 therapy by ultrasound Methods 0.000 claims 1
- 244000005700 microbiome Species 0.000 abstract description 8
- 235000010469 Glycine max Nutrition 0.000 abstract description 5
- 244000068988 Glycine max Species 0.000 abstract description 4
- 238000000751 protein extraction Methods 0.000 abstract description 4
- 238000002474 experimental method Methods 0.000 abstract description 3
- 150000003839 salts Chemical class 0.000 abstract description 3
- 238000000605 extraction Methods 0.000 abstract description 2
- 230000008569 process Effects 0.000 abstract description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 6
- 229940098773 bovine serum albumin Drugs 0.000 description 6
- 238000000855 fermentation Methods 0.000 description 5
- 230000004151 fermentation Effects 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 239000012224 working solution Substances 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 108010077805 Bacterial Proteins Proteins 0.000 description 2
- 108010026552 Proteome Proteins 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 235000013312 flour Nutrition 0.000 description 2
- 239000003205 fragrance Substances 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
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- 239000000047 product Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
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- 230000015572 biosynthetic process Effects 0.000 description 1
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- 230000008859 change Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
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- 238000004451 qualitative analysis Methods 0.000 description 1
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- 238000006467 substitution reaction Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
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- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
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Abstract
The invention belongs to the technical field of new processes, and discloses a sample preparation method for high-salt dilute soy sauce microorganism macroproteomics analysis. According to the invention, through the pretreatment of gauze filtration and the combination and application of various kits, and by utilizing the molecular sieve action of an ultrafiltration tube, salt and impurity are rapidly removed, a high-quality protein sample is obtained, and the polypeptide obtained after alkylation and enzyme digestion can be used for nLC-MS detection and macro-proteomics information analysis of high-salt dilute soy sauce. In the method, the influence of soybeans in a high-salt dilute soy sauce sample on microbial protein extraction is reduced by using gauze filtration pretreatment for the first time, two kits are combined for extraction and concentration for the first time, the operation steps are improved, the protein with higher purity can be obtained, and the rapid desalting and impurity removal are realized by combining the use of an ultrafiltration tube. The method is simple and convenient to operate, high in experiment speed and high in protein quality.
Description
Technical Field
The invention relates to the technical field of new processes, in particular to a sample preparation method for high-salt dilute soy sauce microorganism macroproteomics analysis.
Background
The high-salt liquid soy sauce is an important genre of the traditional Chinese brewed soy sauce and is formed in south China mainly comprising Guangdong. The soy sauce is prepared by taking whole soybeans and flour as main raw materials and naturally brewing the whole soybeans and flour for more than 3 months in the sun-dried night dew, the soy sauce is fermented to form unique fragrance under the combined action of multiple microorganisms, the specific action of each microorganism in different time periods in the soy sauce brewing process is difficult to describe exactly, and particularly, a normal-temperature high-salt dilute fermentation process is adopted, so that the period is long, the influence of the natural environment is large, and the interaction among the microorganisms is more complicated. In the soy sauce fermentation process, the population structure and mutual metabolic regulation of microorganisms play a key role in the formation and quality of the color, the fragrance, the taste and the body state of the soy sauce.
With the development and application of biochemical and molecular biological technologies, detection of 89-99% of uncultured or uncultured microorganisms in the environment is realized by using a non-culture physiological biochemical method and a molecular biological technology, and the microorganisms are more comprehensively and deeply understood. Particularly, the application of the technologies of the metagenome, the macrotranscriptome, the macroproteineome and the macrometabolome enables people to systematically and deeply research the microbial community structure and the functional genes of the fermented food, comprehensively analyze the composition, the change rule and the genetic evolution of the microbial community structure and the functional genes with rich potential, and further uncover the mysterious veil of the high-salt dilute soy microbial community.
Because the fermentation period of the high-salt dilute soy sauce is long and the microbial community is complex, the macro-proteomics research of the high-salt dilute soy sauce is fresh at present. The preparation of proteomics samples is the key to obtain high-quality mass spectrum data, and the suitable protein sample preparation technology can obtain more abundant macro-proteomics information which is fit for the fermentation process of the high-salt dilute soy sauce produced actually.
Disclosure of Invention
Based on this, the present invention aims to provide a sample preparation method for high-salt dilute soy sauce microbe macroproteomics analysis, so as to solve the technical problems of the background art.
In order to achieve the purpose, the invention provides the following technical scheme: according to the invention, through the pretreatment of gauze filtration and the combination and application of various kits, and by utilizing the molecular sieve action of an ultrafiltration tube, salt and impurity are rapidly removed, a high-quality protein sample is obtained, and the polypeptide obtained after alkylation and enzyme digestion can be used for nLC-MS detection and macro-proteomics information analysis of high-salt dilute soy sauce.
The purpose of the invention is realized by the following scheme:
taking 50g of high-salt diluted soy sauce mash and 50ml of water yellow, fully and uniformly mixing, filtering by using gauze, taking filtrate, transferring the filtrate to a 50ml centrifuge tube, centrifuging for 5min at 3000 Xg and 4 ℃, and removing supernatant to obtain bacterial precipitate. Washed 2 times with sterile water.
Adding equal volume of protein extract, and grinding with bead mill at 4 deg.C for 3min at intervals of 20s for 30s each time. The mixture is placed on ice for 1h, and is sucked and uniformly mixed by a gun head for 1 time every ten minutes.
Sonicate on ice for 5min, 20000 Xg, 4 ℃, centrifuge for 15min, transfer the supernatant to a fresh pre-cooled centrifuge tube.
Protein content was determined using BCA kit.
Adding 1/4 volumes of precipitant A into 200ug of protein solution, vortex shaking for 10s, standing on ice for 1h, 20000 Xg, centrifuging for 10min at 4 ℃, adding 600ul of washing solution B, vortex shaking for 10s, standing on ice for 10min, transferring to an ultrafiltration tube, 13500 Xg, centrifuging for 10min at 4 ℃, discarding the filtrate, adding 400ul of first-level water into the ultrafiltration tube, sucking uniformly, 13500 Xg, centrifuging for 10min at 4 ℃, and discarding the filtrate.
Adding 100ul protein extract into ultrafiltration tube to dissolve protein, adding DTT with final concentration of 50mM, keeping the temperature at 60 deg.C for 40min, cooling to room temperature, adding 55mM IAM, reacting in dark for 40min, 13500 Xg, centrifuging at 4 deg.C for 10min, and discarding filtrate.
Adding 400ul of first-stage water into the ultrafiltration tube, sucking and beating uniformly, centrifuging at 13500 Xg and 4 ℃ for 10min, discarding the filtrate, adding 400ul of 25mM NH into the ultrafiltration tube4HCO3Sucking and beating uniformly, centrifuging for 10min at 13500 Xg and 4 ℃, discarding the filtrate, and intercepting the sample protein in an ultrafiltration tube.
100ul of 25mM NH was added to the ultrafiltration tube4HCO3According to the protein: adding trypsin at a ratio of 1:50, performing enzyme digestion for 15h, 13500 Xg, centrifuging at 4 ℃ for 10min, taking filtrate, and performing macro proteome information analysis on the high-salt dilute soy sauce, wherein the filtrate is a prepared polypeptide sample which can be used for nLC-MS detection.
According to the invention, through the pretreatment of gauze filtration and the combination and application of various kits, and by utilizing the molecular sieve action of an ultrafiltration tube, salt and impurity are rapidly removed, a high-quality protein sample is obtained, and the polypeptide obtained after alkylation and enzyme digestion can be used for nLC-MS detection and macro-proteomics information analysis of high-salt dilute soy sauce. In the method, the influence of soybeans in a high-salt dilute soy sauce sample on microbial protein extraction is reduced by using gauze filtration pretreatment for the first time, two kits are combined for extraction and concentration for the first time, the operation steps are improved, the protein with higher purity can be obtained, and the rapid desalting and impurity removal are realized by combining the use of an ultrafiltration tube. The method is simple and convenient to operate, high in experiment speed and high in protein quality.
Drawings
FIG. 1: a BCA standard curve;
FIG. 2: and (4) carrying out nLC-MS detection on the prepared sample to obtain a total ion flow diagram.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.
The materials referred to in the following examples are commercially available.
Determination of protein content:
preparation of a gradient diluted Bovine Serum Albumin (BSA) standard:
a panel of protein standards was prepared according to table 1, and one ampoule of bovine serum albumin standard (BSA) was diluted in a 96-well plate in primary water.
TABLE 1 gradient dilution of Bovine Serum Albumin (BSA) standards
Preparing a BCA working solution: calculating the volume of the required working fluid, and the formula is as follows: (number of standards + number of protein samples to be tested + number of blank controls) × (number of experiment repetitions) × (volume of working solution used for each sample), reagent a and reagent B of the BCA reagent were mixed at 50:1 to prepare a working solution.
The standards were added to the first column of the microplate in order, 25. mu.L/well.
Samples were added to the microplate at (5. mu.L sample + 20. mu.L water)/well and 25. mu.L water was added to the blank control.
BCA working solution was added to each well of sample, blank and standard at 200. mu.L/well.
Incubate at 37 ℃ for 30min with shaking for 30 sec.
The absorbance at 562nm was measured with a microplate reader.
Standard curves were plotted, see fig. 1.
Preparation of a sample:
taking 50g of high-salt diluted soy sauce mash fermented for 60 days and 50ml of water yellow, fully and uniformly mixing, filtering by using gauze, taking filtrate, transferring the filtrate to a 50ml centrifuge tube, centrifuging for 5min at 3000 Xg and 4 ℃, and removing supernatant to obtain bacterial precipitate. Washed 2 times with sterile water.
Adding equal volume of protein extract (Bebo company bacterial protein extraction kit, cat # BB-319907), and grinding with bead mill at 4 deg.C for 3min at an interval of 20s for 30s each time. The mixture is placed on ice for 1h, and is sucked and uniformly mixed by a gun head for 1 time every ten minutes.
Sonicate (150w) on ice for 5min, 20000 Xg, 4 ℃, centrifuge for 15min, and transfer the supernatant to a fresh, precooled centrifuge tube.
Protein content was determined using the BCA kit (Thermo, cat # 23227).
Adding 1/4 volume of precipitant A (product number C510011) into 200ug of protein solution, vortex shaking for 10s, standing on ice for 1h, 20000 Xg, and 4 deg.C, centrifuging for 10min, adding 600ul of washing solution B (product number C510011) into the solution, vortex shaking for 10s, standing on ice for 10min, transferring to ultrafiltration tube (10KD), 13500 Xg, and centrifuging for 10min at 4 deg.C, discarding filtrate, adding 400ul of first-stage water into the ultrafiltration tube, sucking uniformly, 13500 Xg, and centrifuging for 10min at 4 deg.C, and discarding filtrate.
Adding 100ul protein extract (Beeber bacterial protein extraction kit, cat # BB-319907) into ultrafiltration tube to dissolve protein, adding DTT with final concentration of 50mM, keeping at 60 deg.C for 40min, cooling to room temperature, adding 55mM IAM, reacting in dark for 40min, 13500 Xg, centrifuging at 4 deg.C for 10min, and discarding filtrate.
400ul of first-stage water is added into the ultrafiltration tube, the mixture is evenly sucked and beaten, the mixture is centrifuged for 10min at the temperature of 13500 Xg and 4 ℃, and the filtrate is discarded. 400ul of 25mM NH was added to the ultrafiltration tube4HCO3Sucking and beating evenly, centrifuging for 10min at 13500 Xg and 4 ℃, and discarding the filtrate. The ultrafiltration tube was blocked with sample protein.
100ul of 25mM NH was added to the ultrafiltration tube4HCO3According to the protein: adding trypsin, and digesting for 15 h. 13500 Xg, centrifuging at 4 deg.C for 10min, and collecting filtrate to obtain the final productAnd detecting the sample by nLC-MS to obtain a total ion flow diagram, which is shown in figure 2. Through qualitative analysis of the Proteome discover 2.1 software, 731 flora proteins related to high-salt dilute soy sauce fermentation can be identified.
Although embodiments of the present invention have been shown and described, it is intended that the present invention should not be limited thereto, that the particular features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples, and that modifications, substitutions, variations or the like, which are not inventive and may be made by those skilled in the art without departing from the principle and spirit of the present invention and without departing from the scope of the claims.
Claims (3)
1. A sample preparation method for high-salt dilute soy sauce microbial macro-proteomics analysis is characterized in that a high-quality protein sample is obtained by gauze filtering pretreatment, combination and application of various kits and the molecular sieve action of an ultrafiltration tube, desalting and impurity removal, and polypeptides obtained after alkylation and enzyme digestion can be used for nLC-MS detection and high-salt dilute soy sauce macro-proteomics information analysis.
2. The method for preparing the sample for the high-salt dilute soy sauce microbial macro-proteomics analysis according to claim 1, wherein 50g of high-salt dilute soy sauce mash and 50ml of water are taken, fully mixed, filtered by gauze, the filtrate is taken, transferred to a 50ml centrifuge tube, the centrifugal force is 3000 Xg, the temperature is 4 ℃, the centrifugation is carried out for 5min, the supernatant is discarded, the bacterial precipitate is obtained, and the sterile water is adopted for cleaning for 2 times.
3. The sample preparation method for high-salt dilute soy sauce microbial macroproteomics analysis according to claim 2, characterized by comprising the following steps:
step one, adding an isovolumetric protein extracting solution, grinding for 3min at the temperature of 4 ℃ by using a bead mill for 30s every time at intervals of 20s, standing for 1h on ice, and sucking and uniformly mixing for 1 time by using a gun head every ten minutes;
step two, performing ultrasonic treatment on ice for 5min, centrifuging at the centrifugal force of 20000 Xg and the temperature of 4 ℃, centrifuging for 15min, and transferring the supernatant to a precooled new centrifugal tube;
thirdly, determining the protein content by using a BCA kit, wherein the protein concentration of a sample is 0.5 ug/ul;
step four, adding 1/4 volumes of precipitator A into 200ug of protein solution, carrying out vortex oscillation for 10s, standing on ice for 1h, carrying out centrifugal force 20000 Xg at the temperature of 4 ℃ for 10min, adding 600ul of washing solution B, carrying out vortex oscillation for 10s, standing on ice for 10min, transferring to an ultrafiltration tube, carrying out centrifugal force 13500 Xg at the temperature of 4 ℃ for 10min, discarding filtrate, adding 400ul of first-level water into the ultrafiltration tube, uniformly sucking, carrying out centrifugal force 13500 Xg at the temperature of 4 ℃, centrifuging for 10min, and discarding the filtrate;
step five, adding 100ul of protein extracting solution into an ultrafiltration tube to dissolve protein, adding DTT with the final concentration of 50mM, preserving heat for 40min at 60 ℃, cooling to room temperature, adding 55mM IAM, reacting for 40min in a dark place, centrifuging for 13500 Xg at 4 ℃ for 10min, and discarding the filtrate;
step six, adding 400ul of first-level water into the ultrafiltration tube, sucking and beating uniformly, centrifuging for 10min at 13500 Xg and 4 ℃, discarding the filtrate, and adding 400ul of 25mM NH into the ultrafiltration tube4HCO3Sucking and beating uniformly, centrifuging at the centrifugal force of 13500 Xg and the temperature of 4 ℃ for 10min, and discarding the filtrate. Intercepting the flow in the ultrafiltration tube into sample protein;
step seven, adding 100ul of 25mM NH into the ultrafiltration tube4HCO3According to the protein: adding trypsin at a ratio of 1:50, carrying out enzyme digestion for 15h, carrying out centrifugal force 13500 Xg at a temperature of 4 ℃, centrifuging for 10min, taking filtrate, and carrying out high-salt dilute soy sauce macro-proteome information analysis for a prepared polypeptide sample for nLC-MS detection.
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| CN107167353A (en) * | 2017-07-05 | 2017-09-15 | 赛思莱（厦门）生物科技有限公司 | A kind of sample preparation processing method for the grand protein science research of soil |
| CN110308228A (en) * | 2019-08-09 | 2019-10-08 | 陈溪 | A kind of analysis method of sea cucumber protein group |
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| CN107167353A (en) * | 2017-07-05 | 2017-09-15 | 赛思莱（厦门）生物科技有限公司 | A kind of sample preparation processing method for the grand protein science research of soil |
| CN110308228A (en) * | 2019-08-09 | 2019-10-08 | 陈溪 | A kind of analysis method of sea cucumber protein group |
Non-Patent Citations (2)
| Title |
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| 乌日娜等: "豆酱微生物宏蛋白质组提取及分析" * |
| 李丹等: "高盐稀态酱油酿造过程中蛋白质降解规律的研究" * |
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Application publication date: 20220607 |