Characteristic peptide of arca granosa as well as detection method and application thereof

Abstract

The invention relates to characteristic peptide of scapharca granosa as well as a detection method and application thereof. The invention discloses four characteristic peptides of tegillarca granosa, which are respectively FAVNHINR (FAV), FSILENDFDTVNTK (FSI), LTESEQEIASLTR (LTE) and TTLLEDDIAK (TTL), and are used for identifying the tegillarca granosa and measuring the content of the tegillarca granosa. The invention also discloses a liquid chromatography-triple quadrupole mass spectrometry method for simultaneously detecting the characteristic peptides of the tegillarca granosa, and the method can be used for identifying the tegillarca granosa, determining the content of the tegillarca granosa, evaluating the quality of the tegillarca granosa and effectively avoiding the phenomena of counterfeiting and adulteration, is simple and convenient to operate and rapid in detection, has good specificity, sensitivity and stability, and can be used for detecting the characteristic peptides of the tegillarca granosa. The method has a good application value and a wide popularization prospect in quality control of the scapharca granosa medicinal material and related products.

Description

Technical Field

[0001] The present invention relates to the technical field of clam variety identification, and specifically relates to characteristic peptides of clams, their detection methods and applications. Background Art

[0002] The traditional Chinese medicine clam ( Scapharcae and Tegillarcae muscle ) is the meat of animals in the family Arcidae, such as Scapharca broughtonii Scapharca broughtonii (Schrenck), Tegillarca granosa Tegillarca granosa (Linnaeus) Scapharca subcrenata and Arca subcrenata (Lischke), etc. It is recorded that clams have the effects of "treating cold in the heart and abdomen, cold wind in the waist and spine, benefiting the five internal organs, strengthening the stomach, and making people able to eat". Research has found that clams contain rich polysaccharides and polypeptide compounds, with antioxidant, anti-inflammatory, antibacterial and anti-tumor activities. Clams are also foods rich in nutritional components, containing high-quality proteins, minerals, vitamins and various bioactive components. Clams are one of the few bivalve animals containing hemoglobin, with rich and highly absorbable heme iron, and have significant health care effects in dietary iron supplementation.

[0003] The quality standards of clams are included in the "Standards for Traditional Chinese Medicines in Shandong Province" (2022 Edition), including the item of character identification, the items of thin-layer chromatography (TLC), moisture, total ash, acid-insoluble ash inspection, and the determination item of total nitrogen (N) content. There is still room for improvement in the quality standards of clams. For example, the current standards do not cover the detection content of markers for evaluating their quality, and the existing character identification methods rely on expert experience, with certain subjectivity and difficulty in identification. Summary of the Invention

[0004] The purpose of the present invention is to propose characteristic peptides of clams, provide a method for determining the content of the characteristic peptides, and also provide specific applications of the characteristic peptides to make up for the deficiencies of the prior art.

[0005] To achieve the above purpose, the present invention adopts the following technical solutions: The characteristic peptides of clams include: characteristic peptide FAV: FAVNHINR, characteristic peptide FSI: FSILENDFDTVNTK, characteristic peptide LTE: LTESEQEIASLTR, characteristic peptide TTL: TTLLEDDIAK.

[0006] Further, the clams are Scapharca broughtonii, Tegillarca granosa and Arca subcrenata.

[0007] The application of the characteristic peptides of clams in the detection and content determination of clams.

[0008] The application of the characteristic peptides of clams in the identification of clam medicinal materials.

[0009] The method for identifying ark shell based on the characteristic peptides of ark shell is to detect whether any one of the characteristic peptides of ark shell is contained in the sample, and the judgment principle is as follows: (1) If any one or more of the characteristic peptides of ark shell are detected in the sample, it is determined that the test sample contains ark shell; (2) If none of the characteristic peptides of ark shell are detected in the sample, it is determined that the test sample does not contain ark shell.

[0010] The detection method of the characteristic peptides of ark shell includes the following steps: (1) Preparation of test solution: Take samples of three kinds of ark shell medicinal materials, namely Scapharca broughtonii, Tegillarca granosa and Arca subcrenata, for enzymatic hydrolysis, centrifugation, collect the supernatant, freeze-dry to obtain freeze-dried ark shell hydrolysate powder, prepare ark shell hydrolysate solution, and then perform desalting treatment and reconstitution to obtain the test solution; (2) Preparation of reference solution: Use the four characteristic peptides of ark shell, namely FAV, FSI, LTE and TTL, as reference substances, add 0.1% formic acid in water for dissolution, and prepare the reference solution; (3) Detection and analysis: Use liquid chromatography-triple quadrupole mass spectrometry (LC-MS / MS) method for detection and analysis; (4) If any one of the characteristic peptides of ark shell is detected in the sample, it is determined that the test sample contains ark shell; if none of the characteristic peptides of ark shell are detected in the sample, it is determined that the test sample does not contain ark shell.

[0011] Further, in the step (1), the enzymatic hydrolysis conditions are as follows: add PBS buffer solution according to the solid-liquid ratio of 1:10 (w / v, g / mL), add trypsin with a final concentration of 800 U / mL, and perform enzymatic hydrolysis at 50 °C and pH 8.0 for 5 hours; the desalting treatment is to use a desalting column to perform desalting treatment on the sample; finally, reconstitute with 50 μL of 0.1% formic acid aqueous solution.

[0012] Further, in the step (3), the chromatographic conditions are as follows: mobile phase A is 0.1% formic acid aqueous solution, mobile phase B is 0.1% formic acid acetonitrile solution, and the flow rate is set at 0.3 mL / min. The elution gradient is as follows: 0 - 1 min, 95% A; 1 - 2 min, 95 - 80% A; 2 - 8 min, 80 - 60% A; 8 - 10 min, 60 - 10% A; 10 - 11.5 min, 10% A; 11.5 - 11.6 min, 10 - 95% A; 11.6 - 14 min, 95% A. The monitored ion pairs include FAV as m / z 486.0→219.1, FSI as m / z 822.3→362.3, LTE as m / z 739.2→547.4, TTL as m / z 560.1→203.1.

[0013] Furthermore, the detection method also includes the determination of the contents of four characteristic peptides of blood clam, namely FAV, FSI, LTE, and TTL. The content determination is carried out by establishing a concentration gradient curve with four standard characteristic peptides.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The characteristic peptides of blood clam, namely FAV, FSI, LTE, and TTL, provided by the present invention have strong specificity and good stability, and can be widely used in the component detection of blood clam medicinal materials or other related products, accurately identify whether they contain blood clam components and determine the content, effectively avoiding the phenomena of forgery and adulteration. The present invention also discloses an LC-MS / MS detection method based on four characteristic peptides of blood clam. This method is simple to operate, highly specific, sensitive, stable, and reproducible. Compared with traditional methods, this method significantly improves the accuracy and reliability of quality evaluation, has strong practicability, and has broad application prospects and important value in quality control and safety detection in the fields of drugs, foods, etc.

[0015] The present invention can accurately and quickly identify blood clam medicinal materials and evaluate the quality of blood clam, overcoming the deficiencies in existing quality control methods. The present invention can not only improve the quality standards of blood clam medicinal materials and related products, effectively ensure the safety and effectiveness of clinical medication, but also ensure the market order and promote the healthy development of the blood clam industry. Description of the Drawings

[0016] Figure 1 are the secondary fragment mass spectra of FAV, FSI, LTE, and TTL.

[0017] Figure 2 are the extracted ion chromatograms of four standard characteristic peptides FAV, FSI, LTE, and TTL (50 ng / mL).

[0018] Figure 3 are the extracted ion chromatograms of FAV, FSI, LTE, and TTL in Scapharca broughtonii.

[0019] Figure 4 are the extracted ion chromatograms of FAV, FSI, LTE, and TTL in Tegillarca granosa.

[0020] Figure 5 are the extracted ion chromatograms of FAV, FSI, LTE, and TTL in Tegillarca granosa.

[0021] Figure 6 are the extracted ion chromatograms of FAV, FSI, LTE, and TTL in Mactra veneriformis. Detailed Embodiments

[0022] The present invention will be further explained and illustrated below through specific embodiments in combination with the drawings.

[0023] Example 1: Screening of characteristic peptides of three species of ark shell, namely Scapharca broughtonii, Tegillarca granosa and Arca subcrenata Preparation of test sample solution Take 0.1 g of the powder of the medicinal materials of three species of ark shell, namely Scapharca broughtonii, Tegillarca granosa and Arca subcrenata, add 1 mL of PBS buffer solution, and add 800 U of trypsin. Enzymolysis is carried out at 50 °C and pH 8.0 for 5 hours, and centrifuged at 8000 rpm for 10 minutes at 4 °C. Collect the supernatant and freeze-dry to obtain the freeze-dried powder of ark shell enzymolysate. Take the prepared freeze-dried powder of ark shell enzymolysate, dissolve it with water, and prepare an ark shell enzymolysate solution with a concentration of 10 mg / mL. Desalt the sample using a ZIPTIP C18 desalting column produced by Millipore Corporation, and redissolve it with 50 μL of 0.1% formic acid aqueous solution to obtain the test sample solution. Three batches of repeated experiments are carried out on the samples of Scapharca broughtonii, Tegillarca granosa and Arca subcrenata.

[0024] Selection of characteristic ions and sequence speculation LC-MS / MS parameters: Analyze using an EASY-nLC1000 nano ultra-high performance liquid chromatography system equipped with a nano peptide trapping column Acclaim PepMap C18 (100 μm × 20 mm) and a nano peptide analysis column Acclaim PepMap C18 (75 μm × 250 mm). Column temperature: 50 °C; mobile phase A is 0.1% formic acid aqueous solution, mobile phase B is 0.1% formic acid acetonitrile solution, gradient elution; injection volume is 2 μL. Use a Q-Exactive mass spectrometer with an electrospray ionization (ESI) source to collect mass spectrometry information in the positive ion mode under the data-dependent acquisition (DDA) mode.

[0025] Database retrieval and screening of characteristic peptides: Use the mass spectrometry data analysis and retrieval software MaxQuant 2.4.3.0, and use the Uniprot protein database: ark shell ( Arcidae ), including three species Scapharca broughtonii ( Scapharca broughtonii ), Arca subcrenata ( Scapharca subcrenata ), Tegillarca granosa ( Tegillarca granosa ) and ark shells of other genera and species.

[0026] Analyze the common peptides of three species of blood clams, and set the following screening methods to determine the characteristic peptides of blood clam species: (1) Intensity and good response in ≥8 samples among 9 batches of samples; (2) score > 110 in the MQ matching result; (3) The number of amino acids is 8 - 15; (4) No missed cleavage at the C-terminus of arginine and lysine in trypsin digestion; (5) The peptide segments are from multiple different proteins; (6) The protein-protein BLAST analysis result contains two or more species. As Figure 1 shown, the characteristic peptides of blood clams were finally obtained: FAVNHINR (FAV), FSILENDFDTVNTK (FSI), LTESEQEIASLTR (LTE), TTLLEDDIAK (TTL).

[0027] Entrust the synthesis of characteristic peptide reference substances according to the deduced amino acid sequences, and detect the reference substances and the test solution of blood clams simultaneously. The retention times and secondary mass spectrometry information of both are consistent, thus confirming the correctness of the sequence.

[0028] Example 2: Content determination method of characteristic peptides of blood clams Analyze the characteristic peptides FAV, FSI, LTE, and TTL of blood clams in the enzymatic hydrolysate solution of blood clams by LC-MS / MS.

[0029] (1) Sample preparation Preparation of test solution: Take about 0.1 g of the powder of three kinds of blood clam medicinal material samples, add 1 mL of PBS buffer solution, add 800 U of trypsin, enzymatically hydrolyze at 50 °C and pH 8.0 for 5 hours, centrifuge at 8000 rpm for 10 minutes at 4 °C, collect the supernatant, freeze-dry to obtain the freeze-dried powder of blood clam enzymatic hydrolysate. Take the prepared freeze-dried powder of blood clam enzymatic hydrolysate, dissolve it in water, and prepare a 10 mg / mL blood clam enzymatic hydrolysate solution. Use the ZIPTIP C18 desalting column produced by Millipore Corporation to desalt the sample, and finally redissolve it with 50 μL of 0.1% formic acid aqueous solution to obtain the test solution.

[0030] Preparation of reference solution: Weigh precisely four characteristic peptide reference substances of blood clams, dissolve them in 0.1% formic acid water to obtain a stock solution with a concentration of 1 mg / mL, and store it in a -20 °C refrigerator for standby. Precisely measure the stock solution of the characteristic peptide reference substance of blood clams, dilute it step by step with 0.1% formic acid aqueous solution, and add an internal standard to obtain a series of reference solutions with concentrations of 5 ng / mL, 10 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, 400 ng / mL, 500 ng / mL, 600 ng / mL, and the concentration of the internal standard is 250 ng / mL.

[0031] Spiked matrix samples: Take the characteristic peptide reference substance of ark shell, and add it to the matrix of the enzymatic hydrolysis system. Prepare spiked matrix samples of the characteristic peptides of ark shell with concentrations of 10 ng / mL, 300 ng / mL, and 500 ng / mL, and the internal standard concentration is 250 ng / mL.

[0032] Quality control samples: Take the stock solution of the characteristic peptide reference substance of ark shell, serially dilute it with 0.1% formic acid aqueous solution, add the internal standard, and prepare quality control solutions of the characteristic peptides of ark shell at three concentrations of high, medium, and low, with concentrations of 10 ng / mL (LQC), 300 ng / mL (MQC), and 500 ng / mL (HQC), and the internal standard concentration is 250 ng / mL.

[0033] (2)LC-MS / MS content determination method Use an UPLC H-Class PLUS ultra-high performance liquid chromatography system equipped with an Xevo TQ-XS mass detector. The chromatographic column is an ACQUITY UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm), the column temperature is 40°C, mobile phase A is 0.1% formic acid aqueous solution, mobile phase B is 0.1% formic acid acetonitrile solution, and gradient elution is carried out according to Table 1; the injection volume is 4 μL. The mass detector uses ESI, and multiple reaction monitoring (MRM) is carried out in the positive ion mode. The capillary voltage is 3.0 kV, and the desolvation gas temperature is 500°C. Nitrogen is used as the desolvation gas (1000 L / Hr) and the cone gas (150 L / Hr), and the collision gas is argon (0.15 mL / min). The monitored ion pairs are shown in Table 2.

[0034] Table 1 Liquid phase conditions for the determination method of the characteristic peptides of ark shell

[0035] Table 2 Mass spectrometry MRM conditions for the determination method of the characteristic peptides of ark shell

[0036] Note: FAVNHINR ( 13 C6, 15 N4), FSILENDFDTVNTK ( 13 C6, 15 N2), LTESEQEIASLTR ( 13 C6, 15 N4) and TTLLEDDIAK ( 13 C6, 15 N2) are isotope-labeled peptides and are used as internal standards.

[0037] (3)Specificity Such as Figure 3 、Figure 4 and Figure 5 As shown in Figure 5 , in the extracted ion chromatograms of the three clam samples, at the retention time positions corresponding to the reference solutions of the characteristic peptides FAV, FSI, LTE, and TTL of the clam (as shown in Figure 2 ), four chromatographic peaks with consistent retention times were presented; in the extracted ion chromatogram of the clam sample ( Figure 6 ), at the retention time positions corresponding to the reference solutions of the characteristic peptides FAV, FSI, LTE, and TTL of the clam, no corresponding chromatographic peaks were observed. It indicates that the four characteristic peptides of the clam were only detected in the three clams, with strong specificity.

[0038] (4) Linear relationship, detection limit, and quantification limit The standard curves were plotted with the ratios of the peak areas of the quantitative ion pairs of FAV, FSI, LTE, and TTL to the peak area of the internal standard and the corresponding mass concentrations. The calculated lowest detection limit (LOD, S / N≥3), lowest quantification limit (LOQ, S / N≥10), and regression equations are shown in Table 3. The four characteristic peptides of the clam had a good linear relationship within the range of 5 - 600 ng / mL.

[0039] Table 3 Sensitivity and linearity of the method for the determination of the content of the characteristic peptides of the clam

[0040] (5) Matrix effect By comparing the peak areas of the reference solutions of the characteristic peptides of the clam species with those of the spiked matrix samples, the matrix effects of the samples at various concentrations were calculated. The results showed that the matrix effects of FAV, FSI, LTE, and TTL were 95.9% - 104.5%, 100.4% - 111.5%, 88.5% - 103.1%, and 90.7% - 111.4% respectively. It indicates that in the sample determination, the detection signals of the four characteristic peptides of the clam were not significantly interfered with or inhibited.

[0041] (6) Recovery rate By comparing the peak areas of the standards and the quasi - standards after being prepared and processed according to the test solution preparation method under LQC, MQC, and HQC, the recovery rates were calculated. As shown in Table 4, the recovery rates of FAV, FSI, LTE, and TTL were 89.3% - 93.8%, 91.5% - 99.1%, 91.5% - 99.1%, and 94.2% - 108.7% respectively, indicating that this method had good accuracy and was not affected by the sample treatment process.

[0042] Table 4 Matrix effect and recovery rate of the method for the determination of the content of the characteristic peptides of the clam (n = 6)

[0043] (7) Accuracy and precision ​​​​The analysis of accuracy and precision was carried out through intra-day and inter-day experiments at four concentrations: the lowest quantitation limit (LLOQ), LQC, MQC, and HQC concentrations. As shown in Table 5, the intra-day and inter-day precisions (RSD) of the determination results of FAV, FSI, LTE, and TTL contents were 1.3% - 4.2%, 1.3% - 8.2%, 2.7% - 5.6%, and 3.3% - 9.8% respectively, and the accuracies (RE) were 0.0% - 12.8%, -10.3% - 9.8%, -12.2% - 14.9%, and -0.2% - 8.2% respectively, indicating that the detection method has reliable accuracy and good reproducibility.

[0044] Table 5 Precision and accuracy of the determination method for the content of characteristic peptides in ark shell (n = 6)

[0045] (8) Stability When placed at the auto-sampler temperature for 72 h, subjected to 3 cycles of repeated freezing and thawing, and stored at -40 °C for 7 days, the accuracies (RE) of the detection results of FAV, FSI, LTE, and TTL were -1.9% - 6.0%, -11.5% - 10.5%, -8.8% - 10.4%, and -2.2% - 10.6% respectively (Table 6), indicating that the four characteristic peptides of ark shell (FAV, FSI, LTE, TTL) are stable under different storage and detection conditions.

[0046] Table 6. Stability of the determination method for the content of characteristic peptides in ark shell (n = 6)

[0047] Example 3: Identification and content determination of ark shell using the characteristic peptides of ark shell Collect 29 batches of ark shell samples, identify the origin through DNA barcoding. There are 6 batches of Scapharca subcrenata, 12 batches of Scapharca broughtonii, 5 batches of Tegillarca granosa, 2 batches of Mactra veneriformis, and 4 batches with failed detection. Prepare the samples according to the method under (1) Preparation of test solution in Example 1, and detect according to the (1) LC-MS / MS content determination method in Example 2.

[0048] The results showed that the chromatographic peaks of the characteristic peptides FAV, FSI, LTE, and TTL of ark shell were shown in 23 batches of ark shell samples, and the chromatographic peaks of the four characteristic peptides of ark shell did not appear in Mactra veneriformis. Figures 2 - 5 ). The detection results of the characteristic peptides of ark shell in 4 batches of samples (HWZ05, HLYG06, HLYG11, HYT20) with failed DNA barcoding detection are shown in Table 7. The above four characteristic peptides of ark shell were detected in HWZ05, HLYG06, and HLYG11, and these four characteristic peptides were not detected in HYT20, indicating that the HYT20 sample is not ark shell.

[0049] Table 7. Detection of characteristic peptides of 4 batches of clam samples with failed DNA barcoding

[0050] The contents of four characteristic peptides of clam, namely FAV, FSI, LTE, and TTL, in the samples identified by DNA barcoding are shown in Table 8. The contents of the four characteristic peptides FAV, FSI, LTE, and TTL in 23 batches of clam samples are between 0.04 - 0.48 μg / g, 0.11 - 1.07 μg / g, 0.02 - 0.31 μg / g, and 0.04 - 0.25 μg / g, respectively.

[0051] Table 8. Determination of the contents of characteristic peptides of clam in clam samples (n = 3)

[0052] The above embodiments prove that the characteristic peptides provided by the present invention have excellent specificity and stability for clam medicinal materials, with strong specificity, can be used for the quality evaluation of clam, and have good application prospects.

[0053] The specific embodiments described above further elaborate on the purpose, technical solutions, and beneficial effects of the disclosure of the present invention. It should be understood that the above are only specific embodiments of the disclosure of the present invention and are not used to limit the disclosure of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the disclosure of the present invention shall be included within the protection scope of the disclosure of the present invention.

Claims

1. A characteristic peptide of clams, characterized in that: Including characteristic peptide FAV: FAVNHINR, characteristic peptide FSI: FSILENDFDTVNTK, characteristic peptide LTE: LTESEQEIASLTR, characteristic peptide TTL: TTLLEDDIAK.

2. The characteristic peptide of clam according to claim 1, characterized in that The clams are giant clams, mud clams and hairy clams.

3. Use of the characteristic peptide of cockle according to claim 1 in cockle detection and content determination.

4. Use of the characteristic peptide of clam according to claim 1 in identifying clam medicinal materials.

5. A method for identifying cockles based on the characteristic peptide of cockles according to claim 1, characterized in that: The method is to detect whether the sample contains any one of the characteristic peptides of the cockle, and the judgment principle is: (1) If any one of the characteristic peptides of cockle is detected in the sample, it is determined that the sample contains cockle; (2) If any of the characteristic peptides of cockle are not detected in the sample, it is determined that the sample does not contain cockle.

6. A method for detecting the characteristic peptide of clams according to claim 1, characterized in that: The following steps are involved: (1) Preparation of test solution: three kinds of clams, i.e., giant clams, mud clams and hairy clams, were respectively subjected to enzymatic hydrolysis, centrifuged, the supernatant was collected, and freeze-dried to obtain clams enzymatic hydrolysate freeze-dried powder, and clams enzymatic hydrolysate solution was prepared, and then desalted and re-dissolved to obtain the test solution; (2) Preparation of reference solution: using the characteristic peptides of the four clams FAV, FSI, LTE and TTL as reference substances, a reference solution was prepared; (3) Detection and analysis: Detection and analysis are performed using liquid chromatography-triple quadrupole mass spectrometry; (4) If any one of the characteristic peptides of cockle is detected in the sample, it is determined that the sample contains cockle; if any one of the characteristic peptides of cockle is not detected in the sample, it is determined that the sample does not contain cockle.

7. The detection method according to claim 6, characterized in that In the step (1), the enzymatic hydrolysis conditions are: adding PBS buffer at a solid-liquid ratio of 1:10, adding trypsin at a final concentration of 800 U / mL, and enzymatic hydrolysis at 50°C and pH 8.0 for 5 hours; the desalting treatment is to desalt the sample using a desalting column; and finally re-dissolving with 50 μL of 0.1% formic acid aqueous solution.

8. The detection method according to claim 6, characterized in that: In step (3), the chromatographic conditions are: mobile phase A is 0.1% formic acid in water, mobile phase B is 0.1% formic acid in acetonitrile, and the flow rate is set to 0.3 mL / min. The elution gradient is as follows: 0-1 min, 95% A, 1-2 min, 95-80% A, 2-8 min, 80-60% A, 8-10 min, 60-10% A, 10-11.5 min, 10% A, 11.5-11.6 min, 10-95% A, 11.6-14 min, 95% A. The monitored ion pairs include FAV m / z 486.0→219.1, FSI is m / z 822.3→362.3, LTE m / z 739.2→547.4, TTL is m / z 560.1→203.

1.

9. The detection method according to claim 6, characterized in that: The detection method also includes the determination of the contents of the characteristic peptides of four clams, namely, FAV, FSI, LTE and TTL, and the determination is performed by establishing a concentration gradient curve through four characteristic peptide standards.

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