CN115477689A - Earthworm thrombolytic activity marker peptide and application thereof in earthworm thrombolytic activity determination - Google Patents

Abstract

The invention discloses a earthworm thrombolysis activity mark peptide and application thereof in earthworm thrombolysis activity determination, the earthworm thrombolysis activity mark peptide comprises: 1 to 11 portions of limnodrilus salviae miltiorrhizae thrombolytic activity marker peptide, the amino acid sequence of which is shown as SEQ ID NO 1 to SEQ ID NO 11; the amino acid sequences of the Hu earthworm thrombolysis active marker peptides 3 and 11 are shown as SEQ ID NO. 3 and SEQ ID NO. 11. The method screens 11 thrombolytic activity marker peptides from the lumbricus holothurian, the content of which has strong positive correlation with the thrombolytic activity of the lumbricus and can be used for measuring the thrombolytic activity of the lumbricus holothurian; wherein 2 of them have strong correlation with the thrombolytic activity of Hu Di Long, and can be used for determination of the thrombolytic activity of Hu Di Long.

Description

Earthworm thrombolytic activity marker peptide and application thereof in earthworm thrombolytic activity determination

Technical Field

The invention relates to the technical field of traditional Chinese medicine analysis and detection, in particular to earthworm thrombolytic activity marker peptide and application thereof in earthworm thrombolytic activity determination.

Background

Earthworm is a traditional Chinese medicine in China, is recorded in Shen nong Ben Cao Jing, and is commonly used for treating cardiovascular and cerebrovascular diseases such as apoplexy. Its functions are clearing away heat, arresting convulsion, promoting blood circulation, removing obstruction in channels, relieving asthma and promoting urination. The earthworm sources collected in 2020 edition of Chinese pharmacopoeia are dried bodies of Pheretima aspergillum (E.Perrier), pheretima vulgaris Chen, pheretima aglaiensis Pheretima aglaigilli (Michaelsen) or Pheretima aspergillum Michaelsen of Merlacta, the former one is called "Guangdong earthworm", the latter three are called "Shanghai earthworm", and 41 varieties of adult preparation containing earthworm are collected in the first part of 2020 edition of Chinese pharmacopoeia. The medicine has the effects of clearing heat and arresting convulsion of 3, accounting for 7.32 percent of the total preparation, relieving asthma and inducing diuresis of 3 (7.32 percent), calming the liver and suppressing yang, and reducing hypertension of 3 (7.32 percent), the rest 32 medicines mainly have the effects of promoting blood circulation and dredging collaterals of earthworm, and account for 78.04 percent of the total preparation, and is mainly used for patients with symptoms of blood stasis such as difficulty in bending and stretching legs and feet, spasm of hands and feet, soreness and weakness of waist and knees, dark lip color, dark red tongue or ecchymosis and the like in clinical symptoms, and stroke symptoms such as hemiplegia, hemianesthesia, unsmooth mouth and tongue, speaking slurred and the like in severe patients.

Earthworm contains abundant proteins, polypeptides, amino acids, nucleosides and the like, wherein lumbrokinase is a main thrombolytic active substance of the Earthworm, is a kinase with fibrinolytic activity extracted from Earthworm and is also called Earthworm Fibrinolytic Enzyme (EFE). The lumbrokinase standard is a group of enzymes separated from Eisenia foetida of Eisenia fetida of Eisenia foetida, and currently, the determination of the fibrinolytic activity of lumbricus generally takes the lumbrokinase standard as a reference and adopts an agarose plasminogen plate method to determine the fibrinolytic activity by measuring the diameter of a lysosome. The method is used for measuring the total activity of lumbrokinase, and has poor specificity, linearity and stability.

In recent years, based on proteomics research, establishing a quality control method using characteristic peptides as indexes opens up a new situation for improving the quality control standard of traditional Chinese medicines in animals. Patent CN111303263A reports that 5 characteristic polypeptides are used for identifying the species of common earthworm species such as Pheretima aspergillum, pheretima aspergillum and Pheretima buningensis. The difference of proteomes among earthworm species is researched in the early stage of a subject group of an applicant, 6 ion pairs are screened out for identifying the authenticity of medicinal earthworm medicinal materials, the earthworm can be accurately distinguished from the earthworm, common earthworm and the earthworm with the chlamya punctata, and common fake earthworm, namely the earthworm with the earthworm, can be identified. (Gu, Y., zhang, J., sun, J., yu, H., feng, R., mao, X., yang, X., zhou, Y., hu, Q., ji, S. Marker peptide screening and specifications of textile using proteomics, animal. Biological. Chem. [ J ]. 202413 (12): 3167-3176.).

However, the current characteristic peptide identification method can only be used for earthworm species identification, and cannot detect the activity of medicinal materials. The thrombolytic activity of earthworm medicinal materials in the market is uneven, even no activity exists, so that a group of marker peptides capable of accurately reflecting the thrombolytic activity of the earthworm is screened out and an activity determination method is established, and the method has important significance for quality control of the earthworm medicinal materials, specification of earthworm medicinal material market and guarantee of clinical medication safety.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides a earthworm thrombolysis activity marker peptide and application thereof in earthworm thrombolysis activity determination.

In order to achieve the purpose, the invention adopts the technical scheme that:

in a first aspect, the invention provides a group of earthworm thrombolysis activity marker peptides, which comprise

1 to 11 portions of lumbricus crinita thrombolysis activity mark peptide, the amino acid sequence of which is shown as SEQ ID NO. 1 to SEQ ID NO. 11;

the amino acid sequences of the Hu earthworm thrombolysis activity marker peptides 3 and 11 are shown as SEQ ID NO. 3 and SEQ ID NO. 11.

Further, the Hu earthworm is common Megascoleus lumbricus and Megasphaera lumbricus.

Further, the mass spectrum conditions of the thrombolytic activity marker peptides 1-11 are as follows:

the second aspect of the invention provides the application of the earthworm thrombolysis activity marker peptide in earthworm thrombolysis activity determination.

The third aspect of the present invention provides a method for measuring thrombolytic activity of earthworms based on the marker peptide for thrombolytic activity of earthworms, which comprises the following steps:

step one, processing a sample to be detected to obtain a polypeptide mixed solution to be detected;

and step two, detecting the polypeptide mixed solution to be detected by adopting an ultra-high performance liquid chromatography-triple quadrupole mass spectrometer to obtain a mass spectrum of the thrombolytic activity marker peptide of the sample to be detected.

Further, the chromatographic conditions detected by the ultra-high performance liquid chromatography-triple quadrupole mass spectrometer are as follows:

and (3) analyzing the column: CORTECS C18 column, 2.7 μm 2.1X 50mm;

sample injection amount: 1 mul;

flow rate: 0.2ml/min;

mobile phase: the mobile phase A is 0.1 percent formic acid-water solution, the mobile phase B is acetonitrile,

gradient elution: 0 to 10min,2 to 35 percent of the total weight of the crude oil; 30-32min, 35-90% by weight of B.

Further, the mass spectrum conditions detected by the ultra-high performance liquid chromatography-triple quadrupole mass spectrometer are as follows: the spray voltage was 4000V, the gas temperature was 270 ℃, the flow rate was 16L/min, the Nebulizer was 45psi, and the MRM mode was used.

By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:

according to the invention, fresh lumbricus is taken as a research object, non-labeled proteomics research on tissue and body cavity fluid of lumbricus and TMT labeled proteomics research on drying temperature of lumbricus are carried out, and 11 marker peptides related to thrombolytic activity are screened out by systematically comparing changes of up-regulating and down-regulating related proteins and polypeptides, and the content of the marker peptides has strong positive correlation with an activity measurement result.

The method screens 11 thrombolytic activity marker peptides from the lumbricus holothurian, the content of which has strong positive correlation with the thrombolytic activity of the lumbricus and can be used for measuring the thrombolytic activity of the lumbricus holothurian; wherein 2 of them have strong correlation with the thrombolytic activity of Hu Di Long, and can be used for determination of the thrombolytic activity of Hu Di Long.

Drawings

FIG. 1 is a fibrinolytic enzyme profile characterizing fibrinolytic activity of earthworm tissue and body cavity fluid;

FIG. 2 is the effect of different drying temperatures on fibrinolytically active proteins in earthworms;

FIG. 3 is a sample identification non-redundant peptide Heatmap analysis at different drying temperatures;

FIG. 4 is a DMRM chromatogram of a thrombolytic activity marker peptide of lumbricus crinita;

FIG. 5 is a DMRM chromatogram-continuation of the thrombolytic activity marker peptide of lumbricus cricophylla;

FIG. 6 is a DMRM chromatogram of a thrombolysis activity marker peptide of Pheretima aspergillum;

FIG. 7 is a DMRM chromatogram of a thrombolytic activity marker peptide of a common earthworm;

FIG. 8 is a validation of specificity of thrombolytic activity marker peptides;

FIG. 9 shows the content of marker peptide and fibrinolytic activity of Pheretima aspergillum;

FIG. 10 is the correlation coefficient r of the correlation analysis between the marker peptide content of the Hu Di Long herb and the fibrinolysis activity Pearson;

FIG. 11 shows thrombolytic activity and marker peptide content of Hu Di Long medicinal material;

FIG. 12 is the correlation coefficient r of the correlation analysis between the marker peptide content of the Hu Di Long herb and the fibrinolysis activity Pearson;

FIG. 13 is an EIC chart of a Pheretima aspergillum sample according to example 1 of the present invention;

FIG. 14 is an EIC chart of a Pheretima aspergillum sample according to example 2 of the present invention.

Detailed Description

The invention is further described with reference to the following drawings and specific examples, which are not intended to be limiting. It should be noted that the embodiments and features of the embodiments of the present invention may be combined with each other without conflict.

The method screens 11 thrombolytic activity marker peptides from the lumbricus holothurian, the content of which has strong positive correlation with the thrombolytic activity of the lumbricus and can be used for measuring the thrombolytic activity of the lumbricus holothurian; 2 of the above two have strong correlation with the thrombolytic activity of the common lumbricus, and can be used for determination of the thrombolytic activity of the common lumbricus. The specific contents are as follows:

1. screening for signature peptides

Performing enzymolysis on extracted earthworm protein by adopting a shotgun proteome research and TMT (TMT tag proteomics) strategy to obtain a polypeptide mixture, separating by using a chromatographic column, analyzing by adopting a tandem mass spectrum, performing protein identification by searching a database to determine information such as protein information, a peptide fragment sequence, source protein and the like, and screening thrombolytic activity marker peptide by adopting a statistical method.

1.1 extraction and content determination of Lumbricus tissue and body cavity fluid protein

1) Extracting earthworm coelomic fluid and tissue protein: adding 20mg of earthworm coelomic fluid and tissue powder into 50mM NH respectively ₄ HCO ₃ Extracting the extractive solution 400ul with ultrasound at 37 deg.C for 40min, centrifuging, adding 50mM NH into the solid ₄ HCO ₃ Ultrasonic extracting 200 μ l of the extractive solution at 37 deg.C for 30min, centrifuging, and mixing the supernatants to obtain Lumbricus protein.

2) Extracting earthworm protein at different drying temperatures: mixing 40mg of Lumbricus coelomic fluid and tissuePowder, 50mM NH was added separately ₄ HCO ₃ Extracting 800 μ l of the extractive solution with ultrasound at 37 deg.C for 40min, centrifuging, adding 50mM NH into the solid ₄ HCO ₃ Extracting 400 μ l of the extractive solution with ultrasound at 37 deg.C for 30min, centrifuging, mixing the supernatants, adding 400 μ l of the solution into methanol-chloroform (1, v.

3) Reductive alkylation: 100-200. Mu.g of protein was taken, 200. Mu.l of cold tris-phenol was added, respectively, and vortexed at room temperature for 20 minutes. Precipitation was carried out overnight at-20 ℃ with a five-fold amount of 100mM ammonium acetate in methanol. The supernatant was centrifuged, the solid was dissolved in a urea (8M, 1% SDS) solution, and the urea was removed by ultrafiltration. Adding 50mM DTT (dissolved with 50mM ammonium bicarbonate, ready-to-use, final concentration of DTT 25 mM) and standing at 95 deg.C for 30min, adding 50mM MIAA (dissolved with 50mM ammonium bicarbonate, ready-to-use, final concentration of IAA 50 mM) and standing in dark for 40min.

4) And (3) carrying out enzymolysis by trypsin, desalting and enriching: trypsin was dissolved in 1ml of acetic acid at a concentration of 100. Mu.g/ml, and each sample was subjected to enzymatic hydrolysis at 37 ℃ for 18 hours by adding Trypsin (about 1, 50, w/w), and acidified to pH 2-3 with 10% TFA to terminate the reaction. Lyophilized, 200. Mu.l of phosphate buffer (pH 7.8) was added, and Trypsin (about 1, 50, w/w) was added for enzymatic hydrolysis at 45 ℃ for 4 hours. C18 desalting: and (3) buffer solution A: h ₂ O38.4 ml,5% TFA 1.6ml; and (3) buffer solution B: H2O 6.4ml, ACN32ml,10% TFA 1.6ml;50% ACN-activated C18 column, pipetting buffer A, pipetting the sample, desalting buffer A, pipetting buffer B200. Mu.l repeatedly to extract peptide fragments, and repeating once. Spin-drying at room temperature in a vacuum centrifugal concentrator, adding 0.1% (by weight) FA100 μ l for redissolving, centrifuging to remove supernatant, and packaging in plastic liquid phase vial.

1.2 determination of thrombolytic Activity of Pheretima

1) Fibrinolytic enzyme spectrum characterization earthworm protein activity: the plasmin zymogram is a method for measuring fibrinolytic activity based on polyacrylamide gel, wherein fibrinolytic protein is separated according to different molecular weights, the fibrin in the gel is degraded by enzyme in the incubation process, and the fibrinolytic activity is characterized by measuring the specific proteolytic activity of a sample. The protein with fibrinolytic activity showed clear and transparent bands on the fibrinolytic enzyme spectrum. Fibrinogen (final concentration 0.5 mg/mL) and thrombin (0.012 BP/mL) were added to a 12% separation gel prepared in the same manner. After electrophoresis, the gel was destained by soaking in 2.5% Triton X-100 solution for 30min, followed by incubation with PBS buffer (pH 7.4) at 37 ℃ for 30min, staining with coomassie brilliant blue R250 overnight, destaining with methanol-acetic acid-water (4.

The fibrinolytic enzyme spectra of proteins extracted from tissues and body cavity fluid of the lumbricus praecox, the common lumbricus and the chlostephium pectiniferum are shown in figure 1, the body cavity fluid has fibrinolytic activity, has clear bands, is different from the fibrinolytic bands of lumbrokinase, has obvious difference in the distribution of fibrinolytic proteins of different species of body cavity fluid, and is mainly distributed between 15kDa and 55kDa, wherein the fibrinolytic activity protein bands of the lumbricus praecox and the common lumbricus are more abundant between 15kDa and 35 kDa; while the tissue has no fibrinolytic activity. Adopting the tissue and the body cavity fluid of the lumbricus scrophularis as research objects to carry out non-labeled proteomics research and screen the earthworm thrombolysis active marker peptide.

2) Agarose fibrin plate characterization earthworm thrombolytic activity: adding 250mg of agarose gel into a small clean beaker, adding 30ml of PBS buffer solution (pH 7.4), heating by microwave for 1min until the agarose gel is completely dissolved to obtain an agarose solution, and preserving the agarose solution for later use in a water bath at 55 ℃. Taking 50mg of fibrinogen, adding 30ml of PBS buffer solution for dissolving to obtain fibrinogen solution, and preserving heat in a water bath at 55 ℃ for later use. And (3) taking 1ml of 4BP/ml thrombin solution, uniformly mixing the thrombin solution with the agarose solution, slowly adding the preheated fibrinogen solution, uniformly mixing, pouring the mixture into a culture dish with the diameter of 15cm, standing for 1h, and solidifying for later use. Mu.l of earthworm protein solution (the concentration is more than 1 mg/ml) is taken for loading. Preparing a 48000U/ml stock solution by adding 0.5ml water into lumbrokinase control, diluting to obtain a series of concentrations, taking 5 μ l of sample, and drawing a standard curve by taking LogU as a vertical coordinate and a soluble ring area as a horizontal coordinate.

The earthworm samples are prepared by respectively adopting Freeze-drying (FD), drying at 40 ℃, 50 ℃, 60 ℃, 70 ℃,80 ℃, 90 ℃ and 100 ℃ for 12h, and the thrombolytic activity is characterized by adopting a fibrinolytic enzyme spectrum and a fibrinogen plate method. As the drying temperature increased, a decrease in thrombolytic effect was observed for both. The results of fibrinolytic enzyme spectrum show that the fibrinolytic band becomes shallow when the temperature is higher than 70 ℃, and the thrombolytic activity is obviously reduced; but the fibrinolytic protein bands of the samples dried at different temperatures were uniformly distributed (fig. 2).

The plasminogen plate can quantitatively determine the thrombolytic activity, and the research takes the lumbrokinase reference substance as a reference to determine that the fibrinolytic activity is reduced by about 50 percent and 80 percent at 100 ℃ after drying for 12 hours at 80 ℃, and the influence of high-temperature drying on the thrombolytic activity of the lumbricus is larger. The earthworm samples which are freeze-dried and dried at 40 ℃, 60 ℃,80 ℃ and 100 ℃ are adopted to carry out the research and screening of the thrombolytic activity marker peptide in the labeled proteomics.

1.3 chromatographic and Mass Spectrometry conditions

Using an UlltiMate 3000Nano RSLC-Orbitrap Fusion Lumos high resolution mass spectrometer (Thermo Fisher, USA), analytical column Thermo Acclaim PepMap RSLC C182 μm (50 μm × 15 cm), pre-column Thermo Acclaim PepMap C183 μm (75 μm × 2 cm), sample size 1 μ l, flow rate 300nl/min, mobile phase A (0.1% formic acid-water solution), mobile phase B (acetonitrile/0.1% formic acid-water solution = 80/20), gradient elution: 0 to 5min,5% by volume B; 5-90min, 5-80% of B; 91-100min, 80% by weight B; 100-105min, 80% -5%; 105 to 120min,5% by weight of B. Mass spectrum conditions: the spray voltage is 2.0kV, the temperature of the ion transmission capillary is 320 ℃, the cracking mode is high energy induced dissociation (HCD), the normalized collision energy is 30%, and the resolution is set as follows: primary 120K, secondary 30K, parent ion scan range (m/z): 350-1500, and performing HCD fragment scanning on the secondary mass spectrum by selecting primary ions by a 'Top Speed' algorithm by using a dynamic Data Dependent Acquisition (DDA) method. The data acquisition and processing used Xcalibur 4.0 software.

1.4 identification of proteins and preliminary screening of characteristic peptides

1) Proteomics result identification

Protocol discover 2.2 software analysis parameters: introducing Nano LC-Orbirap Fusion Lumos mass spectrum data into PD2.2 software, and adopting Seuqest HT algorithm to carry out MS ² Processing mass spectrogram, taking a theoretical protein database obtained by earthworm transcriptome as a search library, and setting Seuqest HT parameters: trypsin enzyme, two misled cleavages allowed, minipeptide length of6, mass deviation of parent ion: 5ppm, mass deviation of fragment ions: 0.02Daltons. Fixing and modifying: carbamidomethyl,57.02Da (C), variable modification (peptide end): pro-hydroxypro,15.99Da (P); oxidation,15.99Da (M); lysyhroxy-lys 15.99Da (K). Variable modification (protein termination): acetyl 42.01Da (N-terminus). The polypeptide match error rate is determined using the target-decoy strategy in combination with Percolator modeling of correct and incorrect matches. Q-value of 0.01, as determined by Percolator, was used to filter data at the level of peptide match to control false findings.

2) Screening of thrombolytic activity marker peptide

The thrombolytic activity marker peptide screened should have the following characteristics: (1) the number of amino acids is 6-25; (2) the peptide fragment has no modification or fixed modification; (3) Can effectively ionize, and has stable signals and certain strength.

3) Screening of thrombolytic activity marker peptide based on earthworm tissue and body cavity fluid unmarked proteomics research

Based on the purpose of finding a marker of fibrinolytic activity, the invention screens candidate targets from the following two aspects: screening non-redundant peptides with the abundance ratio of E +8, the detection in three biological repeats and the ratio variance (%) of 100 from tissues which are subjected to down-regulation; and secondly, screening from the down-regulated lumbrokinase.

4) Thrombolytic activity marker peptide screened based on TMT marker proteomics research

Analysis of the identified non-redundant peptide, heatmap, using the Proteome scanner 2.4 software showed that the lyophilisation, drying at 40 ℃ and 60 ℃ were grouped together and drying at 80 ℃ and 100 ℃ was grouped together as a cluster (FIG. 3). The Heatmap map has two distinct down-regulation regions, containing 132 non-redundant peptides, and marker peptides related to thrombolytic activity were screened by reference to the following methods: peptides having a ratio of <0.67 dry at 40 ℃ and 60 ℃ of between 0.67 and 1.2 dry and peptides having a ratio of <0.67 dry at 80 ℃ and 100 ℃.

1.5 signature peptide validation

The thrombolytic activity marker peptide preliminarily screened by the Proteome discover software is verified by triple quadrupole mass spectrometry. Ultra performance liquid chromatography-triple quadrupole mass spectrometer (Agilent 1290/Agilent 6495), analytical column CORTECS C18 chromatography column (2.7 μm 2.1 × 50 mm), sample size 1 μ l, flow rate 0.2ml/min, mobile phase a (0.1% formic acid-water solution), mobile phase B (acetonitrile), gradient elution: 0 to 10min,2 to 35 percent of the total weight of the crude oil; 30-32min, 35-90% by weight of B. Mass spectrum conditions: the spray voltage was 4000V, gas temperature 270 deg.C, flow rate 16L/min, nebulizer 45psi, multiplex reaction detection mode (MRM) was used, and Mass Hunter software was used for data acquisition and processing. The specificity of the preliminarily screened characteristic peptide is verified, and the result shows that 11 marker peptides can distinguish the activity of the tissue and the body cavity fluid of the earthworm. Further validation in other species: 4 the mark peptide can distinguish the activity of the Bractenopharynia pectinifera tissue and the coelomic fluid, and 5 the mark peptide can distinguish the activity of the popular cyclothymia pectinifera tissue and the coelomic fluid. The results are shown in FIGS. 5-7.

TABLE 1 earthworm thrombolytic activity marker peptide sequences and Mass Spectrometry conditions

2. Methodological verification of thrombolytic activity marker peptide content determination method

The invention takes the synthetic LongshuanshuanShuactingmark peptide as a contrast, and examines the linearity, the range, the precision, the repeatability, the stability, the accuracy and the like of the method. The results were as follows:

2.1 specificity test

In order to investigate the specificity of the established method, the specificity was investigated by comparing it with the retention time of the synthetic marker peptide control ion pair, indicating that each synthetic marker peptide, blank solvent was not interfered, see fig. 8. The result shows that the specificity of the establishing method is good.

2.2 Standard Curve

Accurately weighing an appropriate amount of synthetic peptide reference substance, preparing mixed reference substance solutions with the concentrations of 5 mu g/ml by using water, accurately weighing the appropriate amounts of the mixed reference substance solutions into measuring bottles with certain volumes respectively to prepare a series of standard solutions, respectively sucking 1 mu l of each reference substance solution for sample injection analysis, drawing a standard curve by taking the concentration (mu g/ml) of the reference substance as a horizontal coordinate X and the peak area as a vertical coordinate Y, and calculating to obtain a regression equation (table 2).

TABLE 2 Linear relationship

2.3 accuracy

9 samples were taken and 11 thrombolytically active marker peptide controls corresponding to the original amounts of 50% (low concentration level), 100% (medium concentration level) and 150% (high concentration level) were precisely added before the enzymatic hydrolysis, and samples were prepared according to the enzymatic hydrolysis method in the test preparation for determining the sample recovery rate, and the accuracy of the method was examined, and the results of the sample recovery rates of the 11 thrombolytically active marker peptides are shown in tables 6-6. Wherein the ranges of PEP.1, PEP.2, PEP.3 and PEP.7 are respectively 84.3-113.1%, 99.6-118.9%, 83.9-117.5% and 85.5-108.5, and the RSD is less than 11%, which indicates that the method has good accuracy. PEP.4, PEP.5, PEP.6, PEP.8, PEP.9, PEP.10 and PEP.11, wherein the ranges are respectively 76.4-123.9%, 102.2-149.4%, 99.7-138.8%, 80.3-133.0%, 111.6-215.3%, 81.2-133.4% and 101.3-199.7%, and the RSD value is higher. Wherein PEP.5 and PEP.11 have higher recovery rate in low-concentration sample loading, but have better recovery rate in medium-high concentration sample loading.

2.4 precision

Precisely measuring 1 μ l of the mixed control solution, carrying out sample injection analysis for 6 times, and recording peak areas, wherein the results are shown in Table 3. The results of6 consecutive analyses showed RSD of pep.3 (GEFPWQLSMTR) of 11.82, indicating that the reproducibility of the method was essentially correct. The RSD of the areas of the rest 10 thrombolytic activity marker peptides is less than 8 percent, and the parallelism of the control solution is good.

TABLE 3 examination of precision

2.5 reproducibility

40mg of earthworm samples were weighed out in 6 copies, precisely weighed, prepared and measured according to the preparation method of the test solution shown in the text, and the results are shown in Table 4. The relative standard deviation of the marker peptide PEP.3 in 6 samples is 10.43 percent, which indicates that the repeatability of the method is basically possible. The relative standard deviation of peak area/sample weight (mg) of the remaining 10 marker peptides was less than 6.0%, indicating that the method was more reproducible (table 4).

TABLE 4 repeatability test

2.6 stability

Taking the test solution, injecting 1 mul of sample at 0, 4, 8, 12, 16 and 24 times respectively, analyzing, recording peak areas, and showing the result in Table 5, wherein the relative standard deviation of the sample peak areas is less than 6.0%, and the test solution is basically stable within 24 hours.

TABLE 5 test article solution stability survey

3. Analysis of correlation between thrombolytic activity marker peptide and fibrinolytic activity

The content of 11 thrombolytic activity marker peptides in 39 samples was determined by established method, including 18 batches of Hu earthworm (A1-A16 is popular Cyclotellularia, wherein A1-A4 is homemade medicinal material, A17, A18 are Cyclotellularia Williamsii), 15 batches of Guang earthworm (B1-B5 is homemade medicinal material) 6 batches of counterfeit product (C1-C6). And determining the thrombolytic activity of 39 lot of earthworm medicinal materials by adopting an agarose fibrin plate method, and determining the content of a thrombolytic activity marker peptide in the earthworm medicinal materials by adopting a Pearson correlation coefficient method and the correlation of fibrinolytic activity.

3.1 analysis of correlation between thrombolytic activity marker peptide of Pheretima aspergillum and fibrinolytic activity

When the contents of thrombolytic activity marker peptides and fibrinolytic activities in different lumbricus crinita samples are analyzed (fig. 9), lumbricus has no fibrinolytic activity when the detection of PEP.1 (TDASNILPNTLQK), PEP.3 (GEFPWQLSMTR), PEP.4 (VISTDECNR), PEP.5 (CQDSGDSGGPLSVK), PEP.7 (ATFDATIV), PEP.8 (YAINVIGR), PEP.9 (WPLDYFIK), PEP.10 (VLYPSSGTAQDYSK) and PEP.11 (VTMTPAPPGLIYR) marker peptides, or PEP.2 (TSASLNTLQK) and PEP.6 (GSVIAGVGVVR) is lower than 1.198 and 4.757 mug/g.

As can be seen from FIG. 10, the correlation coefficient values of the fibrinolytic activity Pearson of 11 thrombolytic activity marker peptides screened from lumbricus exuberculus and the fibrinolytic activity of lumbricus exculus medicinal material are all greater than 0.83 (P < 0.01) and are significantly and positively correlated, wherein the Pearson correlation coefficient values of PEP.3, PEP 7, PEP 8, PEP 9, PEP 10 and PErson 11 are greater than 0.90 (P < 0.01). The thrombolytic activity marker peptide screened from the earthworm drug is related to the thrombolytic activity thereof. In addition, the r value of the correlation coefficient among the 11 thrombolytic activity marker peptides screened out is between 0.790 and 0.999 (P is less than 0.01), and the correlation is obviously positive. Indicating that the selected marker peptides of thrombolytic activity have different degrees of influence.

3.2 correlation analysis of thrombolytic activity marker peptide of Hu earthworm and fibrinolytic activity

Analyzing the content and fibrinolytic activity of the thrombolytic activity marker peptides in different samples of the common crinis ciliaris (figure 11), detecting active 5 thrombolytic activity marker peptides in four batches of self-made medicinal materials, and detecting active medicinal material marker peptides PEP.3 (GEFPWQLSMTR) and PEP.11 (VTPAPPGLIYR) in the Hu earthworm medicinal materials (common crinis ciliaris and Williams disease); although one batch of the counterfeit products have fibrinolytic activity, 3 batches of the counterfeit products have no PEP.3 and PEP.11 detected in the counterfeit products; the other 3 marker peptides are not obvious in the rule of the medicinal materials.

Further adopts Pearson correlation coefficient method to determine correlation between thrombolytic activity marker peptide content and fibrinolytic activity in Lumbricus medicinal material. As can be seen from FIG. 12, the thrombolytic activity marker PEP.3 has significant correlation with the fibrinolytic activity of a common lumbricus drug (r =0.635, P < 0.01), and PEP.11 has significant correlation with the fibrinolytic activity of a common lumbricus drug (r =0.878, P < 0.01). The thrombolytic activity marker peptide PEP.3 has different degrees of influence from PEP.11 (r =0.677, P < 0.01). The thrombolytic activity marker peptides PEP.3 and PEP.11 screened from the lumbricus praecox can be used for characterizing the thrombolytic activity of the lumbricus vulgare.

The 11 thrombolytic activity marker peptides have strong correlation, and the contents of the marker peptides in medicinal materials have mutual influence, so that 2 marker peptides with representatives are screened for quality control of the medicinal materials. The correlation coefficient of PEP.7 and other marker peptides is more than 0.90 (P is less than 0.01), the correlation is very strong, the precision, the repeatability and the accuracy are good, and the PEP can be selected as a quality control polypeptide. In addition, the protein of PEP.1 which has strong correlation with the medicinal materials of the earthworms trepangae (r =0.865, P < -0.01) is lumbrokinase and fibrinolytic activity protein, is not detected in the inactive earthworms trepangae, has better precision, repeatability and accuracy, and can be selected as earthworm quality control polypeptide. The marker peptides PEP.7 and PEP.1 (belonging protein is fibrinolytic active protein) are applied to the quality control and detection of the activity of the earthworm medicinal material, both can be detected in the active earthworm, and the inactive earthworm and the counterfeit product are not detected.

5 of 11 thrombolytic activity marker peptides screened from the lumbricus aspergillum can distinguish the tissue and body cavity fluid of the popular lumbricus aspergillum, and the content of the 5 marker peptides in the lumbricus medicine is further determined, and the result shows that the marker peptides PEP.3 and PEP.11 have strong correlation with the thrombolytic activity of the lumbricus aspergillum, and no thrombolytic activity is detected in the counterfeit product. Is applied to the quality control and the detection of the activity of the Hu earthworm medicine.

Example 1

The earthworm samples are purchased in the market and identified as the earthworm with reference to a DNA bar code molecular identification method of traditional Chinese medicinal materials of four parts 9107 in 2020 edition of Chinese pharmacopoeia.

Taking 40mg of Guangdong earthworm medicinal material powder, and adding 50mM NH ₄ HCO ₃ Extracting 800 μ l of the extractive solution with ultrasound at 37 deg.C for 40min, centrifuging, adding 50mM NH into the solid ₄ HCO ₃ Ultrasonic extracting 400 μ l of the extractive solution at 37 deg.C for 30min, centrifuging, and mixing the supernatants to obtain Lumbricus protein. The activity is measured by an agarose plasminogen plate, and the thrombolysis activity per milligram of the medicinal material is 2395.6U.

100-200. Mu.g of protein was taken, 200. Mu.l of cold tris-phenol was added, respectively, and vortexed at room temperature for 20 minutes. Precipitation was carried out overnight at-20 ℃ with a five-fold amount of 100mM ammonium acetate in methanol. The supernatant was centrifuged, the solid was dissolved in a urea (8M, 1% SDS) solution, and the urea was removed by ultrafiltration. Adding 500mM DTT 10 μ l (dissolved with 50mM ammonium bicarbonate, ready-to-use, final concentration of DTT 25 mM) and standing at 95 deg.C for 30min, adding 500mM IAA20 μ l (dissolved with 50mM ammonium bicarbonate, ready-to-use, final concentration of IAA 50 mM) and standing in the dark for 40min.

Trypsin was dissolved in 1ml of acetic acid at a concentration of 100. Mu.g/ml, and each sample was subjected to enzymolysis at 37 ℃ for 18 hours by adding Trypsin (about 1, 50, w/w), and then acidified to pH 2-3 with 10% TFA to terminate the reaction. Lyophilized, 200. Mu.l of phosphate buffer (pH 7.8) was added, and Glu-C endonuclease (about 1, w/w) was added for enzymatic hydrolysis at 45 ℃ for 4 hours. C18 desalting: and (3) buffer solution A: h ₂ O38.4 ml,5% TFA 1.6ml; and (3) buffer solution B: h ₂ O6.4ml, ACN32ml,10% TFA 1.6ml;50% ACN-activated C18 column, pipetting buffer A, sampling the pipetted sample, desalting buffer A, pipetting 200. Mu.l buffer B repeatedly to extract peptide fragments, and repeating once. Spin-drying at room temperature in a vacuum centrifugal concentrator, adding 0.1% (by weight) FA100 μ l for redissolving, centrifuging to remove supernatant, and packaging in plastic liquid phase vial.

Ultra performance liquid chromatography-triple quadrupole mass spectrometer (Agilent 1290/Agilent 6495), analytical column CORTECS C18 chromatography column (2.7 μm 2.1 × 50 mm), sample size 1 μ l, flow rate 0.2ml/min, mobile phase a (0.1% formic acid-water solution), mobile phase B (acetonitrile), gradient elution: 0 to 10min,2 to 35 percent of the total weight of the crude oil; 30-32min, 35-90% of B. Mass spectrum conditions: the spray voltage was 4000V, gas temperature 270 ℃, flow rate 16L/min, nebulizer 45psi, MRM mode was used, and Mass Hunter software was used for data acquisition and processing. The obtained MRM chromatogram is shown in FIG. 13, and can simultaneously detect 11 chromatographic peaks of the thrombolytic activity marker peptide, wherein the contents of PEP.1-PEP.11 are respectively 1.294, 4.948, 1.774, 2.287, 2.306, 12.156, 0.509, 3.333, 1.550, 1.253 and 1.651 mug/g.

Example 2

The lumbricus sample is purchased in the market and identified as the lumbricus crinis by referring to DNA bar code molecular identification method of four parts 9107 Chinese medicinal materials in 2020 edition of Chinese pharmacopoeia.

Taking 40mg of Guangdong earthworm medicinal material powder, adding 50mM NH ₄ HCO ₃ Extracting 800 μ l of the extractive solution with ultrasound at 37 deg.C for 40min, centrifuging, adding 50mM NH into the solid ₄ HCO ₃ Ultrasonic extracting 400 μ l of extractive solution at 37 deg.C for 30min, and separatingAnd (4) merging the supernatant fluid of the hearts to obtain the earthworm protein. The activity was measured by agarose plasminogen plate with thrombolysis activity of 0U per mg drug.

100-200. Mu.g of protein was added to 200. Mu.l of cold tris-phenol, respectively, and vortexed at room temperature for 20 minutes. Precipitation was carried out overnight at-20 ℃ with a five-fold amount of 100mM ammonium acetate in methanol. Centrifuging, removing supernatant, dissolving the solid in urea (8M, 1% SDS) solution, and ultrafiltering to remove urea. Adding 500mM DTT 10 μ l (dissolved with 50mM ammonium bicarbonate, ready-to-use, final concentration of DTT 25 mM) and standing at 95 deg.C for 30min, adding 500mM IAA20 μ l (dissolved with 50mM ammonium bicarbonate, ready-to-use, final concentration of IAA 50 mM) and standing in the dark for 40min.

Trypsin was dissolved in 1ml of acetic acid at a concentration of 100. Mu.g/ml, and each sample was subjected to enzymolysis at 37 ℃ for 18 hours by adding Trypsin (about 1, 50, w/w), and then acidified to pH 2-3 with 10% TFA to terminate the reaction. Lyophilized, 200. Mu.l of phosphate buffer (pH 7.8) was added, and Glu-C endonuclease (about 1, w/w) was added for enzymatic hydrolysis at 45 ℃ for 4 hours. C18 desalting: and (3) buffer solution A: h ₂ O38.4 ml,5% TFA 1.6ml; and (3) buffer solution B: h ₂ O6.4ml, ACN32ml,10% TFA 1.6ml;50% ACN-activated C18 column, pipetting buffer A, sampling the pipetted sample, desalting buffer A, pipetting 200. Mu.l buffer B repeatedly to extract peptide fragments, and repeating once. Spin-drying at room temperature in a vacuum centrifugal concentrator, adding 0.1% (by weight) FA100 μ l for redissolving, centrifuging to remove supernatant, and packaging in plastic liquid phase vial.

Ultra performance liquid chromatography-triple quadrupole mass spectrometer (Agilent 1290/Agilent 6495), analytical column CORTECS C18 column (2.7 μm 2.1 × 50 mm), sample size 1 μ l, flow rate 0.2ml/min, mobile phase a (0.1% formic acid-water solution), mobile phase B (acetonitrile), gradient elution: 0 to 10min,2 to 35 percent of the total weight of the crude oil; 30-32min, 35-90% of B. Mass spectrum conditions: the spray voltage was 4000V, gas temperature 270 ℃, flow rate 16L/min, nebulizer 45psi, MRM mode was used, and Mass Hunter software was used for data acquisition and processing. The resulting MRM chromatogram is shown in figure 14, the contents of PEP.1-PEP.11 are respectively 0, 00, 3.116, 0 and 0. Mu.g/g.

While the invention has been described with reference to a preferred embodiment, 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 spirit and scope of the invention.

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Claims (7)

1. A group of earthworm thrombolysis activity marker peptides is characterized by comprising

1 to 11 portions of limnodrilus salviae miltiorrhizae thrombolytic activity marker peptide, the amino acid sequence of which is shown as SEQ ID NO 1 to SEQ ID NO 11;

the amino acid sequences of the Hu earthworm thrombolysis active marker peptides 3 and 11 are shown as SEQ ID NO. 3 and SEQ ID NO. 11.

2. The earthworm thrombolytically active marker peptide according to claim 1, wherein the lumbricus is a Pantoea punctata and a Lumipia willebrand.

3. The earthworm thrombolytic activity marker peptide according to claim 1, wherein the mass spectrum conditions of the thrombolytic activity marker peptides 1-11 are as follows:

4. use of the earthworm thrombolysis activity marker peptide according to any one of claims 1 to 3 in an earthworm thrombolysis activity assay.

5. A method for measuring thrombolytic activity of Pheretima based on the marker peptide for thrombolytic activity of Pheretima of any one of claims 1 to 3, comprising the steps of:

step one, processing a sample to be detected to obtain a polypeptide mixed solution to be detected;

and step two, detecting the polypeptide mixed solution to be detected by adopting an ultra-high performance liquid chromatography-triple quadrupole mass spectrometer to obtain a mass spectrogram of the thrombolytic activity marker peptide of the sample to be detected.

6. The method for determining the thrombolytic activity of the earthworm according to claim 5, wherein the chromatographic conditions of the ultra-high performance liquid chromatography-triple quadrupole mass spectrometer are as follows:

and (3) analyzing the column: CORTECS C18 column, 2.7 μm 2.1X 50mm;

sample injection amount: 1 mul;

flow rate: 0.2ml/min;

mobile phase: the mobile phase A is 0.1 percent formic acid-water solution, the mobile phase B is acetonitrile,

gradient elution: 0 to 10min,2 to 35 percent of the total weight of the crude oil; 30-32min, 35-90% by weight of B.

7. The method for determining the thrombolytic activity of the earthworm according to claim 5, wherein the mass spectrometric conditions of the ultra-high performance liquid chromatography-triple quadrupole mass spectrometer are as follows: the spray voltage was 4000V, gas temperature 270 deg.C, flow rate 16L/min, nebulizer 45psi, using MRM mode.

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