CN108572214B - Method for large-scale proteomics identification based on silkworm tissue samples - Google Patents

Abstract

The invention relates to a method for large-scale proteomics identification based on silkworm tissue samples, which comprises silkworm proteomics sample pretreatment, graded peptide fragment mass spectrometry on-machine detection and silkworm protein database construction. By optimizing a protein extraction method and adopting a high pH classification method to classify the peptide segment into 8 grades, the silkworm fat body sample can be extracted as much as possible, and the identification number of the protein is increased; the blockage of a sample spray needle caused by overlarge sample loading amount is prevented by optimizing the sample loading amount and the chromatographic gradient time; the detection time is shortened by optimizing the chromatographic gradient time; a Streamline database containing 21878 protein sequences is established, the database can identify more protein numbers, redundant sequences are removed, and the later proteomic data analysis is facilitated. The invention establishes a stable and efficient silkworm proteomics identification platform, and has important significance for large-scale identification of silkworm proteomics.

Description

Method for large-scale proteomics identification based on silkworm tissue samples

Technical Field

The invention belongs to the field of proteomics, and relates to a method for carrying out large-scale proteomic identification based on a silkworm tissue sample.

Background

In recent years, the development of proteomics is greatly promoted by the appearance of high-resolution mass spectrometers, and the research of silkworm proteomics also enters a new stage. Nevertheless, the methods used in the studies are different, so that it is difficult to correlate and compare the results of the studies, and the number of proteins identified in the studies and the scientific problems explained are very different, so that the expression of the proteins in the body of silkworms at various stages and tissues cannot be comprehensively understood. Currently, only mice and humans are animals that possess the full spectrum of proteins, but both species belong to mammals. Silkworm belongs to lepidoptera insects, and is a completely metamorphotic insect after four different development stages of eggs, larvae, pupae and moths in life. The identification of the complete proteomics spectrum of the silkworm can provide a reference template for understanding the growth and development of insects and even invertebrates and the biological functions of each organ. Therefore, the method has important significance for large-scale proteomic identification of the silkworms.

For large-scale proteomics identification of silkworms, a stable and efficient proteomics platform is needed. At present, some reports of silkworm proteome identification by using an ultra-high resolution mass spectrometer are available, although the researches lay a foundation for silkworm proteome research, the number of identified proteins is still to be improved, and the method cannot be used for large-scale proteome identification of silkworm tissue samples. Currently, methods for increasing the number of proteomic identifications mostly adopt grading methods, and human cell lines are mostly adopted in the development process of the methods. The scholars compare the advantages and disadvantages of different grading techniques, and the selected material is the Hela cell line. However, no report is found on a method for improving the proteome identification number of the silkworm tissue samples.

Disclosure of Invention

In view of the above, the present invention aims to provide a method for large-scale proteomic identification based on bombyx mori tissue samples.

In order to achieve the purpose, the invention provides the following technical scheme:

a method for large-scale proteomics identification based on silkworm tissue samples comprises the following steps:

(1) pretreatment of silkworm proteomics samples: adding 100mM DTT/8M urea solution into silkworm fat, putting the sample on ice, grinding the sample by using a G50 tissue grinder to fully dissolve the sample, centrifuging and collecting supernatant; performing enzymolysis on the sample by using an FASP enzymolysis method, dividing the peptide fragment into 8 grades by using a high pH grading method, and freeze-drying each component by using a vacuum freeze-drying instrument;

(2) and (3) carrying out mass spectrum detection on the classified peptide fragments: dissolving the peptide fragment with 50 μ L of 0.1% formic acid solution, loading 8 μ L, and effective chromatographic gradient time of 120 min;

(3) matching data base of silkworm proteomics data: carrying out redundancy removal processing on 4 Silkworm protein databases of NCBI, SilkDB, Silkworm and Uniprot by using a 90% threshold value to obtain a database Streamline, and searching the database by using MaxQuant as original data.

Preferably, in the step 1), 200 μ L of 100mM DTT/8M urea solution is added to each mg of silkworm fat in the amount of 100mM DTT/8M urea solution; grinding for 3 min; the full dissolution is oscillation for 5min by a horizontal oscillator; the centrifugation was 12000g, centrifugation 15min at 25 ℃.

Preferably, the performing enzymolysis on the sample by the FASP enzymolysis method specifically comprises:

1) adding the sample into a 10kDa ultrafiltration tube, adding 8M urea solution, uniformly mixing, placing in a centrifuge at 12000g, and centrifuging for 20 min;

2) adding 8M urea at 12000g, and centrifuging for 20 min;

3) repeating the step 2) twice;

4) adding 1M DTT and 8M urea solution, sealing the pipe orifice with a sealing film, and incubating at 37 ℃ for 2 h;

5) tearing the sealing film, adding 1M iodoacetamide solution, mixing uniformly, and incubating for 1h in a dark place;

6) centrifuging at 12000g for 20 min;

7) adding 8M urea solution, mixing uniformly, and centrifuging at 12000g for 20 min;

8) repeating the step 7) twice;

9) 50mM NH was added₄HCO₃Centrifuging the solution at 12000g for 20 min;

10) repeating the step 9) for three times;

11) the outer pipe of the ultrafiltration pipe is replaced by a new outer pipe to prevent pollution;

12) adding 10 mu g/mL trypsin solution into an ultrafiltration tube, sealing the tube opening with a sealing film, placing in a constant-temperature incubator at 37 ℃ for incubation for 36h, and carrying out enzymolysis on the protein into peptide fragments;

13) tearing off the sealing film, and centrifuging at 12000g for 20 min;

14) add 40. mu.L of 50mM NH to the ultrafiltration tube₄HCO₃Centrifuging the solution at 12000g for 20 min;

15) freeze-concentrate at 4 ℃ until the sample is completely dry.

Preferably, the step of dividing the peptide fragment into 8 grades by using the high pH fractionation method specifically comprises:

1) taking off the white plastic sleeve from the bottom of the spin column, and placing the spin column in a centrifuge tube;

2) centrifuging at 5000g for 2min, and removing the solution in the spin column;

3) taking off a red cap at the top end of the spin column, loading 300 mu L of acetonitrile into the spin column, covering the red cap, putting the spin column into a centrifuge tube, placing the centrifuge tube in a centrifuge for 5000g for centrifugation for 2min, washing the column and removing the acetonitrile;

4) washing the rotating column twice with trifluoroacetic acid with volume fraction of 0.1%;

5) adding a trifluoroacetic acid solution with the volume fraction of 0.1% into the freeze-dried peptide fragment sample, and vortexing for 30s to dissolve the peptide fragment;

6) placing the spin column in a new centrifuge tube, loading the sample into the column, covering with a red cover, placing in a centrifuge at 3000g, and centrifuging for 2min to obtain a flow-through centrifuge tube;

7) putting the spin column into a new centrifugal tube, adding ultrapure water, and centrifuging for 2min at 3000g to obtain a cleaning solution;

8) preparing an elution solution such as an eluent shown in table 1, putting a spin column into a new centrifugal tube, adding the eluent with the tube number of 1, covering a red cover, and centrifuging for 2min at 3000 g;

9) collecting the eluent in a centrifuge tube, opening a red cap, and adding 300 mu L of the solution with the tube number of 2 into a spin column;

repeating the steps 8) and 9), and collecting different gradient eluates in 8 centrifuge tubes.

Preferably, the mass spectrometric detection of the fractionated peptide fragments comprises:

adding 0.1% formic acid water into each component peptide fragment, shaking for 30s for dissolution, and centrifuging at 12000g for 10 min; sucking the supernatant into a sample loading bottle, and separating peptide fragments by adopting a Thermo Fisher Scientific EASY-nLC 1000 nanoliter flow rate system, wherein the volume of an equilibrium pre-column is 10 mu L, the flow rate is 3.5 mu L/min, the volume of an equilibrium analysis column is 5 mu L, and the flow rate is 0.25 mu L/min; the sample loading amount is 8 muL, the sample suction flow rate is 8 muL/min, 18 muL of 0.1 percent formic acid water is pushed into a pre-column and an analytical column by using the flow rate of 3.5 muL/min, and the peptide fragment separation is carried out by using a gradient 0.1 percent formic acid acetonitrile solution, and the flow rate is 250 nL/min; the effective chromatographic gradient time is 120min gradient, and 100 mul of formic acid acetonitrile with volume fraction of 0.1% is used for cleaning an automatic sample loading ring;

detecting by adopting a Q active mass spectrometer, wherein the detection time is 0-140min, the spray voltage is 2.3kV, the temperature of an ion transmission tube is 275 ℃, the ion mode is a positive ion mode, the default charge is two, the resolution of full scan is 70000, the AGCtarget is 1e6, Maximum IT is 20ms, the scanning range is 300-1800 m/z, the secondary scanning resolution is 17500, the AGC target is 1e5, and Maximum IT is 60ms, acquiring data by adopting a Top 10 principle, and the dynamic exclusion time is set to be 30 s; the ambient temperature of the mass spectrum is 22 ℃, and the dehumidifier controls the air humidity of the mass spectrum working environment to be 50%.

The invention has the beneficial effects that: the invention discloses a method for large-scale proteomics identification based on silkworm tissue samples, which has the following advantages compared with the traditional liquid nitrogen grinding method:

1) the sample is ground on ice by adopting a G50 tissue grinder, and on the basis of not influencing the identification of the number of proteins, the method has the advantages of time and labor saving, safety, economy and the like; the sample is extracted by using 100mM DTT/8M urea solution, so that the silkworm fat body sample can be extracted as much as possible;

2) the invention adopts 8 mu L sample loading, and can prevent the condition that the sample injection needle is blocked due to overlarge sample loading amount. The detection time of the silkworm proteomics mass spectrum reported in the prior art is more than 180min, the method adopts 120min effective chromatographic gradient time to carry out mass spectrum on-machine detection, and the detection time of 60min can be reduced for each needle;

3) the established bombyx mori Streamline database contains 21878 protein sequences, the database can identify more protein numbers, and redundant sequences are removed, so that the subsequent proteomic data analysis is facilitated;

4) the peptide fragment is classified into 8 grades by adopting a high pH grade method, the proteomics identification number can be greatly increased, the detection time can be greatly reduced, and the classification method does not need to perform a desalting process, so that the sample pretreatment time is shortened.

Drawings

In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:

FIG. 1 is a process for integrating and removing redundant sequences of databases of 4 silkworm.

FIG. 2 shows a comparison of different milling methods (A: protein electrophoretogram; B: number of identified proteins).

FIG. 3 shows the number of proteins identified in different extraction solutions

FIG. 4 shows the effect of loading volume (A: number of proteins identified by different loading volumes; B: abundance of proteins identified by different loading volumes).

FIG. 5 shows the effect of time gradients (A: the number of proteins identified by different time gradients; B: the abundance of proteins identified by different time gradients).

FIG. 6 shows the results of peptide fractionation and protein fractionation.

Detailed Description

Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

Example 1 method for Large-Scale proteomic identification based on Bombyx mori tissue samples

(1) Pretreatment of silkworm fat body proteomics samples:

a. sample extraction

1) Placing 2mg of silkworm fat sample in 1.5mL centrifuge tube, adding 400 μ L100mM DTT/8M urea solution, placing on ice, and grinding for 3min with Calud G50 tissue grinder;

2) oscillating for 5min by a horizontal oscillator to fully dissolve the sample in the solution;

3)12000g, centrifuging for 15min at 25 ℃, taking the supernatant and placing in a new centrifuge tube;

4) the protein concentration of the sample was measured using the Bradford protein quantitation kit.

b. Sample pretreatment

1) Sucking 300 mu g of sample, adding the sample into a 10kDa ultrafiltration tube, supplementing 8M urea solution to 200 mu L, blowing and sucking the solution for a plurality of times by using a liquid transfer gun, uniformly mixing the solution, and placing the mixture in a centrifuge at 12000g of room temperature (18-25 ℃) for centrifuging for 20 min;

2) then 200. mu.L of 8M urea was added, and 12000g of the mixture was centrifuged at room temperature (18-25 ℃) for 20 min.

3) Repeating the step 2) twice, (when the solution at the bottom of the outer tube of the ultrafiltration tube is close to the bottom of the inner tube of the ultrafiltration tube, pouring the solution at the outer tube);

4) adding 4 μ L of 1M DTT and 150 μ L of 8M urea solution, sealing the tube opening with a sealing film, and incubating at 37 deg.C for 2 h;

5) tearing the sealing film, adding 15 mu L of 1M iodoacetamide solution, uniformly mixing, and incubating for 1h at room temperature (18-25 ℃) in the dark;

6) centrifuging at 12000g for 20min at room temperature (18-25 ℃);

7) adding 200 mu L of 8M urea solution, uniformly mixing for several times through blowing and sucking, and then centrifuging for 20min at 12000g of room temperature (18-25 ℃);

8) repeating the step 7) twice;

9) add 200. mu.L of 50mM NH₄HCO₃Centrifuging the solution at 12000g for 20min at room temperature (18-25 ℃);

10) repeating the step 9) for three times;

11) the outer pipe of the ultrafiltration pipe is replaced by a new outer pipe to prevent pollution;

12) adding 400 mu L of 10 mu g/mL trypsin solution into an ultrafiltration tube, sealing the tube opening with a sealing film, placing in a constant-temperature incubator at 37 ℃ for incubation for 36h, and carrying out enzymolysis on the protein into peptide fragments;

13) tearing off the sealing film, and centrifuging at 12000g at room temperature (18-25 ℃) for 20 min;

14) add 40. mu.L of 50mM NH to the ultrafiltration tube₄HCO₃Centrifuging the solution at 12000g for 20min at room temperature (18-25 ℃);

15) freeze-concentrate at 4 ℃ until the sample is completely dry.

c. High pH spin column fractionation

1) Taking off the white plastic sleeve from the bottom of the spin column, and placing the spin column in a 2.0mL centrifuge tube;

2) centrifuging at 5000g for 2min, and removing the solution in the spin column;

3) taking off a red cap at the top end of the spin column, loading 300 mu L of ACN into the spin column, covering the red cap, putting the spin column into a 2.0mL centrifuge tube, placing the spin column in a centrifuge for 5000g for centrifugation for 2min, washing the column and removing CAN;

4) the spin column was washed twice with 300. mu.L of 0.1% TFA as described in 3);

5) adding 300 μ L of 0.1% TFA solution to the lyophilized peptide fragment sample, and vortexing for 30s to dissolve the peptide fragment;

6) placing the spin column in a new 2.0mL centrifuge tube, loading 300 μ L of sample into the column, covering with a red cover, placing in a centrifuge 3000g, centrifuging for 2min, and obtaining flow-through in the centrifuge tube;

7) putting the spin column into a new 2.0mL centrifuge tube, loading 300 mul MilliQ water, and centrifuging at 3000g for 2min to obtain a washing solution;

8) preparing an elution solution from acetonitrile and 0.1% TFA according to the following table 1, placing the spin column into a new 2.0mL centrifuge tube, loading 300 μ L of an eluent with the tube number 1, covering a red cover, and centrifuging for 2min at 3000 g;

9) collecting the eluent in a 1.5mL centrifuge tube, opening a red cap, and adding 300 mu L of the solution in the No. 2 tube into the spin column;

10) repeating the steps 8) and 9), and collecting different gradient eluents in 8 centrifugal tubes with the volume of 1.5 mL;

the centrifuge tube was placed in a vacuum freeze concentrator and drained.

TABLE 1 gradient of elution solution configuration in high pH spin column fractionation

d. Classified peptide fragment mass spectrum detection on computer

Each peptide fragment was dissolved by adding 50. mu.L of 0.1% formic acid water to each fraction, followed by shaking for 30 seconds, and centrifugation at 12000g for 10min at room temperature. Pipette 12. mu.L of supernatant into the loading flask. The separation of peptide fragments was performed using a Thermo Fisher Scientific EASY-nLC 1000 nanoliter flow rate system with an equilibrium pre-column volume of 10. mu.L, a flow rate of 3.5. mu.L/min, an equilibrium analytical column volume of 5. mu.L, and a flow rate of 0.25. mu.L/min. The loading was 8. mu.L, the flow rate of the draw was 8. mu.L/min, and an 18. mu.LA phase solution (0.1% formic acid) was pushed onto a pre-column (100. mu. m.times.2 cm, NanoViper, C18, 5. mu.m,

) And an analytical column (50. mu. m.times.15 cm, NanoViper, C18, 2 μm,

) In the step (2), the peptide fragment was separated by gradient phase B solution (0.1% acetonitrile formate) at a flow rate of 250 nL/min. The effective chromatographic gradient time was a 120min gradient. The autosampler loop was washed with 100 μ L B phase.

Detecting with Q active mass spectrometer for 0-140min, spray voltage of 2.3kV, and ion transfer tube temperature of 275 deg.C. The ion mode is a positive ion mode, the default charge is two, the resolution of full scan is 70000, AGCtarget is le6, Maximum IT is 20ms, the scanning range is 300 to 1800m/z, the secondary scanning resolution is 17500, AGC target is le5, Maximum IT is 60ms, the Top 10 principle is adopted to acquire data, and the dynamic exclusion time is set to be 30 s.

The ambient temperature of the mass spectrum is 22 ℃, and the dehumidifier controls the air humidity of the mass spectrum working environment to be 50%.

3. Fat body proteomics data matching

The silkworm protein sequence database was downloaded from the website of table 2. The data in 4 databases of NCBI (29619), SilkDB (14623), Silkworm (16329) and Uniprot (18319) are merged (75584) by using an online software CD-HIT 90% threshold, and then are subjected to deduplication (33515) and redundancy removal (21878), so that a database Streamline (figure 1) with redundancy removed is obtained. The library search parameters are as follows: the variable modification is selected from oxidation (M) and Acetyl (Protein N-term), the restriction enzyme cutting site is Trypsin/P, the maximum leakage cutting site is two, the maximum charge number is 7, the Protein FDR is 0.01, and the minimum peptide segment length is 6.

The obtained data was free of contaminating sequence data and reverse sequence data to obtain raw proteomics data, which was co-constructed to 3944 proteins (table 3).

TABLE 2 download of websites for silkworm databases

TABLE 3 identification of proteins in adipose bodies using large-scale proteomics identification method (iBAQ values top 20)

Optimizing conditions:

(1) determining a grinding mode:

in order to find the best grinding mode, fat body proteins of five-year-old three-day-old silkworms are extracted by two methods, namely liquid nitrogen extraction and tissue grinder extraction, and the protein electrophoresis detection result is shown in fig. 2. The results showed that the protein amounts were comparable for both milling modes and that the tissue mill extracted only slightly increased protein amounts. Since some tissue samples of silkworms are small, if protein is extracted by means of liquid nitrogen grinding, the loss of the samples is caused. The tissue grinder can grind samples in a 1.5mL centrifuge tube, and protein samples are hardly lost, but heat is easily generated during grinding, and protein extraction is affected. As can be seen by comparison, although the ground sample of the tissue grinder generates heat during the grinding process, the number of the identified proteins is not reduced, but is slightly increased. The reason for this may be that the protein degradation itself is not much, and the principle of mass spectrometric detection is to match specific peptide fragments, which are not completely degraded into amino acids although the protein is degraded. Since the tissue grinder will not reduce the number of proteins, the tissue grinder can grind the protein sample when the amount of the processed protein sample is small. This avoids loss of protein and does not affect the number of proteins identified.

(2) Extraction reagent optimization

The results are shown in FIG. 3, in which bombyx mori three-day-old fatty body materials were extracted with 4% SDS, 4% SDS/100mM DTT, 8M urea, and 100mM DTT/8M urea, respectively, using conventional protein extraction reagents, protein samples were treated with FASP mass spectrometry, and mass spectrometry was performed using LC-MS/MS. As a result, it was found that the number of proteins identified by different extraction reagents was different, and that the number of proteins identified by the 100mM DTT/8M urea solution was the largest, and 1811 proteins were identified. The results also show that the extraction method of the SDS solution and the extraction method of the urea solution are the same in most of the identified protein types, and different extraction reagents can extract some specific proteins. Therefore, the 115 proteins are improved by using the extraction reagent of the invention.

(3) Sample application time and volume optimization

By comparing the number of proteins identified by different loading volumes, as can be seen in FIG. 4, it is not the case that the larger the loading volume the greater the number of proteins identified. When a certain saturation value is reached, the number of identified proteins no longer increases. The amount of the sample is preferably 6 to 8. mu.g (8. mu.L). In this case, the abundance of the protein is also substantially at a critical value, which is related to the corresponding signal value. The time for detecting the gradient is not longer as much as possible, and the optimal gradient time is 2h (figure 5) for silkworm samples, and the abundance value of the protein is similar. And the peptide fragments which are too much to be loaded on the mass spectrum can not be combined with the analytical column, thereby causing waste, easily causing the blockage of the analytical column and influencing the service life of the column and a machine. It follows that by comparing the amounts of the different species, we can find that 322 proteins can be elevated. Through comparison of different gradient times, it was found that 224 proteins could be elevated.

(4) Comparison of protein fractionation and peptide fractionation

By comparing the number of proteins identified by protein fractionation, peptide fractionation and non-fractionation (FIG. 6), it can be seen that the number of proteins identified can be increased in both protein fractionation and peptide fractionation. The number of proteins identified by peptide fragment fractionation is more than that identified by protein fractionation, and the operation process of peptide fragment fractionation is relatively easy. In summary, reverse-phase column fractionation is a desirable fractionation method.

The specific method for protein fractionation is as follows:

similarly, selecting a fat body sample of the fifth day of age, grinding the sample by using a G50 tissue grinder, extracting a reagent of 8M urea 100mM DTT solution, selecting a Bradford quantitative kit to quantify protein, and loading 300 mu G of protein into 12% protein gel for electrophoresis; and (3) removing the protein glue, dyeing the protein glue by using Coomassie brilliant blue until the whole glue surface becomes dark blue, and removing the protein glue by using a decoloring solution until the protein glue is colorless. The glue is scanned into an electronic file for storage by an Image scanner. Lanes were cut into 9 portions according to protein abundance with a clean scalpel and placed in 1.5mL centrifuge tubes, respectively.

Pretreatment of mass spectrum: cutting the classified protein adhesive tape into cubes with the size of about 1 cubic millimeter by using a scalpel, and filling the cubes into a clean 1.5mL centrifuge tube; 50% acetonitrile 100mM NH was charged₄HCO₃Adding 200 mu L of the leaching solution into each centrifugal tube, leaching for 10min, sucking the washing solution, and repeating the step twice; pumping the gel block to dryness by using a vacuum freezing concentrator; with 10mM DTT/50mM NH₄HCO₃Immersing the small rubber blocks, placing in a 56 ℃ metal bath for constant temperature warm bath for 1h, and then sucking out the immersion liquid; with 55mM iodoacetamide/50 mM NH₄HCO₃Immersing the small gel block, incubating in a dark room for 30min, and sucking off the immersion liquid; with 200. mu.L of 10mM NH₄HCO₃After 10min of immersion, the immersion liquid was aspirated. Then soaking and washing with 200 μ L acetonitrile solution for 10min, and removing the soaking solution; repeating the steps once; pumping the gel block to dryness by using a vacuum freezing concentrator; adding 10 ng/microliter of trypsin solution into the dried small gel block, and removing the redundant enzyme solution after the gel block is completely swelled; 10mM NH was added₄HCO₃Covering the solution with small rubber blocks; placing in a constant temperature incubator at 37 ℃ overnight, and performing enzymolysis on the protein to obtain peptide fragments; approximately equal volume of 60% acetonitrile/5% formic acid solution was added and shaken ultrasonically for 10 min. After the flash separation, collecting the supernatant in a new centrifuge tube; this procedure was repeated and the supernatants from both collections were vacuum freeze-concentrated to dryness.

Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.

Claims (4)

1. A method for large-scale proteomics identification based on silkworm tissue samples is characterized by comprising the following steps:

(1) pretreatment of silkworm proteomics samples: adding 100mM DTT/8M urea solution into silkworm fat, putting the sample on ice, grinding the sample by using a G50 tissue grinder to fully dissolve the sample, centrifuging and collecting supernatant; performing enzymolysis on the sample by using an FASP enzymolysis method, dividing the peptide fragment into 8 grades by using a high pH grading method, and freeze-drying each component by using a vacuum freeze-drying instrument;

the method for dividing the peptide fragment into 8 grades by using a high pH grading method specifically comprises the following steps:

1) taking off the white plastic sleeve from the bottom of the spin column, and placing the spin column in a centrifuge tube;

2) centrifuging at 5000g for 2min, and removing the solution in the spin column;

3) taking off a red cap at the top end of the spin column, loading 300 mu L of acetonitrile into the spin column, covering the red cap, putting the spin column into a centrifuge tube, placing the centrifuge tube in a centrifuge for 5000g for centrifugation for 2min, washing the column and removing the acetonitrile;

4) washing the spin column twice with 300 μ L of 0.1% trifluoroacetic acid;

5) adding 300 mu L of trifluoroacetic acid solution with volume fraction of 0.1% into the freeze-dried peptide fragment sample, and vortexing for 30s to dissolve the peptide fragment;

6) placing the spin column in a new centrifuge tube, loading the sample into the column, covering with a red cover, placing in a centrifuge at 3000g, and centrifuging for 2min to obtain a flow-through centrifuge tube;

7) putting the spin column into a new centrifugal tube, adding ultrapure water, and centrifuging for 2min at 3000g to obtain a cleaning solution;

8) preparing an elution solution with tube numbers of 1-8, putting the spin column into a new centrifuge tube, adding the eluent with the tube number of 1, covering a red cover, and centrifuging for 2min at 3000 g;

the elution solution of the tube number 1-tube number 8 is specifically as follows:

tube number 1: acetonitrile 50. mu.L, 0.1% trifluoroacetic acid 950. mu.L; tube number 2: 75 μ L, 925 μ L of 0.1% trifluoroacetic acid; tube number 3: acetonitrile 100. mu.L, 0.1% trifluoroacetic acid 900. mu.L; tube number 4: 125 mul acetonitrile, 875 mul 0.1% trifluoroacetic acid; tube number 5: acetonitrile 150. mu.L, 0.1% trifluoroacetic acid 850. mu.L; tube number 6: acetonitrile 175. mu.L, 0.1% trifluoroacetic acid 825. mu.L; tube number 7: acetonitrile 200. mu.L, 0.1% trifluoroacetic acid 800. mu.L; tube number 8: acetonitrile 500. mu.L, 0.1% trifluoroacetic acid 500. mu.L;

9) collecting the eluent in a centrifuge tube, opening a red cap, and adding 300 mu L of the solution with the tube number of 2 into a spin column;

10) according to the steps 8) and 9), sequentially adding elution solutions with tube numbers of 3-8, and collecting different gradient eluents in 8 centrifuge tubes;

(2) and (3) carrying out mass spectrum detection on the classified peptide fragments: dissolving each stage of peptide fragment in 50 μ L of 0.1% formic acid solution with sample loading amount of 8 μ L and effective chromatographic gradient time of 120 min;

(3) bombyx mori proteomics database: and removing redundant sequences of NCBI, SilkDB, Silkworm and Uniprot 4 Silkworm protein databases by using a 90% threshold value to obtain a database Streamline, and searching the database by using MaxQuant as original data.

2. The method of claim 1 for large scale proteomic identification based on bombyx mori tissue samples, wherein the method comprises the steps of: in the step 1), 200 mu L of 100mM DTT/8M urea solution is added into 100mM DTT/8M urea solution per milligram of silkworm fat; grinding for 3 min; the full dissolution is oscillation for 5min by a horizontal oscillator; the centrifugation was 12000g, centrifugation 15min at 25 ℃.

3. The method for large-scale proteomics identification based on silkworm tissue samples according to claim 1, wherein the FASP enzymolysis method specifically comprises the following steps:

1) adding the sample into a 10kDa ultrafiltration tube, adding 8M urea solution, uniformly mixing, placing in a centrifuge of 12000g, and centrifuging for 20 min;

2) adding 8M urea at 12000g, and centrifuging for 20 min;

3) repeating the step 2) twice;

4) adding 1M DTT/8M urea solution, sealing the pipe orifice with a sealing film, and incubating at 37 deg.C for 2 h;

5) tearing the sealing film, adding 1M iodoacetamide solution, mixing uniformly, and incubating for 1h in a dark place;

6) centrifuging at 12000g for 20 min;

7) adding 8M urea solution, mixing uniformly, and centrifuging at 12000g for 20 min;

8) repeating the step 7) twice;

9) 50mM NH was added₄HCO₃Centrifuging the solution at 12000g for 20 min;

10) repeating the step 9) for three times;

11) the outer pipe of the ultrafiltration pipe is replaced by a new outer pipe to prevent pollution;

12) adding 400 mu L of 10 mu g/mL trypsin solution into an ultrafiltration tube, sealing the tube opening with a sealing film, placing in a constant-temperature incubator at 37 ℃ for incubation for 36h, and carrying out enzymolysis on the protein into peptide fragments;

13) tearing off the sealing film, and centrifuging at 12000g for 20 min;

14) add 40. mu.L of 50mM NH to the ultrafiltration tube₄HCO₃Centrifuging the solution at 12000g for 20 min;

15) freeze-concentrate at 4 ℃ until the sample is completely dry.

4. The method for large-scale proteomics identification based on silkworm tissue samples according to claim 1, wherein the classification peptide fragment mass spectrometry is specifically as follows:

adding 0.1% formic acid water into each component peptide fragment, shaking for 30s for dissolution, and centrifuging at 12000g for 10 min; sucking the supernatant into a sample loading bottle, and separating peptide fragments by adopting a Thermo Fisher Scientific EASY-nLC 1000 nanoliter flow rate system, wherein the volume of an equilibrium pre-column is 10 mu L, the flow rate is 3.5 mu L/min, the volume of an equilibrium analysis column is 5 mu L, and the flow rate is 0.25 mu L/min; the sample loading amount is 8 mu L, the sample suction flow rate is 8 mu L/min, 18 mu L of formic acid water with volume fraction of 0.1 percent is pushed into a pre-column and an analytical column by using the flow rate of 3.5 mu L/min, and the peptide section is separated by using the formic acid acetonitrile solution with volume fraction of 0.1 percent in a gradient manner, and the flow rate is 250 nL/min; the effective chromatographic gradient time is 120min gradient, and 100 mul of formic acid acetonitrile with volume fraction of 0.1% is used for cleaning an automatic sample loading ring;

detecting by adopting a Q active mass spectrometer, wherein the detection time is 0-140min, the spray voltage is 2.3kV, the temperature of an ion transmission tube is 275 ℃, the ion mode is a positive ion mode, the default charge is two, the resolution of full scan is 70000, the AGCtarget is 1e6, Maximum IT is 20ms, the scanning range is 300-1800 m/z, the secondary scanning resolution is 17500, the AGC target is 1e5, and Maximum IT is 60ms, acquiring data by adopting a Top 10 principle, and the dynamic exclusion time is set to be 30 s;

the ambient temperature of the mass spectrum is 22 ℃, and the dehumidifier controls the air humidity of the mass spectrum working environment to be 50%.

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