阅读说明：本技术 一种真菌蛋白酶解肽指纹图谱的建立方法 (Method for establishing fungal proteolytic peptide fingerprint ) 是由 张萍 魏锋 马双成 康帅 李娟� 刘薇 李明华 陆兔林 于 2021-09-26 设计创作，主要内容包括：本发明公开了一种真菌蛋白酶解肽指纹图谱的建立方法,包括真菌蛋白的提取；对所述真菌蛋白液进行纯化与酶解；对酶解后的所述真菌蛋白液进行UPLC-MS分析,按质谱-色谱条件下的方法建立质谱指纹图谱,并进行方法学考察；所述质谱指纹图谱为进一步寻找冬虫夏草及各发酵虫草制剂的特征离子提供了大数据支持。本发明构建的方法具有操作简单、灵敏度高、准确性好等优点,能从质谱的整体特征上把握各样品真菌蛋白酶解肽的质量情况,为冬虫夏草及各发酵虫草菌粉和制剂的质量控制、真伪鉴别提供了新的科学方法。(The invention discloses a method for establishing a fungal protein enzymolysis peptide fingerprint, which comprises the steps of extracting fungal protein; purifying and performing enzymolysis on the fungal protein liquid; carrying out UPLC-MS analysis on the fungus protein liquid after enzymolysis, establishing a mass spectrum fingerprint spectrum according to a method under the mass spectrum-chromatographic condition, and carrying out methodology investigation; the mass spectrum fingerprint provides big data support for further searching characteristic ions of cordyceps sinensis and various fermented cordyceps sinensis preparations. The method constructed by the invention has the advantages of simple operation, high sensitivity, good accuracy and the like, can grasp the quality condition of the fungal proteolysis peptide of each sample from the overall characteristics of the mass spectrum, and provides a new scientific method for quality control and authenticity identification of cordyceps sinensis and fermented cordyceps sinensis powder and preparations.)

1. The method for establishing the fingerprint of the fungal proteolytic enzyme peptide is characterized by comprising the following steps

Extracting fungal protein A;

b, purifying and performing enzymolysis on the fungal protein liquid;

c, performing UPLC-MS-MS analysis on the fungus protein liquid after enzymolysis, and recording a mass spectrum fingerprint spectrum according to a method under the mass spectrum-chromatographic condition;

and D, carrying out methodology investigation on the mass spectrum fingerprint, verifying the feasibility of the established mass spectrum fingerprint method, and providing big data support for further searching characteristic ions of cordyceps sinensis and various fermented cordyceps sinensis preparations and further identifying authenticity.

2. The method for establishing the fingerprint of the fungal proteolytic peptide according to claim 1, wherein the method for extracting the fungal protein comprises the following steps: taking about 10mg of sample powder, adding a proper amount of deionized water, uniformly mixing by vortex, centrifuging, removing supernatant, and extracting the mycoprotein by a kit method. Extracting for 2 times, mixingMixing the supernatant, centrifuging, collecting supernatant, measuring protein concentration, placing the supernatant in a centrifuge tube with inner liner, centrifuging, removing filtrate, and adding 0.05M NH into the centrifuge tube₄HCO₃Washing the appropriate amount of solution for 2 times, centrifuging respectively, discarding filtrate, inverting the inner lining centrifuge tube, centrifuging by reverse ultrafiltration, adding 0.05M NH into the filtrate₄HCO₃The solution is diluted to a solution with the concentration of 1mg/mL and is mixed evenly for standby.

3. The method for establishing the fungal proteolytic peptide fingerprint spectrum according to claim 1, wherein the method for purifying and hydrolyzing the protein extract comprises the following steps: mixing the sample with a loading Buffer, heating for 5min, standing at room temperature, loading 20 μ L of sample per well, electrifying for 10min at 100V, purifying the sample in 16% polyacrylamide gel separator, dyeing with Coomassie brilliant blue, decolorizing with acetic acid, cutting and cutting blue strip into small pieces, washing with ultrapure water, adding 10mM DTT to immerse the gel pieces, reacting at 56 deg.C, sucking out the liquid, adding 55mM iodoacetamide solution, reacting at room temperature in a dark place. 0.05M NH was added₄HCO₃the/ACN (1:1) solution was decolorized until the gum mass became colorless, the liquid was aspirated and dried in vacuo. Adding trypsin at a ratio of 1:20(W/W), standing to swell the colloidal particles, adding 0.05M NH₄HCO₃Performing enzymolysis at 37 deg.C for 18h, adding 50% ACN (containing 5% TFA) into the micelle, incubating at 37 deg.C for 1h, dissolving with 0.1% formic acid, centrifuging, and collecting supernatant for UPLC-MS-MS analysis.

4. The method for establishing the fungal proteolytic peptide fingerprint spectrum according to claim 1, wherein the mobile phase of the chromatographic conditions of the chromatography-mass spectrometry combined detection is as follows: a: 0.1% formic acid solution; b: 0.1% formic acid-acetonitrile solution; flow rate: 300 nL/min. The gradient elution procedure was as follows:

5. the method for establishing the fungal proteolytic peptide fingerprint spectrum according to claim 1, wherein a nano electrospray ion source (NSI) positive ion monitoring mode is adopted in mass spectrum conditions of chromatography-mass spectrum combined detection, the spraying voltage is 2.2kV, the flow rate of sheath gas (N2) is 60arb (about 400kPa), the scanning range (m/z) is 350-1550, and the primary mass spectrum adopts an Orbitrap detector, Orbitrap Resolution: 120000, secondary mass scan is DIA scan, using an ion hydrazine detector, and secondary mass collision energy is 35%.

6. The method for establishing the fingerprint of the fungal proteolytic peptide according to claim 1, wherein the methodological investigation of the mass spectrum fingerprint comprises (1) precision investigation: taking 10mg of bailing capsule content, extracting fungal protein liquid, purifying and performing enzymolysis, repeatedly injecting for 6 times according to a mass spectrum-chromatography condition, and recording a mass spectrum fingerprint. Because the purpose of establishing the fingerprint is to search the different characteristic peptide components of each sample, the identification of each hydrolyzed polypeptide is retention time and accurate mass, 7 ions (330.1976, 508.2743, 615.3333, 577.2941, 218.2128, 802.4413 and 246.2455) with representative retention time in the fingerprint are selected to extract chromatographic peaks, the retention time and the accurate mass of the 7 ion chromatographic peaks are calculated, an extracted ion chromatogram is shown in figure 1, and the result shows that the retention time and the RSD of the accurate mass of each ion chromatographic peak are both less than 1.0 percent and meet the technical requirements of the fingerprint. (2) And (3) repeatability inspection: taking 10mg of bailing capsules, taking 6 parts in total, extracting fungal protein liquid, purifying and carrying out enzymolysis, injecting according to a mass spectrum-chromatography method, and recording a mass spectrum fingerprint. And (4) selecting the 7 ion extraction chromatographic peaks in the same way, and calculating the retention time and the accurate mass of the 7 ion chromatographic peaks. The result shows that the retention time and the RSD of the accurate mass of each spectrum peak are both less than 1.0 percent, and the technical requirements of the fingerprint spectrum are met. (3) And (3) stability investigation: taking 10mg bailing capsules, extracting fungal protein liquid, purifying and performing enzymolysis, respectively measuring for 0, 2, 4, 8, 12 and 24 hours according to a mass spectrum-chromatography method, and recording mass spectrum fingerprint. The 7 ion extraction chromatographic peaks are selected, and the result shows that the 7 ion chromatographic peaks can be detected within 24 hours, the retention time and the RSD (remote resolution ratio) of the accurate mass of each chromatographic peak are less than 1.0%, which indicates that the sample is stable within 24 hours and meets the technical requirement of qualitative identification of the fingerprint.

Technical Field

The invention belongs to the technical field of traditional Chinese medicine component identification, and particularly relates to a method for establishing a fungal protein enzymolysis peptide fingerprint.

Background

The traditional Chinese medicine has complex components, and the requirement of quality control of the traditional Chinese medicine is not met by taking a certain component as a control index. The traditional Chinese medicine fingerprint can judge the truth and quality of traditional Chinese medicinal materials and can also judge the quality of the traditional Chinese medicine prescription preparation and the rationality of the preparation process. Compared with a quality control method using a single component as a control index, the fingerprint can comprehensively reflect the quality of a sample from the whole chemical components, so that the aim of effectively controlling the quality is fulfilled, and the disadvantage that the effective components of the traditional Chinese medicine are mostly unclear is overcome.

Chemometrics, which is a subject of combining statistical, mathematical and computer-technical methods and principles to process chemical data, can optimize chemical measurement processes and extract chemical information useful for analysis from the measured chemical data to the greatest extent, is characterized in that multivariate analysis methods are introduced into the field of chemical research, which is a concept that is open to the ideas of traditional Chinese medicine administration, and thus, it is feasible to apply chemometrics to the research of traditional Chinese medicine. The application of fingerprint spectrum in the quality control of Chinese medicine is in the research of chemometrics. The method of modern instrument analysis and the chemometric method are reasonably combined and utilized, useful information is effectively extracted, and the method has profound influence on improving and improving the quality control method of the traditional Chinese medicine and promoting the modernization process of the traditional Chinese medicine.

The natural Cordyceps has similar chemical components with fermented Cordyceps powder, such as sterols, nucleosides, Cordyceps polysaccharides, proteins and amino acids, mannitol, stearic acid, palmitic acid, vitamins, organic acids and microelements. Fungal proteins are one of the active ingredients and have been studied very little. In order to investigate the characteristics and the differences of fungal protein of cordyceps sinensis and the fermented cordyceps sinensis powder and preparation, mass spectrum fingerprint spectrums of polypeptide components obtained after fungal protein enzymolysis in all samples are analyzed and established under the same conditions, methodology investigation is carried out, an effective method is established for quality control of all the fermented cordyceps sinensis powder and preparation, and technical support is provided for comprehensive evaluation of the quality of all the fermented cordyceps sinensis powder and preparation. Based on the analysis, a method for establishing a fungal proteolysis peptide fingerprint is provided.

Disclosure of Invention

The invention provides a method for establishing a fungal proteolysis peptide fingerprint.

The invention comprises the following steps:

extracting fungal protein A;

b, purifying and performing enzymolysis on the fungal protein liquid;

c, performing UPLC-MS analysis on the fungus protein liquid after enzymolysis, and recording a mass spectrum fingerprint spectrum according to a method under the mass spectrum-chromatographic condition;

and D, carrying out methodology investigation on the mass spectrum fingerprint, verifying the feasibility of the established mass spectrum fingerprint method, and providing big data support for further searching characteristic ions of cordyceps sinensis and various fermented cordyceps sinensis preparations and further identifying authenticity.

Further, the method for extracting the fungal protein comprises the following steps: taking about 10mg of sample powder, adding deionized water, uniformly mixing by vortex, centrifuging, removing supernatant, and extracting the mycoprotein by a kit method. Extracting for 2 times, mixing supernatants, centrifuging, collecting supernatant, measuring protein concentration, placing supernatant in a centrifuge tube with inner liner, centrifuging, discarding filtrate, and adding 0.05M NH₄HCO₃Washing the appropriate amount of solution for 2 times, centrifuging respectively, discarding filtrate, inverting the inner lining centrifuge tube, centrifuging by reverse ultrafiltration, adding 0.05M NH into the filtrate₄HCO₃The solution is diluted to a solution with the concentration of 1mg/mL and is mixed evenly for standby.

Further, the method for purifying and performing enzymolysis on the protein extracting solution comprises the following steps: mixing the sample with the loading Buffer, addingHeating for 5min, standing at room temperature, loading 20 μ L of the solution to each well, electrifying at 100V for 10min, purifying the sample by running the sample into 16% polyacrylamide gel separator, staining with Coomassie brilliant blue, decolorizing with acetic acid, cutting off blue band and cutting into small pieces, washing with ultrapure water, adding 10mM DTT to the gel piece, reacting at 56 deg.C, sucking out the liquid, adding 55mM iodoacetamide solution, and reacting at room temperature in a dark place. 0.05M NH was added₄HCO₃the/ACN (1:1) solution was decolorized until the gum mass became colorless, the liquid was aspirated and dried in vacuo. Adding trypsin at a ratio of 1:20(W/W), standing to swell the colloidal particles, adding 0.05M NH₄HCO₃Performing enzymolysis at 37 deg.C for 18h, adding 50% ACN (containing 5% TFA) into the micelle, incubating at 37 deg.C for 1h, dissolving with 0.1% formic acid, centrifuging, and collecting supernatant for UPLC-MS-MS analysis.

Further, the mobile phase of the chromatography condition of the chromatography-mass spectrometry combined detection is as follows: a: 0.1% formic acid solution; b: 0.1% formic acid-acetonitrile solution; flow rate: 300 nL/min. The gradient elution procedure was as follows:

further, a nano electrospray ionization source (NSI) positive ion monitoring mode is adopted in mass spectrum conditions of chromatography-mass spectrometry detection, the spraying voltage is 2.2kV, the flow rate of sheath gas (N2) is 60arb (about 400kPa), the scanning range (m/z) is 350-1550, and an Orbitrap detector, Orbitrap Resolution: 120000, secondary mass scan is DIA scan, using an ion hydrazine detector, and secondary mass collision energy is 35%.

Further, performing methodology investigation on the mass spectrum fingerprint, including (1) precision investigation: taking 10mg of bailing capsule content, extracting fungal protein liquid, purifying and performing enzymolysis, repeatedly injecting for 6 times according to a mass spectrum-chromatography condition, and recording a mass spectrum fingerprint. Because the purpose of establishing the fingerprint is to search the different characteristic peptide components of each sample, the identification of each hydrolyzed polypeptide is retention time and accurate mass, 7 ions (330.1976, 508.2743, 615.3333, 577.2941, 218.2128, 802.4413 and 246.2455) with representative retention time in the fingerprint are selected to extract chromatographic peaks, the retention time and the accurate mass of the 7 ion chromatographic peaks are calculated, an extracted ion chromatogram is shown in figure 1, and the result shows that the retention time and the RSD of the accurate mass of each ion chromatographic peak are both less than 1.0 percent and meet the technical requirements of the fingerprint. (2) And (3) repeatability inspection: taking 10mg of bailing capsules, taking 6 parts in total, extracting fungal protein liquid, purifying and carrying out enzymolysis, injecting according to a mass spectrum-chromatography method, and recording a mass spectrum fingerprint. And (4) selecting the 7 ion extraction chromatographic peaks in the same way, and calculating the retention time and the accurate mass of the 7 ion chromatographic peaks. The result shows that the retention time and the RSD of the accurate mass of each spectrum peak are both less than 1.0 percent, and the technical requirements of the fingerprint spectrum are met. (3) And (3) stability investigation: taking 10mg bailing capsules, extracting fungal protein liquid, purifying and performing enzymolysis, respectively measuring for 0, 2, 4, 8, 12 and 24 hours according to a mass spectrum-chromatography method, and recording mass spectrum fingerprint. The 7 ion extraction chromatographic peaks are selected, and the result shows that the 7 ion chromatographic peaks can be detected within 24 hours, the retention time and the RSD (remote resolution ratio) of the accurate mass of each chromatographic peak are less than 1.0%, which indicates that the sample is stable within 24 hours and meets the technical requirement of qualitative identification of the fingerprint.

The invention has the beneficial effects that:

the construction method has the advantages of simple operation, high sensitivity, good accuracy and the like, can grasp the quality condition of fungal proteolysis peptide of each sample from the integral characteristics of mass spectrum, and provides a new scientific method for quality control and authenticity identification of cordyceps sinensis and fermented cordyceps sinensis powder and preparations.

Drawings

FIG. 1 is a schematic diagram of an ion chromatogram for extracting a fungal proteolytic peptide fingerprint spectrum of the invention;

FIG. 2 is a mass spectrum of total ion flux polypeptide LC/MS/MS of a Cordyceps sinensis sample of a fungal proteolysis peptide fingerprint spectrum of the present invention;

FIG. 3 is a Bailing capsule total ion flux polypeptide LC/MS/MS mass spectrogram of fungal proteolysis peptide fingerprint of the present invention;

FIG. 4 is a mass spectrum of total ion flux polypeptide LC/MS/MS of the Xinganbao capsule of the fungal proteolytic peptide fingerprint spectrum of the invention;

FIG. 5 is a mass spectrum of total ion flux polypeptide LC/MS/MS of Jinshuibao capsule of the fungal proteolysis peptide fingerprint of the present invention;

FIG. 6 is a LC/MS/MS mass spectrum of Zhiling capsule total ion flux polypeptide of the fungal proteolysis peptide fingerprint of the present invention;

FIG. 7 is a mass spectrum of total ion flux polypeptide LC/MS/MS of Ningxinbao capsule of the fungal proteolytic peptide fingerprint of the invention;

Detailed Description

The principles and features of this invention are described below in conjunction with examples which are set forth to illustrate, but are not to be construed to limit the scope of the invention.

Instrument and reagent

The instrument comprises the following steps: ultra high performance liquid chromatography (Thermo corporation, usa); orbitrap Fusion mass spectrometer (Thermo corporation, usa); EPS-301 electrophoresis apparatus (General Electric company, U.S.); vacuum centrifugal concentrator.

Reagent: polyacrylamide, coomassie brilliant blue, acetic acid, Dithiothreitol (DTT), Iodoacetamide (IAM), trifluoroacetic acid (TFA), ammonium bicarbonate, and the like, are all chemically or analytically pure; both formic acid and acetonitrile were chromatographically pure (Fisher, USA); deionized water is Millipore ultrapure water; trypsin is sequence pure.

Chromatographic conditions

A chromatographic column: ZorbaxSB-C₁₈(150mm×75mm，3μm)；

Mobile phase: a: 0.1% formic acid solution; b: 0.1% formic acid-acetonitrile solution; the gradient elution procedure was as follows:

flow rate: 300 nL/min.

Conditions of Mass Spectrometry

By using nano-electrosprayIon source (NSI) positive ion monitor mode, spray voltage 2.2kV, sheath gas (N)₂) The flow rate is 60arb (about 400kPa), the scanning range (m/z) is 350-1550, and the primary mass spectrum adopts an Orbitrap detector, Orbitrap Resolution: 120000, secondary mass scan is DIA scan, using an ion hydrazine detector, and secondary mass collision energy is 35%.

Sample solution preparation

Fungal protein extraction

Taking about 10mg of sample powder, adding deionized water, uniformly mixing by vortex, centrifuging, removing supernatant, and extracting mycoprotein by a kit method. Extracting for 2 times, mixing supernatants, centrifuging, collecting supernatant, measuring protein concentration, placing supernatant in a centrifuge tube with inner liner, centrifuging, discarding filtrate, and adding 0.05M NH₄HCO₃Washing the appropriate amount of solution for 2 times, centrifuging respectively, discarding filtrate, inverting the inner lining centrifuge tube, centrifuging by reverse ultrafiltration, adding 0.05M NH into the filtrate₄HCO₃The solution is diluted to a solution with the concentration of 1mg/mL and is mixed evenly for standby.

Fungus protein liquid purification and enzymolysis

Mixing the sample with loading Buffer, heating for 5min, standing at room temperature, loading 20 μ L per well, and electrifying at 100V for 10min to allow the sample to run into 16% polyacrylamide separation gel for purification. Coomassie blue staining, acetic acid destaining, cutting blue bands and cutting into small pieces, ultra pure water washing. 10mM DTT-impregnated gel was added thereto and reacted at 56 ℃ to aspirate the liquid, and 55mM iodoacetamide solution was added thereto and reacted at room temperature in the absence of light. 0.05M NH was added₄HCO₃the/ACN (1:1) solution was decolorized until the gum mass became colorless, the liquid was aspirated and dried in vacuo. Adding trypsin at a ratio of 1:20(W/W), standing to swell the colloidal particles, adding 0.05M NH₄HCO₃Appropriate amount of enzyme was digested at 37 ℃ for 18h, 50% ACN (containing 5% TFA) was added to the gel particles, and incubated at 37 ℃ for 1 h. After 0.1% formic acid is dissolved, the supernatant is centrifuged and analyzed by UPLC-MS-MS.

Methodology investigation

Precision investigation:

10mg of bailing capsule is taken, fungal protein liquid is extracted, purified and subjected to enzymolysis, sample introduction is repeated for 6 times according to a mass spectrum-chromatography condition, and a mass spectrum fingerprint spectrum is recorded. Because the purpose of establishing the fingerprint map in the experiment is to search different characteristic peptide components of each sample, the identification of each hydrolyzed polypeptide is retention time and accurate mass, 7 ions (330.1976, 508.2743, 615.3333, 577.2941, 218.2128, 802.4413 and 246.2455) with representative retention time in the fingerprint map are selected to extract chromatographic peaks, the retention time and accurate mass of the 7 ion chromatographic peaks are calculated, and an ion chromatogram map is extracted and shown in figure 1. The result shows that the retention time and the RSD of the accurate mass of each ion chromatographic peak are both less than 1.0 percent, and the technical requirements of the fingerprint spectrum are met.

Repeatability survey

Taking 10mg of bailing capsules, taking 6 parts in total, extracting fungal protein liquid, purifying and carrying out enzymolysis, injecting according to a mass spectrum-chromatography method, and recording a mass spectrum fingerprint. And (4) selecting the 7 ion extraction chromatographic peaks in the same way, and calculating the retention time and the accurate mass of the 7 ion chromatographic peaks. The result shows that the retention time and the RSD of the accurate mass of each spectrum peak are both less than 1.0 percent, and the technical requirements of the fingerprint spectrum are met.

And (3) stability investigation:

taking 10mg bailing capsules, extracting fungal protein liquid, purifying and performing enzymolysis, respectively measuring for 0, 2, 4, 8, 12 and 24 hours according to a mass spectrum-chromatography method, and recording mass spectrum fingerprint. The 7 ion extraction chromatographic peaks are selected, and the result shows that the 7 ion chromatographic peaks can be detected within 24 hours, the retention time and the RSD (remote resolution ratio) of the accurate mass of each chromatographic peak are less than 1.0%, which indicates that the sample is stable within 24 hours and meets the technical requirement of qualitative identification of the fingerprint.

Data acquisition

And (3) respectively introducing the sample solutions after enzymolysis into a mass spectrometer to obtain primary and secondary mass spectrograms of the total ion flow of each sample, wherein the primary mass spectrograms are as shown in the following figures 2-7. Respectively, the total ion flow polypeptide mass spectrogram of the cordyceps sinensis, the bailing capsule, the Xinganbao capsule, the Jinshuibao capsule, the Zhiling capsule and the Ningxinbao capsule.

The experiment obtains the total ion flow polypeptide fingerprint of each sample through chromatography-mass spectrometry combined detection, and aims to lay a foundation for searching for polypeptide components with different characteristics among the samples in the next step. Therefore, 7 ions with representative retention time are selected from the fingerprint as detection indexes, the retention time and the accurate quality of 7 ion chromatographic peaks are inspected, and the methodology inspection of the total ion flow polypeptide fingerprint is carried out.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can substitute or change the technical solution of the present invention and the inventive concept within the technical scope of the present invention.