A method for extracting and analyzing proteins in ancient murals

Abstract

The application discloses an extraction method and an analysis method of proteins in ancient murals, and relates to the technical field of biology.The application provides an extraction method of proteins, which can effectively extract collagen from ancient murals through the steps of acid demineralization, washing, reduction alkylation and enzymolysis of powder samples from ancient murals, and provides technical support for scientific archaeological research, scientific cognition of ancient murals and mural protection and restoration research, and has the advantages of good extraction effect, simple operation, high repeatability and the like.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a method for extracting and analyzing proteins from ancient murals. Background Technology

[0002] Ancient murals are precious and fragile, possessing extremely high historical, artistic, scientific, and socio-cultural value. Many ancient murals worldwide are listed as World Heritage Sites, such as the Mogao Grottoes murals in Dunhuang, China; the Cave of Original Sin murals in Italy; and the Lascaux prehistoric murals in France.

[0003] For a long time, understanding and interpreting the materials and processes used in the production of ancient murals has been the main focus of mural analysis and research worldwide. However, due to the diversity of mural types, the richness of materials, and the complexity of processes, coupled with limitations in analytical techniques and sample quantities, and the fact that most ancient murals suffer from severe degradation, the analysis of the composition of the cementing materials in the pigment layers of murals has always been a difficult problem in the field of cultural relic protection. Most analytical techniques are still unable to accurately obtain comprehensive information on the proteins in the cementing materials, and it is even more difficult to determine and accurately identify the animal origin of the proteins. This, in turn, greatly limits the realization of the ancient mural protection concept based on "raw materials and original processes" and the development of protection and restoration techniques for fragile ancient murals.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide a method for extracting and analyzing proteins from ancient murals.

[0006] This invention is implemented as follows:

[0007] In a first aspect, embodiments of the present invention provide a method for extracting proteins from ancient murals, comprising: obtaining a powder sample derived from a pigment layer of an ancient mural; subjecting the powder sample to acidic demineralization, washing, reductive alkylation, and enzymatic hydrolysis to obtain crude protein peptide fragments.

[0008] Secondly, embodiments of the present invention provide a method for analyzing proteins in ancient murals, the method comprising: analyzing peptides extracted by the extraction method described in the foregoing embodiments.

[0009] Thirdly, embodiments of the present invention provide a product for extracting and / or analyzing proteins in ancient murals, comprising reagents for performing the extraction method or the analysis method described in the foregoing embodiments.

[0010] Fourthly, embodiments of the present invention provide the application of reagents for implementing the extraction method or analytical method described in the foregoing embodiments in the preparation of products for the extraction or analysis of proteins in ancient murals.

[0011] The present invention has the following beneficial effects:

[0012] This invention provides an extraction method that effectively extracts collagen from ancient murals through steps such as acidic demineralization, washing, reductive alkylation, and enzymatic hydrolysis. This provides technical support for scientific archaeology research, scientific understanding of ancient murals, and research on mural conservation and restoration. The extraction method has advantages such as good extraction effect, simplicity, and high reproducibility.

[0013] This invention, through extensive and innovative experiments and continuous optimization of experimental parameters, ultimately provides an extraction method that yields high-quality collagen protein in large quantities. Furthermore, the mass spectrometry analysis provides a large amount of data and highly reliable identification information, enabling its effective application in the characterization and understanding of materials used in ancient murals and in guiding the conservation and restoration of murals. Detailed Implementation

[0014] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0015] This invention proposes a method for extracting collagen from the cementing material of ancient mural pigment layers and obtaining the informational characteristics of collagen, providing technical support for scientific archaeology research, scientific understanding of ancient murals, and research on mural protection and restoration; compared with other methods, it has advantages such as good extraction effect, simple operation, and strong repeatability.

[0016] The extraction method of this invention is specifically applicable to any type of mural that incorporates protein-containing materials such as egg white, casein, or animal glue during its production process. This includes dry and wet murals created using different techniques and preserved in environments such as grottoes, temples, churches, and tombs. Examples include the murals of the Mogao Grottoes in Dunhuang (a World Heritage Site), the Bingling Temple Grottoes in Yongjing, the Maijishan Grottoes in Tianshui, and the Beishiku Temple Grottoes in Qingyang (a National Key Cultural Relics Protection Unit). These ancient murals span over a thousand years, and their materials and techniques varied at different times. In principle, the extraction method of this invention is not limited by the preservation period of the mural, but the lower the degree of protein degradation in the mural, the higher the accuracy of the results.

[0017] On one hand, embodiments of the present invention provide a method for extracting proteins from ancient murals, which includes: obtaining a powder sample from a flaky sample originating from the damaged edge of the pigment layer of an ancient mural, which is then manually crushed by a micro-grinding pestle; subjecting the powder sample to acidic demineralization (the purpose of which is to dissolve minerals, loosen the material, and facilitate the release of proteins from the material), washing (washing away unreacted trifluoroacetic acid), reduction, alkylation, and enzymatic hydrolysis to obtain crude protein peptide fragments.

[0018] In some embodiments, the acid demineralization includes mixing the powder sample with an acid solution. The acid solution includes either a trifluoroacetic acid solution or a hydrochloric acid solution.

[0019] In some embodiments, when the acid solution is a trifluoroacetic acid solution, the amount of trifluoroacetic acid added is: 0.25 mL of a trifluoroacetic acid solution with a volume fraction of 5% to 15% for every 30 mg of the powder sample. Specifically, the volume fraction of trifluoroacetic acid in the trifluoroacetic acid solution can be any one or a range between any two of 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, and 15%.

[0020] In some embodiments, the mixing conditions with the acid solution include: 4–10°C, 20–30 h (low temperature is acceptable, but time must be more than 20 h). Specifically, the temperature can be any one or any two of 0°C, 2°C, 4°C, 6°C, 8°C, and 10°C; the time can be any one or any two of 10 h, 12 h, 14 h, 16 h, 18 h, 20 h, 22 h, 24 h, 26 h, 28 h, and 30 h.

[0021] In some embodiments, the acid demineralization further includes centrifuging the mixed product to obtain a solid material for subsequent washing. Optionally, the centrifugation conditions are as follows: a rotation speed of 10,000–14,000 rpm for 1–5 minutes. Specifically, the rotation speed can be any one or a range between any two of 10,000 rpm, 11,000 rpm, 12,000 rpm, 13,000 rpm, and 14,000 rpm; the centrifugation time can be any one or a range between any two of 1 minute, 2 minutes, 3 minutes, 4 minutes, and 5 minutes.

[0022] In some embodiments, the powder sample is derived from the damaged edge of the ancient mural, and the mass of the powder sample can be 10–50 mg, specifically within any one or any two of the following ranges: 10 mg, 12 mg, 14 mg, 16 mg, 18 mg, 20 mg, 22 mg, 24 mg, 26 mg, 28 mg, 30 mg, 32 mg, 34 mg, 36 mg, 38 mg, 40 mg, 42 mg, 44 mg, 46 mg, 48 mg, and 50 mg. The powder sample is taken from the damaged edge of the mural, and the sampling process will not cause significant damage to the precious ancient mural.

[0023] In some embodiments, the particle size of the powder sample is ≤0.5-10mm, specifically less than any one of 0.5mm, 1mm, 2mm, 3mm, 4mm, 5mm, 6mm, 7mm, 8mm, 9mm, and 10mm, preferably less than 0.5mm.

[0024] In some embodiments, the washing solution used for washing includes: 50–150 mM Tris-HCl, with a pH of 8–9. The concentration of Tris-HCl can specifically be any one or a range between any two of the following: 50 mM, 60 mM, 80 mM, 100 mM, 120 mM, 140 mM, and 150 mM; the pH can specifically be any one or a range between any two of the following: 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, and 9.

[0025] In some embodiments, the number of washes is 2 to 5. Specifically, the number of washes can be any one or a range between 2, 3, 4, and 5.

[0026] In some embodiments, each washing step includes: mixing the product after acid demineralization or the precipitate after the previous washing with the washing liquid, allowing it to stand, and then centrifuging to obtain the precipitate for subsequent reductive alkylation or subsequent washing.

[0027] In some embodiments, the centrifugation conditions for the washing process include: a rotation speed of 10,000–14,000 rpm and a duration of 1–5 minutes. Specifically, the rotation speed can be any one or a range between any two of 10,000 rpm, 11,000 rpm, 12,000 rpm, 13,000 rpm, and 14,000 rpm; and the duration can be any one or a range between any two of 1 minute, 2 minutes, 3 minutes, 4 minutes, and 5 minutes.

[0028] In some embodiments, the reductive alkylation includes mixing the washed product with a lysis buffer.

[0029] In some embodiments, the lysis buffer comprises the following components and their final concentrations: 4–6 M guanidine hydrochloride, 5–15 mM tris(2-carboxyethyl)phosphine (TCEP), 30–50 mM 2-chloroacetamide (CAA), and 50–150 mM Tris-HCl, with a pH of 8–9. Specifically, the final concentration of guanidine hydrochloride in the lysis buffer can be any one or any two of 4 M, 5 M, and 6 M; the final concentration of tris(2-carboxyethyl)phosphine can be any one or any two of 5 mM, 6 mM, 8 mM, 10 mM, 12 mM, 14 mM, and 15 mM; and the final concentration of 2-chloroacetamide can be 30 mM, 32 mM, 34 mM, 36 mM, 38 mM, 40 mM, 42 mM, or 44 mM. The concentration of Tris-HCl can be any one or any two of the following: 46mM, 48mM, and 50mM; the concentration of Tris-HCl can be any one or any two of the following: 50mM, 60mM, 80mM, 100mM, 120mM, 140mM, and 150mM; and the pH can be any one or any two of the following: 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, and 9.

[0030] In some embodiments, the mixing conditions with the lysis buffer include: 80–100°C for 15–40 min (under these time and temperature ranges, the reduction reaction is relatively complete). Specifically, the temperature can be any one or any two of the following: 80°C, 82°C, 84°C, 86°C, 88°C, 90°C, 92°C, 94°C, 96°C, 98°C, and 100°C; and the time can be any one or any two of the following: 15 min, 16 min, 18 min, 20 min, 22 min, 24 min, 26 min, 28 min, 30 min, 32 min, 34 min, 36 min, 38 min, and 40 min.

[0031] In some embodiments, the reductive alkylation further includes sonicating the product mixed with the lysis solution.

[0032] In some embodiments, the ultrasound conditions include: 40–50°C, 15–40 min (this time and temperature have limited impact on the final result). Specifically, the temperature can be any one or any two of 40°C, 42°C, 44°C, 46°C, 48°C, and 50°C; the time can be any one or any two of 15 min, 16 min, 18 min, 20 min, 22 min, 24 min, 26 min, 28 min, 30 min, 32 min, 34 min, 36 min, 38 min, and 40 min.

[0033] In some embodiments, the enzymatic hydrolysis includes: reducing the alkylated product and mixing it with trypsin.

[0034] In some embodiments, the enzymatic hydrolysis further includes: first mixing the reduced alkylated product with a diluent, and then mixing it with the trypsin.

[0035] In some embodiments, the amount of trypsin added is: 1 part trypsin is added for every (30-80) parts of protein in the reduced alkylation product. The specific mass ratio of protein to trypsin can be any one or any two of the following: 30:1, 40:1, 50:1, 60:1, 70:1, and 80:1. Too little enzyme will lead to incomplete digestion, while too much enzyme will result in inter-enzymatic cleavage, reducing enzyme efficiency and contaminating the protein sample itself. Within the scope defined in this application, trypsin can more effectively digest protein.

[0036] In some embodiments, the diluent comprises the following components and their final concentrations: 70%–90% acetonitrile (by volume) and 50–150 mM Tris-HCl at a pH of 8–9. Specifically, the volume fraction of acetonitrile in the diluent can be any one or any combination of 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, and 90%. The Tris-HCl is as described in the foregoing embodiments and will not be repeated.

[0037] In some embodiments, the conditions for mixing with trypsin include: 30–37°C for 18–24 hours. Specifically, the temperature can be any one or any two of the following: 25°C, 26°C, 28°C, 30°C, 32°C, 34°C, 36°C, 38°C, and 40°C, preferably 37°C (the optimal activity temperature of the enzyme); the time can be any one or any two of the following: 18 hours, 20 hours, 22 hours, and 24 hours.

[0038] In some embodiments, the extraction method further includes: separating and purifying the enzymatically hydrolyzed product.

[0039] In some embodiments, the separation and purification includes acidifying and desalting the enzymatically hydrolyzed product.

[0040] In some embodiments, the acidification includes: adding an acidifying solution to the enzymatically hydrolyzed product to adjust the pH of the solution to 1.5–2.5 (this range is the optimal acidification condition), centrifuging, and then using the supernatant for desalting. Specifically, the pH of the solution can be adjusted to any one or any two of the following: 1.5, 1.6, 1.8, 2.0, 2.2, 2.4, and 2.5.

[0041] In some embodiments, the acidifying solution comprises a trifluoroacetic acid solution with a volume fraction of 1% to 10%. The volume fraction of trifluoroacetic acid can specifically be any one or any two of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and 10%.

[0042] In some embodiments, the acidification further includes: first concentrating the enzymatically hydrolyzed product, and then acidifying it.

[0043] In some embodiments, the concentration includes centrifugal rotary concentration.

[0044] In some embodiments, the temperature of the centrifugal rotary concentration is 55–65°C. Specifically, this temperature can be any one or a range between any two of 55°C, 56°C, 58°C, 60°C, 62°C, 64°C, and 65°C.

[0045] In some embodiments, the desalting includes: separating the acidified product using a solid-phase extraction membrane, and eluting with an eluent to obtain peptides.

[0046] In some embodiments, the solid-phase extraction membrane includes: Empore C 18 SPE membrane.

[0047] In some embodiments, the eluent comprises the following components and their final concentrations: 70%–90% acetonitrile by volume and 0.05%–0.15% acetic acid by volume. Specifically, the volume fraction of acetonitrile in the eluent can be any one or any two of 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, and 90%; the volume fraction of acetic acid can be any one or any two of 0.05%, 0.06%, 0.08%, 0.1%, 0.12%, 0.14%, and 0.15%.

[0048] In some embodiments, the number of washes is 1 to 5. Specifically, the number of washes can be any one or a range between any two of 1, 2, 3, 4, and 5.

[0049] In some embodiments, the extraction method further includes concentrating the eluted product.

[0050] On the other hand, embodiments of the present invention provide a method for analyzing proteins in ancient murals, the method comprising: analyzing peptides extracted by the extraction method described in any of the foregoing embodiments.

[0051] In some embodiments, the analysis includes mass spectrometry.

[0052] In some embodiments, the mass spectrometry analysis includes: dissolving the separated and purified peptides in an aqueous solution containing 0.05% to 0.15% formic acid (by volume), centrifuging, and collecting the supernatant for mass spectrometry data collection. In the aqueous solution, the volume fraction of formic acid can specifically be any one or a range between any two of 0.05%, 0.06%, 0.08%, 0.1%, 0.12%, 0.14%, and 0.15%.

[0053] In some embodiments, the analysis method further includes processing the collected mass spectrometry data to obtain information about proteins in ancient murals.

[0054] In some embodiments, PEAKS software can be used to analyze and process the mass spectrometry data. Specific settings are as follows: enzyme is set to trypsin, maximum allowable number of missed cleavages is set to 3, primary mass tolerance is set to 10 ppm, secondary mass tolerance is set to 0.02 Da, no fixed modification is used, methionine oxidation, asparagine and glutamine deamidation, and lysine and proline hydroxylation are set to variable modification.

[0055] On the other hand, embodiments of the present invention also provide a product for extracting and / or analyzing proteins in ancient murals, comprising reagents for performing the extraction method or the analysis method described in any of the foregoing embodiments.

[0056] Furthermore, embodiments of the present invention also provide the application of reagents for implementing the extraction method or analytical method described in any of the foregoing embodiments in the preparation of products for the extraction or analysis of proteins in ancient murals.

[0057] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0058] Example 1

[0059] A method for extracting and analyzing collagen from ancient murals, comprising the following steps:

[0060] (1) A pigment layer sample of about 25 square millimeters was obtained from the edge of the ancient mural in Cave C275 of Mogao Grottoes in Dunhuang. It was crushed with a micro-grinding pestle (≤0.5mm) and about 30mg of material was obtained.

[0061] (2) Acidic demineralization: Add 0.25 mL of 10% trifluoroacetic acid to the pretreated material, shake and incubate at 4°C for 24 h, then centrifuge at 12000 rpm for 3 min, and take the solid material for later use.

[0062] (3) Washing: Add 1 mL of 100 mM Tris-HCl pH=8.5 washing solution to the above solid material, vortex for 1 min, let stand for 2 min, then centrifuge at 12000 rpm for 3 min, take the precipitate and add more washing solution, repeat the above steps 3 times to obtain a precipitate without trifluoroacetic acid.

[0063] (4) Reductive alkylation: Add 0.1 mL of lysis buffer consisting of 6 M guanidine hydrochloride, 10 mM TCEP, 40 mM CAA, and 100 mM Tris-HCl at pH 8.5 to the above precipitate (each concentration is the final concentration of each component), seal the container, and then incubate at 95 °C for 30 min, followed by sonication in a water bath at 45 °C for 30 min.

[0064] (5) Enzymatic hydrolysis: After cooling the above mixed solution to room temperature, add 0.9 mL of a diluent prepared by mixing 80% ACN and 100 mM Tris-HCl (pH = 8.5) (each concentration is the final concentration of each component). Measure the protein concentration in the solution using a BCA protein concentration assay kit. Then, add trypsin at a protein to trypsin mass ratio of 50:1, vortex to mix, seal, and incubate at 37°C with shaking for 20 h. During this process, the protein is digested into smaller peptides.

[0065] (6) Acidification: The peptide solution in step (5) above is concentrated to a volume of 0.1 mL in a centrifugal rotary concentrator at 60 °C. Then, 0.02 mL of 5% trifluoroacetic acid solution is added. The result is compared with the color comparison card of the wide pH test paper. The pH value of the enzymatic hydrolysis solution is adjusted to 2. Then, the precipitate is removed by centrifugation at 12000 rpm for 30 min. The supernatant is taken for later use.

[0066] (7) Desalination: Use 3M Empore C 18 The peptides in the supernatant from step (6) were separated using an SPE membrane, and then eluted with a solution prepared by mixing 0.1 mL of 80% acetonitrile and 0.1% acetic acid. This process was repeated three times. The peptides were then dried by rotary evaporation in a centrifugal concentrator at 60°C.

[0067] (8) Mass spectrometry analysis: The peptide fragments were dissolved in an aqueous solution containing 0.1% formic acid, and the concentration of the peptide fragments in the solution was determined using a BCA protein concentration assay kit. The peptide fragment solution was centrifuged at 12000 rpm for 30 min, and 1 μg of peptide fragments was placed in a mass spectrometry sample vial for mass spectrometry data collection.

[0068] (9) Data processing: The above mass spectrometry data were processed using PEAKS software and bioinformatics technology. The parameters were set as follows: the enzyme was set to trypsin, the maximum number of uncut samples was set to 3, the first-level mass tolerance was set to 10 ppm, the second-level mass tolerance was set to 0.02 Da, no fixed modification was used, methionine oxidation, asparagine and glutamine deamidation, and lysine and proline hydroxylation were set as variable modifications. Detailed information on the collagen contained in the sample was then obtained.

[0069] Example 2

[0070] A method for extracting and analyzing collagen from ancient murals in Cave C275 of the Mogao Grottoes in Dunhuang is roughly the same as in Example 1, except that the mass ratio of protein to trypsin in step (5) is 30:1.

[0071] Example 3

[0072] A method for extracting and analyzing collagen from ancient murals in Cave C275 of the Mogao Grottoes in Dunhuang is roughly the same as in Example 1, except that the mass ratio of protein to trypsin in step (5) is 80:1.

[0073] Comparative Example 1

[0074] A method for extracting and analyzing collagen from ancient murals in Cave C275 of the Mogao Grottoes in Dunhuang is largely the same as in Example 1, except that step (2) is omitted.

[0075] Comparative Example 2

[0076] A method for extracting and analyzing collagen from ancient murals in Cave C275 of the Mogao Grottoes in Dunhuang is largely the same as in Example 1, except that step (3) is omitted.

[0077] The extracted protein peptides prepared from Examples 1-3 (Samples 1-3) and Comparative Examples 1-2 (Samples 4-5) of this invention were analyzed using PEAKS software and bioinformatics techniques. The results are shown in Table 1 below.

[0078] Table 1. Protein components identified in the samples

[0079]

[0080] The analytical results of Examples 1-3 show that when the amount of trypsin added is too small, the protein digestion is incomplete, resulting in a reduced number of identified proteins and lower coverage (reliability). Furthermore, the analytical results of Examples 1 and Comparative Examples 1-2 show that omitting any step in the sample processing will affect the extraction and identification of collagen in the ancient mural samples.

[0081] Experimental Example 1

[0082] Using the method provided in Example 1, a 25mm sample was extracted from the damaged edge of an ancient mural in Cave 256 of the Mogao Grottoes in Dunhuang. 2 The sample consisted of approximately 30 mg of pigment layer material from the left and right sides. The protein components identified in the sample are shown in Table 2.

[0083] Table 2 shows the protein components identified in the samples.

[0084]

[0085]

[0086] Experimental Example 2

[0087] Using the method provided in Example 1, a 25mm sample was extracted and analyzed from the damaged edge of the ancient mural in Cave 127 of Maijishan Grottoes. 2 The sample of the mural with applied gold leaf, approximately 30 mg in size, is shown in Table 3. The protein composition identified in the sample is shown in Table 3.

[0088] Table 3 shows the protein components identified in the samples.

[0089]

[0090] This invention, through extensive and creative experiments, continuously optimized experimental parameters, including the reagents used and their amounts, acidic demineralization temperature, acidic demineralization time, mixing conditions with the lysis buffer and heating time, acidification conditions, the mass of trypsin added, enzymatic hydrolysis temperature, enzymatic hydrolysis time, and the number of peptide elutions. Ultimately, the specific method provided by this invention was obtained. Arbitrary adjustments to the mentioned parameters will affect the extraction and identification of collagen in ancient mural samples. Furthermore, mass spectrometry analysis provides a large amount of data and highly reliable identification information, thus enabling its effective application in the characterization and understanding of materials used in the creation of ancient murals and in guiding mural conservation and restoration work.

[0091] It should be noted that in the description of this specification, the referenced technical terms should be understood in their ordinary meaning as to be understood by one of ordinary skill in the art. Furthermore, the steps and numerical values ​​set forth in these embodiments do not limit the scope of the invention. Unless otherwise specified, any specific value should be interpreted as merely exemplary and not as a limitation; therefore, other examples of exemplary embodiments may have different values.

[0092] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. For those skilled in the art, the present invention can have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for extracting proteins from ancient murals, characterized in that, It includes: A powder sample derived from the pigment layer of an ancient mural was obtained. The powder sample was subjected to acid demineralization, washing, reductive alkylation, and enzymatic hydrolysis to obtain crude protein peptide fragments. The powder sample originated from the damaged edge of an ancient mural. The protein in question is collagen. The acid demineralization process includes: mixing the powder sample with an acid solution, centrifuging the mixture to obtain a solid material for subsequent washing; the acid solution is a trifluoroacetic acid solution, and the amount of trifluoroacetic acid added is: 0.1~0.5 mL of a 5%~10% trifluoroacetic acid solution per 30 mg of the powder sample; the mixing conditions include: 0~10℃, 10~30 h; Each washing step includes: mixing the product after acid demineralization or the precipitate after the previous washing with the washing solution, allowing it to stand, and then centrifuging to obtain the precipitate for subsequent reduction alkylation or subsequent washing. The washing solution used is 50~150 mM Tris-HCl with a pH of 8~9. The reductive alkylation includes: mixing the washed product with a lysis buffer; the components and final concentrations of the lysis buffer are as follows: 4-6 M guanidine hydrochloride, 5-15 mM tris(2-carboxyethyl)phosphine, 30-50 mM 2-chloroacetamide, and 50-150 mM Tris-HCl at a pH of 8-9; the mixing conditions with the lysis buffer include: 80-105°C for 15-40 min; the reductive alkylation also includes: sonicating the product mixed with the lysis buffer; the sonication conditions include: 40-50°C for 15-40 min. The enzymatic hydrolysis includes: first mixing the reduced alkylated product with a diluent, and then mixing it with trypsin; the components and final concentrations of the diluent are as follows: 70%~80% acetonitrile and 50~150 mM Tris-HCl, pH 8~9; the amount of trypsin added is: 1 part trypsin for every 30~50 parts of protein in the reduced alkylated product; the mixing conditions with trypsin include: 30~37℃, 18~24 h; The extraction method further includes: separating and purifying the enzymatically hydrolyzed product; the separation and purification includes: acidifying and desalting the enzymatically hydrolyzed product; The acidification includes: adding an acidifying solution to the enzymatic hydrolysis product to adjust the pH of the solution to 1.5-2.5, and taking the supernatant after centrifugation for desalting; The desalting process includes: separating the acidified product using a solid-phase extraction membrane, and eluting with an eluent to obtain peptides; the eluent comprises the following components and final concentrations: 70%–90% acetonitrile (v / v) and 0.05%–0.15% acetic acid (v / v); the solid-phase extraction membrane comprises: Empore C... 18 SPE membrane.

2. The extraction method according to claim 1, characterized in that, The centrifugation conditions during the acid demineralization process are as follows: rotation speed of 10,000~14,000 rpm, 1~5 min.

3. The extraction method according to claim 1, characterized in that, The washing process is repeated 2 to 5 times.

4. The extraction method according to claim 1, characterized in that, The centrifugation conditions for the washing process include: a rotation speed of 10,000 to 14,000 rpm for 1 to 5 minutes.

5. The extraction method according to any one of claims 1 to 4, characterized in that, The acidification solution is a trifluoroacetic acid solution with a volume fraction of 1% to 10%.

6. The extraction method according to claim 5, characterized in that, The acidification process further includes: first concentrating the enzymatically hydrolyzed product, and then acidifying it.

7. The extraction method according to claim 6, characterized in that, The concentration includes: centrifugal rotary concentration.

8. The extraction method according to claim 7, characterized in that, The temperature for centrifugal rotary concentration is 55~65℃.

9. The extraction method according to claim 1, characterized in that, The number of washes is 2 to 5.

10. The extraction method according to claim 1, characterized in that, The extraction method further includes concentrating the eluted product.

11. A method for analyzing proteins in ancient murals, characterized in that, The analytical method includes analyzing the peptides extracted by the extraction method according to any one of claims 1 to 10.

12. The analytical method according to claim 11, characterized in that, The analysis includes mass spectrometry.

13. The analytical method according to claim 12, characterized in that, The mass spectrometry analysis includes: dissolving the peptide in an aqueous solution containing 0.05%~0.15% formic acid by volume, centrifuging, and then collecting the supernatant for mass spectrometry data collection.

14. The analytical method according to claim 13, characterized in that, The analytical method further includes processing the collected mass spectrometry data to obtain information about proteins in ancient murals.

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