DNA modification detection method using gel electrophoresis coupled with liquid chromatography-mass spectrometry

Abstract

This invention provides a DNA modification detection method using gel electrophoresis coupled with liquid chromatography-mass spectrometry (LC-MS / MS), comprising the following steps: separating a DNA sample to be tested by gel electrophoresis to obtain a gel strip containing the target DNA, wherein the electrophoresis buffer used in the gel electrophoresis method does not contain EDTA; enzymatically digesting the target DNA in the gel strip into single deoxynucleosides / deoxynucleotides by in-gel DNA digestion to obtain enzymatic digestion products; and detecting the enzymatic digestion products by liquid chromatography-mass spectrometry to obtain the results of DNA chemical modifications (including epigenetic modifications and DNA damage adducts). The detection method of this invention can accurately quantify the chemical modification information in different DNA components separated by gel electrophoresis.

Description

Technical Field

[0001] This invention relates to the field of biochemical analysis technology, and in particular to a method for detecting DNA modifications using gel electrophoresis coupled with liquid chromatography-mass spectrometry (LC-MS / MS). Background Technology

[0002] LC-MS / MS is a widely used technique for the precise identification and quantification of various DNA chemical modifications. It is a powerful tool for determining the diversity and potential biological significance of epigenetic DNA modifications. For decades, DNA chemical modification-mediated biological processes have been a subject of extensive research. One of the most abundant DNA modifications in mammals—5-methylcytosine (5mC)—plays a crucial role in embryonic development and physiological processes. Furthermore, it has a profound impact on gene expression in various diseases. Before mass spectrometry detection, DNA must first be extracted from cells or tissues, then enzymatically digested and dephosphorylated to monodeoxynucleotides. To measure the abundance of DNA modifications, a series of optimizations were performed on steps such as DNA extraction, DNA hydrolysis, nucleoside derivatization, and the chromatographic mobile phase to improve the sensitivity and accuracy of mass spectrometry detection.

[0003] However, directly digesting extracted genomic DNA with enzymes and then performing mass spectrometry analysis only provides information on the abundance of DNA modifications at the whole-genome level or the overall sample. To perform more precise analysis and quantification of DNA modifications in low-abundance non-chromatin DNA on chromatin genomic DNA and in subchromatin structures (such as mitochondrial DNA, outer circular DNA, and nuclear matrix attachment regions), it is necessary to separate the target DNA from other contaminating DNAs. Generally, the separation process for these samples is complex and yields low output, leading to impurities in the product. The recovered samples often contain residual non-target DNA. Furthermore, some samples may themselves consist of DNA molecules of different sizes and conformations. Directly digesting these incompletely separated DNA samples in solution may result in discrepancies between the DNA modification abundance detected by mass spectrometry and the true values. Summary of the Invention

[0004] In view of this, the main objective of the present invention is to provide a method for detecting DNA modifications by gel electrophoresis coupled with LC-MS / MS, in order to at least partially solve at least one of the aforementioned technical problems.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows:

[0006] A method for detecting DNA modification by gel electrophoresis coupled with LC-MS / MS includes the following steps: separating the DNA sample to be tested by gel electrophoresis to obtain a gel strip containing the target DNA, wherein the electrophoresis buffer used in the gel electrophoresis does not contain EDTA; digesting the target DNA in the gel strip into single deoxynucleosides and / or single deoxynucleotides by in-gel DNA digestion to obtain the digestion product; and detecting the digestion product by LC-MS / MS to obtain the DNA chemical modification result.

[0007] Based on the above technical solution, the DNA modification detection method using gel electrophoresis coupled with liquid chromatography-mass spectrometry of the present invention has at least one or a part of the following beneficial effects:

[0008] This invention utilizes gel electrophoresis to separate DNA samples, resulting in good purification of the target DNA before enzymatic digestion and achieving excellent separation yield. The gel electrophoresis procedure is also relatively simple. Furthermore, it has been found that gel strips containing the target DNA obtained through gel electrophoresis under EDTA-free conditions are suitable for direct in-gel enzymatic digestion to obtain deoxynucleosides or deoxynucleotides for LC-MS / MS detection, leading to more accurate DNA chemical modification results. Attached Figure Description

[0009] Figure 1 This is a schematic flowchart of the DNA modification detection method using gel electrophoresis coupled with LC-MS / MS according to an embodiment of the present invention;

[0010] Figure 2 Images of circular plasmid DNA and 293T genomic DNA stained on agarose gel at 470 nm wavelength before and after digestion in Example 1 of this invention;

[0011] Figure 3 The results of EDTA inhibition of DNA digestion in the gel in the commonly used electrophoresis buffer in Example 2 of the present invention are shown. A is the agarose gel electrophoresis result of the DNA substrate, B is the gel electrophoresis result of the DNA digestion product after agarose gel electrophoresis with different EDTA contents, and C is the mass spectrometry detection result of the DNA digestion product after agarose gel electrophoresis with different EDTA contents.

[0012] Figure 4 To add Mg to the intragel enzymatic hydrolysis system in Example 3 of the present invention 2+ The results show the effect of the content of dC nucleosides on nuclease activity, where A represents the chromatographic peaks of dC nucleosides after enzymatic hydrolysis in gel under different MgCl2 conditions, B represents the mass spectrometry concentration of dC nucleosides after enzymatic hydrolysis in gel under different MgCl2 conditions, and C represents the normalized mass spectrometry content of dC nucleosides after enzymatic hydrolysis in gel under different MgCl2 conditions.

[0013] Figure 5 In Example 4 of this invention, the in-gel DNA digestion method combined with LC-MS / MS detection method was used to determine the 5mC and 6mA content on circular plasmid DNA and the 5mC modification content on 293T cell genomic DNA, respectively, and compared with the mass spectrometry detection results after direct enzymatic digestion. Wherein, A is the chromatographic peak of dC nucleoside, B and C are the mass spectrometry detection results of four nucleosides on circular plasmid DNA and 293T cell genomic DNA, D is the mass spectrometry detection result of 5mC on circular plasmid DNA, E is the mass spectrometry detection result of 6mA on circular plasmid DNA, and F is the mass spectrometry detection result of 5mC on 293T cell genomic DNA. Detailed Implementation

[0014] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0015] Gel electrophoresis is widely used for nucleic acid separation due to its powerful separation capabilities. With technological advancements, gel electrophoresis can distinguish DNA molecules of different sizes and conformations. Leveraging the advantages of gel electrophoresis, Sanger et al. developed first-generation gene sequencing technology. Furthermore, gel electrophoresis based on microfabrication platforms can even achieve rapid detection of single nucleotide polymorphisms. However, there are currently no reports of coupling gel electrophoresis with mass spectrometry to detect DNA modifications.

[0016] In the process of developing this invention, it was discovered that DNA separated by gel electrophoresis can be digested within the gel using an in-gel enzymatic digestion method, which can then be combined with mass spectrometry for the detection of various DNA modifications. However, the conventionally used agarose gel electrophoresis buffer contains EDTA, which inhibits enzyme activity, thereby reducing the efficiency of in-gel DNA digestion. This may affect the accuracy of subsequent mass spectrometry analysis and evaluation. By removing EDTA from the gel electrophoresis buffer, the efficiency of in-gel digestion is improved while maintaining the resolution of electrophoretic separation. Furthermore, it was found that adding Mg to the enzymatic digestion solution... 2+ The ions increased the release of nucleosides during enzymatic digestion, thereby further enhancing the signal detected by mass spectrometry.

[0017] Specifically, according to some embodiments of the present invention, a method for detecting DNA modifications using gel electrophoresis coupled with liquid chromatography-mass spectrometry is provided. Figure 1 This is a schematic flowchart of the DNA modification detection method using gel electrophoresis coupled with LC-MS / MS according to an embodiment of the present invention, as shown below. Figure 1 As shown, the detection method of the present invention includes the following steps S101 to S103:

[0018] In step S101, the DNA sample to be tested is separated by gel electrophoresis to obtain a gel strip containing the target DNA. The electrophoresis buffer used in the gel electrophoresis does not contain EDTA.

[0019] In step S102, the target DNA in the gel strip is enzymatically digested into a single deoxynucleoside and / or a single deoxynucleotide by in-gel DNA digestion to obtain the enzymatic digestion product;

[0020] In step S103, the enzymatic hydrolysis product is detected by liquid chromatography-mass spectrometry to obtain the DNA chemical modification results.

[0021] According to embodiments of the present invention, before enzymatically digesting complex DNA samples, the present invention first separates them using gel electrophoresis, and then performs in-gel DNA enzymatic digestion on the target DNA corresponding to specific bands according to experimental needs, followed by mass spectrometry detection. This can reduce sample loss while obtaining precise modification information of complex and small amounts of DNA components after gel electrophoretic separation.

[0022] It should be noted beforehand that the term "DNA chemical modification" in this article includes, but is not limited to, DNA epigenetic modification and / or DNA damage adducts. DNA epigenetic modification refers to reversible and heritable changes in gene function without altering the DNA sequence, such as DNA methylation modification. DNA damage adducts refer to complexes formed by the binding of DNA fragments with chemical substances. When these chemical substances bind to DNA, the DNA is damaged, causing biological processes such as DNA replication to malfunction.

[0023] In this article, the term "deoxynucleoside" refers to the dephosphorylation product of "deoxynucleotide," which is the basic unit of DNA, mainly composed of a nitrogenous base, deoxyribose, and phosphate.

[0024] According to an embodiment of the present invention, in step S101, agarose is used as the medium in gel electrophoresis, and the electrophoresis buffer is any EDTA-free buffer solution suitable for electrophoresis, including but not limited to buffer solutions with a pH of 7.0 to 9.0, such as Tris-acetic acid with a pH of 8.0. Experiments have shown that agarose gel electrophoresis is more conducive to subsequent in-gel enzymatic digestion than polyacrylamide gel electrophoresis (PAGE), exhibiting a stronger mass spectrometry signal, presumably due to the larger pore size of agarose compared to polyacrylamide. Furthermore, by removing EDTA, which is commonly used in traditional agarose gel electrophoresis buffers, the efficiency of in-gel enzymatic digestion is improved while maintaining the resolution of electrophoretic separation.

[0025] According to embodiments of the present invention, the gel strip containing the target DNA is obtained by cutting the target DNA according to its molecular size, and the mass of the gel strip is as small as possible, for example, less than 0.1g, such as about 0.05g. If the mass of the gel strip is too large, it may lead to: 1) an increase in the reaction time of subsequent enzymatic digestion in the gel; 2) the enzymatic digestion product, a single deoxynucleoside or a single deoxynucleotide, remaining in the gel and difficult to release into the buffer, resulting in a low mass spectrometry detection signal.

[0026] According to an embodiment of the present invention, in step S102, the enzymatic hydrolysis solution used in the in-gel DNA hydrolysis method includes a buffer solution with a pH of 7.0-9.0 and a nuclease. Further, the buffer solution in this step includes, but is not limited to, Tris-hydrochloric acid with a pH of 8.0. More preferably, the nuclease may include endonuclease and exonuclease. Endonuclease may be, for example, Benzonase, deoxyribonuclease I (DNase I), nuclease S1, etc., and exonuclease may be, for example, snake venom phosphodiesterase (SVP), exonuclease V (recBCD), etc. The combination of the two can greatly improve the hydrolysis efficiency.

[0027] According to an embodiment of the present invention, alkaline phosphatase may also be added to the above-mentioned enzymatic hydrolysate for dephosphorylation of DNA after nuclease hydrolysis to form monodeoxynucleotides.

[0028] According to embodiments of the present invention, the above-mentioned enzymatic hydrolysate further includes 1-5 mM MgCl2, the concentration of which may be, for example, 1 mM, 1.5 mM, 2.0 mM, 2.5 mM, 3.0 mM, etc. MgCl2 is added to the enzymatic hydrolysate. 2+ Ions increase the release of nucleosides or nucleotides during enzymatic digestion, thereby further enhancing the signal for mass spectrometry detection.

[0029] According to an embodiment of the present invention, step S102 specifically includes sub-steps S1021 to S1022.

[0030] In sub-step S1021, the gel strip is immersed in an enzymatic digestion solution containing Tris-hydrochloric acid, nuclease, and MgCl2, and incubated for 8–24 hours to obtain the first digestion solution. In sub-step S1022, alkaline phosphatase is added to the first digestion solution, and the mixture is incubated for 0.5–1 hour to obtain the enzymatic digestion product containing a single deoxynucleotide. It can be understood that without the addition of alkaline phosphatase, i.e., without sub-step S1022, the enzymatic digestion product containing a single deoxynucleotide is obtained.

[0031] According to an embodiment of the present invention, before performing liquid chromatography-mass spectrometry detection on the enzymatic hydrolysis product in step S103, the detection method of the present invention further includes: ultrafiltration of the enzymatic hydrolysis product to remove the protease in the enzymatic hydrolysis product so as to avoid interference with subsequent LC-MS / MS detection.

[0032] According to an embodiment of the present invention, in step S103, the detection parameters of LC-MS / MS can be conventionally selected as needed. Since this is not the focus of the present invention, it will not be described in detail here.

[0033] According to embodiments of the present invention, the results of DNA chemical modification include, but are not limited to, 5-methylcytosine (5mC), N... 6 -Methyladenine (6mA), 5-hydroxymethylcytosine (5hmC), 5-aldehyde cytosine (5fC), 5-carboxycytosine (5caC), 5-aldehyde uracil (5fU), 3-methylcytosine (3mC), N 1 Abundance information for at least one of methyladenine (1mA) and 8-hydroxy-2-deoxyguanosine (8-oxo-dG). The abundance information here refers to the relative content of DNA-modified and unmodified nucleosides / nucleotides. Using the abundance information of DNA modifications as the detection result allows for more accurate analysis and quantification of DNA modifications compared to absolute concentrations.

[0034] The following detailed description provides several specific embodiments to illustrate the technical solutions of the present invention. It should be noted beforehand that, unless otherwise defined, the technical terms used in the following embodiments have the same meaning as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods, unless otherwise specified, are conventional methods.

[0035] Example 1: Feasibility Study of In-Gel DNA Enzymatic Digestion

[0036] Circular plasmid DNA and 293T cell genomic DNA were used as DNA substrates. Both were purified samples, serving as the target DNA. Using a gel imaging system, stained agarose gel strips containing the two DNA substrates were excised, ensuring the strips were as small as possible, weighing approximately 0.05 grams. The strips were then immersed in 50 μL of enzymatic digestion buffer containing 10 mM Tris-HCl (pH 8.0), 2 mM MgCl2, 1.0 U Benzonase, and 0.02 U SVP (snake venom phosphodiesterase). The samples were then incubated at 37°C for 8 hours, followed by the addition of 5.0 U CIP (alkaline phosphatase) and incubation at 37°C for 1 hour. Simultaneously, enzymatic digestion buffers without added nucleases served as negative controls for both DNA substrates.

[0037] Figure 2 Images of plasmid DNA and 293T genomic DNA stained on agarose gel at 470 nm wavelength before and after digestion in Example 1 of this invention are shown below. Figure 2 As shown, without the addition of nuclease to the hydrolysate, the DNA substrate remained in the gel and did not diffuse into the hydrolysate over time. However, with the addition of nuclease to the hydrolysate, both DNA substrates were essentially hydrolyzed within about 1 hour. This indicates that the nuclease enters the gel to hydrolyze the DNA substrate, verifying the feasibility of the in-gel DNA hydrolysis method.

[0038] Example 2: Study on the effect of EDTA on in-gel DNA digestion

[0039] Genomic DNA from 293T cells was used as the DNA substrate, and the purified sample was used. Agarose gel electrophoresis was performed on the DNA substrate using electrophoresis buffer (40 mM Tris-acetic acid-EDTA, pH 8.0) containing 0–10 mM EDTA. With the support of a gel imaging system, the agarose gel strip containing the DNA substrate was removed, ensuring the strip was as small as possible, with a mass of approximately 0.05 g. The gel strip was then added to 50 μL of enzymatic digestion buffer containing 10 mM Tris-HCl (pH 8.0), 1 mM MgCl2, 1.0 U Benzosenase, and 0.02 U SVP (snake venom phosphodiesterase). The sample was then incubated at 37°C for 8 hours, followed by the addition of 5.0 U CIP (alkaline phosphatase) and incubation at 37°C for 1 hour.

[0040] Undigested DNA substrates were subjected to gel electrophoresis. The products of DNA digestion in agarose gels with different EDTA concentrations, along with the corresponding gel strips, were placed into the gel wells for further gel electrophoresis to detect the digestion status and to analyze the digestion products by LC-MS / MS. Figure 3 The EDTA in the commonly used electrophoresis buffer in this invention inhibits DNA digestion within the gel, as shown in the example. Figure 3 As shown in Figures A and B, with the increase of EDTA content in the electrophoresis buffer, more undigested products are produced. At an EDTA content of 10.0 mM, the DNA substrate is hardly digested, while... Figure 3 As shown in Figure C, the LC-MS / MS detection signals of the five nucleosides (dC, dA, 5mC, dG, and dT) became weaker with increasing EDTA content in the electrophoresis buffer. At an EDTA content of 5.0, the five nucleosides were almost not detected by mass spectrometry, indicating that the inhibition of nuclease activity by EDTA is not conducive to the realization of the in-gel enzymatic digestion method.

[0041] Example 3 Mg 2+Study on the impact of in-gel DNA hydrolysis

[0042] Genomic DNA from 293T cells was used as the DNA substrate; this was a purified sample. Gel strips were obtained by EDTA-free agarose gel electrophoresis using 40 mM Tris-acetic acid, pH 8.0, ensuring the gel strips were as small as possible, approximately 0.05 g in weight. The gel strips were then immersed in 50 μL of enzymatic digestion solution containing 10 mM Tris-HCl (pH 8.0), 0–2 mM MgCl2, 1.0 U Benzonase, and 0.02 U SVP (snake venom phosphodiesterase). The sample was then incubated at 37°C for 8 hours, followed by the addition of 5.0 U CIP (alkaline phosphatase) and incubation at 37°C for 1 hour. Finally, the digested solution was carefully transferred to an ultrafiltration tube (MWcutoff: 3 kDa, Pall, Port Washington, NY, USA) using a pipette tip, avoiding contact with the gel. The ultrafiltration tubes were centrifuged at 14,000g for 15 minutes at 4°C to remove proteases involved in DNA digestion, yielding the enzymatic digestion products. The enzymatic digestion products were analyzed by LC-MS / MS. Additionally, a control group was prepared by directly digesting the DNA substrate solution using LC-MS / MS.

[0043] Figure 4 To add Mg to the intragel enzymatic hydrolysis system in Example 3 of the present invention 2+ The effect of the content on nuclease activity, such as Figure 4 As shown in Figure B, with the increase of Mg in the enzymatic hydrolysis system within the gel... 2+ The higher the content of Mg, the higher the detection signal intensity of LC-MS / MS, indicating that Mg 2+ The presence of [a specific substance] can partially restore nuclease activity and improve the efficiency of in-gel digestion. For example... Figure 4 As shown in Figures B-C, compared with the direct solution enzymatic digestion of DNA substrate, the concentration of nucleoside dC obtained by the in-gel DNA digestion method is lower, but with the increase of Mg... 2+ The higher the concentration used, the closer it gets to the dC concentration of the direct solution enzymatic digestion method for DNA substrates.

[0044] Example 4: DNA digestion in gel followed by LC-MS / MS detection of 5mC and 6mA modifications on circular plasmid DNA and 293T cell genomic DNA.

[0045] Circular plasmid DNA and 293T cell genomic DNA were used as DNA substrates, and these were purified samples. Gel strips were obtained by EDTA-free gel electrophoresis using 40 mM Tris-acetic acid, pH 8.0, ensuring the gel strips were as small as possible, weighing approximately 0.05 g. The gel strips were then immersed in 50 μL of enzymatic digestion buffer containing 10 mM Tris-HCl (pH 8.0), 2 mM MgCl2, 1.0 U Benzonase, and 0.02 U SVP (snake venom phosphodiesterase). Next, the samples were incubated at 37°C for 8 hours, followed by the addition of 5.0 U CIP (alkaline phosphatase) and incubation at 37°C for 1 hour. Finally, the digested solution was carefully transferred to an ultrafiltration tube (MWcutoff: 3 kDa, Pall, Port Washington, NY, USA) using a pipette tip, avoiding contact with the gel. The ultrafiltration tubes were centrifuged at 14,000g for 15 minutes at 4°C to remove proteases involved in DNA digestion, yielding the enzymatic hydrolysate. The hydrolysate was analyzed by LC-MS / MS. Additionally, a control group was prepared by directly digesting the DNA substrate solution with LC-MS / MS.

[0046] Figure 5 The gel-based DNA digestion method of Example 4 of this invention, combined with LC-MS / MS detection, was used to determine the 5mC and 6mA content on circular plasmid DNA and the 5mC modification content on 293T cell genomic DNA, respectively. Figure 5 As shown, LC-MS / MS analysis revealed that even at lower Mg levels... 2+ At ionic concentrations, satisfactory digestion efficiency (over 60%) can be achieved by gel electrophoresis to separate DNA without EDTA buffer followed by in-gel digestion. Furthermore, the in-gel DNA digestion method does not exhibit any preference for the release of the six nucleosides (dA, dT, dC, dG, 5mC, and 6mA).

[0047] The abundances of 5mC and 6mA in plasmid DNA and the abundance of 5mC in the 293T genome were calculated. The abundances of 5mC and 6mA in plasmid DNA were 1.2% and 1.3%, respectively, while the abundance of 5mC in 293T cell genomic DNA was 3.0%. These values ​​were obtained through in-gel digestion. Interestingly, they are consistent with results obtained directly through in-solution digestion without gel electrophoresis separation. These results demonstrate that combining EDTA-free in-gel digestion with LC-MS / MD analysis can accurately quantify DNA modifications. This novel method will be applicable to the characterization and analysis of complex DNA samples.

[0048] In summary, this invention, using circular plasmid DNA and 293T cell genomic DNA as substrates, demonstrated the feasibility of in-gel enzymatic digestion. After in-gel digestion, the 6mA and 5mC contents detected by LC-MS / MS were consistent with those of the control group directly digested in buffer. This provides a simple and reliable detection method for accurately quantifying chemical modification information in different DNA components, such as subchromatin structure.

[0049] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting DNA modification using gel electrophoresis coupled with liquid chromatography-mass spectrometry, comprising the following steps: DNA samples to be tested are separated by gel electrophoresis to obtain gel strips containing the target DNA, wherein the electrophoresis buffer used in the gel electrophoresis method does not contain EDTA; The target DNA in the gel strip is digested into single deoxynucleosides and / or single deoxynucleotides by an in-gel DNA digestion method to obtain the digestion product. The digestion solution used in the in-gel DNA digestion method includes a buffer solution with a pH of 7.0-9.0, nuclease, and 1-5 mM MgCl2. The enzymatic hydrolysis products were analyzed by liquid chromatography-mass spectrometry to obtain the DNA chemical modification results, which included 5-methylcytosine and N... 6 -Methyladenine, 5-hydroxymethylcytosine, 5-aldehyde cytosine, 5-carboxycytosine, 5-aldehyde uracil, 3-methylcytosine, N 1 Abundance information of at least one of methyladenine and 8-hydroxy-2-deoxyguanosine.

2. The detection method according to claim 1, wherein, The gel electrophoresis method uses agarose as the medium, and the electrophoresis buffer is any buffer solution that does not contain EDTA and can be used for electrophoresis.

3. The detection method according to claim 1, wherein, The nucleases include endonucleases and exonucleases.

4. The detection method according to claim 3, wherein, The endonuclease is Benzonase, deoxyribonuclease I, or nuclease S1; the exonuclease is snake venom phosphodiesterase or exonuclease V.

5. The detection method according to claim 1, 3, or 4, wherein, The enzymatic hydrolysate also contains alkaline phosphatase.

6. The detection method according to claim 5, wherein, The target DNA in the gel strip is digested into single deoxynucleosides and / or single deoxynucleotides using an in-gel DNA digestion method, yielding the following digestion products: The gel strip is immersed in an enzymatic hydrolysate containing Tris-hydrochloric acid, the nuclease, and MgCl2, and incubated for 8-24 hours to obtain the first digestion solution. The alkaline phosphatase was added to the first digestion solution and incubated for 0.5 to 1 hour to obtain the enzymatic hydrolysis product.

7. The detection method according to claim 1, wherein, Before performing liquid chromatography-mass spectrometry (LC-MS) detection on the enzymatic hydrolysis products, the detection method further includes: The enzymatic hydrolysate is subjected to ultrafiltration to remove the protease in the enzymatic hydrolysate.

Images