Method for determining content of corydalus cornutus polypeptide

Abstract

The invention discloses a method for determining the content of corydalus cornutus polypeptide, which belongs to the technical field of biology, and comprises the following steps: detecting and determining corydalus cornutus characteristic polypeptide sequences as LIRF and LEI by adopting mass spectrometry, and naming the corydalus cornutus characteristic polypeptide sequences as PSC-1 and PSC-2; and preparing a corydalus cornutus solution, preparing a reference substance solution, establishing a standard curve and determining the polypeptide content. The method for determining the content of the polypeptide LIRF and the polypeptide LEI in the corydalus cornutus is established on the basis of the LC-MS technology, has the characteristics of easiness in reference substance synthesis, simplicity in operation, high sensitivity, accuracy in determination and the like, can be used for quality evaluation research of the corydalus cornutus, and provides reference for quality control of the corydalus cornutus.

Description

Technical Field

[0001] The present invention relates to the determination and detection of biological polypeptides, in particular to a method for determining the content of Treptilodon polypeptides. Background Art

[0002] Megaloptera corydalidae, a dried insect of the family Corydalidae, order Macroptera, class Insecta, is a plant with a main component, including proteins, oils, organic acids, peptides, and polysaccharides. It boasts benefits such as tonifying qi and kidney, removing dampness and relieving pain, and strengthening the body's constitution. It is primarily used in folk medicine to treat enuresis in children and frequent urination in the elderly. Modern research on the basic principles of pharmacological activity has revealed that Megaloptera corydalidae is rich in protein, of which 4.87% is water-soluble. However, a review of the literature reveals that research on its components is limited, and studies on its quality are incomplete. Further research is needed on the substance of Megaloptera corydalidae.

[0003] Polypeptides are formed by the dehydration condensation of less than 50 amino acids and are widely present in nature. According to their sources, they are divided into natural polypeptides and protein hydrolyzed polypeptides. They have significant biological activity and their molecular weight is generally less than 10 kDa. Polypeptides are protein hydrolysis products with specific amino acid sequences. They can be used as markers for quality control of animal drugs and have the advantages of strong specificity, good stability and quantitative analysis. At present, the detection based on characteristic polypeptides can be applied to the identification of traditional Chinese medicine and has been included in the 2020 edition of the "Chinese Pharmacopoeia". The use of polypeptides for the identification of traditional Chinese medicine plays an important role. Focusing on the characteristic polypeptide components, an HPLC-MS method for the determination of the content of characteristic polypeptides is established, which can provide a scientific basis for the quality evaluation of sandworms. Therefore, the present invention will extract polypeptides from sandworms, establish a method for the determination of the content of sandworm polypeptides based on HPLC-MS, and provide a scientific basis for the quality research of sandworms. Summary of the Invention

[0004] The purpose of the present invention is to solve the above-mentioned background technical problems and provide a method for determining the content of Treptospira polypeptide.

[0005] In order to achieve the above purpose, the technical solutions adopted are: A method for determining the content of a Psoralea sp. polypeptide is characterized by comprising the following steps: first, using mass spectrometry to determine that the characteristic polypeptide sequences of Psoralea sp. are LIRF and LEI, respectively, and are named PSC-1 and PSC-2; (1) Preparation of the Sandworm Solution: Weigh the defatted Sandworms into a conical flask, add water and shake well, adjust the pH and add pepsin, heat in a 37°C water bath for enzymatic hydrolysis, then inactivate in a boiling water bath, cool to room temperature, centrifuge and collect the supernatant to obtain the Sandworm Solution; (2) Preparation of reference solution: Weigh the reference substances PSC-1 and PSC-2 of the Treptilophora spp. peptide reference substances respectively, dissolve them in ultrapure water to obtain the reference solution stock solution; (3) Establishment of a standard curve: Prepare the mother solution of the reference substance in step (2) into different mass concentrations and inject it into the LC-MS, export the mass spectrometry data, and use Skyline software for data analysis. Draw a standard curve with mass concentration as the horizontal axis and peak area as the vertical axis; (4) Content determination: According to the method and detection conditions of step (3), the solution of the crawling sandworm in step (1) was prepared and injected into the LC-MS, the mass spectrometry data was exported and imported into the Skyline software to calculate the content of PSC-1 and PSC-2 in the crawling sandworm solution.

[0006] Furthermore, in the step (1), 0.15 g of defatted Sandworms were weighed into a conical flask, 9 mL of water was added and shaken, the pH was adjusted to 2, protease was added so that the enzyme activity was 450 U / mL, and the solution was enzymatically hydrolyzed in a water bath at 37°C for 60 min, then taken out, and pepsin was inactivated in a boiling water bath for 10 min. After being placed at room temperature, the solution was centrifuged at 8000 rpm for 5 min, 100 μL of the supernatant was taken out and added to 900 μL of methanol, and the solution was centrifuged at 4°C and 12000 rpm for 15 min to precipitate large proteins, and then the solution was filtered through a 0.22 μm organic filter membrane to obtain the Sandworm solution.

[0007] Furthermore, in steps (2) and (3), 2 mg of PSC-1 and PSC-2 reference substances were weighed, respectively, dissolved in 2 mL of ultrapure water, ultrasonically shaken, and mixed to prepare a 1 mg / mL reference substance solution stock solution; the reference substance solution stock solution was further diluted to 20, 40, 80, 100, 200, 400, 800, and 1000 ng / mL.

[0008] Furthermore, in step (3) and step (4), the detection conditions include: Chromatographic conditions: Thermo Kinetex C18 column (2.1 × 150 mm, 2.6 μm); mobile phase A: 0.1% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile; flow rate, 0.2 mL / min; gradient elution: 0-0.5 min, 8% B, 1-8 min, 8%-70% B, 8-9.5 min, 70%-8% B, 9.5-10 min, 8% B; column temperature, 25°C; injection volume, 1 μL; Mass spectrometry conditions: Positive ion multiple reaction monitoring (MRM) mode was used: curtain gas 40 psi; ionization voltage 5500 V; ion source temperature 500°C; nebulizer gas 50 psi; auxiliary heater gas 50 psi. The reaction parameters for the Treptospira peptide are as follows: Analyte Precursor ion m / z Product m / z Precursor RT Collision energy (Volts) Declustering potential (Volts) PSC-1 548.2 401.2 5.06 39 100 PSC-2 374.2 243.3 5.07 20 20 .

[0009] Furthermore, in step (3) and step (4), the parameters of Skyline are set as follows: the mass spectrometry parameters Molecule List Name, Precursor Name, Precursor m / z, Precursor charge, Precursor RT, Precursor CE, Product m / z, and Product Charge of PSC-1 and PSC-2 are imported into Skyline software to establish a qualitative analysis method, and the mass spectrometry data in .wiff format are imported to establish a quantitative analysis method through a standard curve.

[0010] Furthermore, the ion pair information of the LIRF is 548.2→401.2; the ion pair information of the LEI is 374.2→243.3 The beneficial effects of adopting the above scheme are as follows: the present invention establishes a method for determining the content of polypeptide LIRF and polypeptide LEI in sandworm based on LC-MS technology. This method has the characteristics of easy synthesis of reference substances, simple operation, high sensitivity, and accurate determination. It can be used for quality evaluation research of sandworm and provide a reference for quality control of sandworm. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 It is the detection spectrum of PSC-1 in the content determination method of the Treptospira polypeptide of the present invention; Figure 2 It is the detection spectrum of PSC-2 in the content determination method of the Treptospira polypeptide of the present invention; Figure 3 It is a linear graph of LIRF content determination in the present invention; Figure 4 It is a linear graph for the determination of LEI content in the present invention. DETAILED DESCRIPTION

[0012] The technical solutions of the present invention are described clearly and completely below in conjunction with specific embodiments of the present invention. The described embodiments are only a portion of the embodiments of the present invention, not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative work are within the scope of protection of the present invention.

[0013] Example 1: LC-MS-based method for determining the content of Treptospira polypeptides 1 Materials and Methods 1.1 Instrument AB Qtrap 4000 high-performance liquid chromatography-mass spectrometry (AB SCIEX, USA); 1-16R desktop high-speed refrigerated centrifuge (Hunan Kecheng Instrument Equipment Co., Ltd.); LPD2500 multi-tube vortex mixer (Laput Scientific Instrument (Beijing) Co., Ltd.); FB2085 1 / 100,000 electronic balance (Shanghai Sunny Hengping Scientific Instrument Co., Ltd.); B-260 CNC constant temperature water bath (Shanghai Yarong Biochemical Instrument Factory).

[0014] 1.2 Drug testing The PSC-1 (LIRF) and PSC-2 (LEI) peptides of the creeping worm were further synthesized by Sangon Biotech (Shanghai) Co., Ltd. (purity > 98%).

[0015] The dried bodies of Psoralea corylifolia were purchased from the ecological breeding base in Yuxi City, Yunnan Province.

[0016] 1.3 Reagents Acetonitrile (mass spectrometry grade, Beijing J&K Technology Co., Ltd.), formic acid (mass spectrometry grade, Thermo Fisher Scientific (China) Co., Ltd.), methanol (mass spectrometry grade, Beijing J&K Technology Co., Ltd.), hydrochloric acid (analytical grade, Chongqing Chuandong Chemical (Group) Co., Ltd.), pepsin 1:10000 (Beijing Solebold Technology Co., Ltd.).

[0017] 1.4 Methods 1.4.1 Chromatographic conditions Thermo Kinetex C18 column (2.1 × 150 mm, 2.6 μm); mobile phase A: 0.1% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile; flow rate, 0.2 mL / min; gradient elution: 0-0.5 min, 8% B, 1-8 min, 8%-70% B, 8-9.5 min, 70%-8% B, 9.5-10 min, 8% B; column temperature, 25°C; injection volume, 1 μL.

[0018] 1.4.2 Mass spectrometry conditions Positive ion multiple reaction monitoring (MRM) mode: curtain gas 40 psi; ionization voltage 5500 V; ion source temperature 500°C; nebulizer gas 50 psi; auxiliary heater gas 50 psi. The reaction parameters for the peptides from Treptospira spp. and Mylabris blister beetles are as follows: Analyte Precursor ion m / z Product m / z Precursor RT Collision energy (Volts) Declustering potential (Volts) PSC-1 548.2 401.2 5.06 39 100 PSC-2 374.2 243.3 5.07 20 20 .

[0019] 1.4.3 Preparation of Sandworm Solution Weigh 0.15 g of defatted D. spp. in a conical flask, add 9 mL of water and shake well, adjust the pH to 2, add protease to an enzyme activity of 450 U / mL, and incubate in a water bath at 37°C for 60 min before removing the sample. Boil the sample in a boiling water bath for 10 min to inactivate pepsin. After standing at room temperature, centrifuge at 8000 rpm for 5 min, remove 100 μL of the supernatant and add it to 900 μL of methanol. Centrifuge at 4°C and 12000 rpm for 15 min to precipitate large proteins, then remove the sample and filter through a 0.22 μm organic filter membrane to obtain the D. spp. solution. 1.4.4 Preparation of reference solution Accurately weigh the PSC-1 (LIRF) and PSC-2 (LEI) reference substances, dissolve them in ultrapure water, ultrasonically oscillate, and shake to obtain single reference substance stock solutions with concentrations of 1.00 mg / mL and 1.04 mg / mL, respectively. The reference substance stock solutions were diluted with methanol to prepare mixed reference substance solutions of 20, 40, 80, 100, 200, 400, 800, and 1000 ng / mL, and filtered through a 0.22 μm microporous filter before injection.

[0020] 2 Methodological research (1) Repeatability study Prepare the solution of Psoralea sp. using the method described in 1.4.3. Prepare six replicates, inject them into the LC-MS for analysis, and record the chromatographic peaks and peak areas. Calculate the relative standard deviation (RSD) of the peak areas for the Psoralea sp. peptides PSC-1 and PSC-2. The RSD values for both peptides were less than 5%, indicating that both methods established in this study have good reproducibility. (2) Stability investigation Prepare the Psoralea sp. solution according to the method described in 1.4.3. Prepare six replicates and inject them into LC-MS for analysis at 0, 4, 12, 24, and 48 hours. Record the peak area for each group and calculate the relative standard deviation (RSD) of the peak area for the Psoralea sp. peptides PSC-1 and PSC-2 over 48 hours. The RSD values for both peptides were less than 5%, indicating that the PSC-1 and PSC-2 peptides were stable over 48 hours.

[0021] (3) Instrument precision inspection Take the mixed reference solution of PSC-1 and PSC-2 peptides from 1.4.4 and inject it six times in succession. Check the RSD values of the peak areas. If the RSD values are all less than 5%, it indicates good instrument stability.

[0022] (4) Linearity and range Take the mixed reference solution of PSC-1 and PSC-2 peptides from 1.4.4 and dilute it with methanol solution to obtain mixed solutions of different mass concentrations. Inject them into LC-MS for determination and draw standard curves with the mass concentration of PSC-1 and PSC-2 peptides as the abscissa (X) and the peak area as the ordinate (Y).

[0023] Table 1 Regression equation and linear range of peptide component content .

[0024] (5) Detection limit and quantification limit The standard solution from 1.4.4 was diluted gradually and injected into the LC-MS for analysis. The mass spectrometer software performed S / N analysis. The detection limit was determined to be a signal-to-noise ratio (S / N) ≥ 3, and the quantification limit was determined to be a signal-to-noise ratio (S / N) ≥ 10. For the PSC-1 standard solution, the detection limit (S / N = 4.2) was 15 ng / mL, and the quantification limit (S / N = 11.5) was 20 ng / mL. For the PSC-2 standard solution, the detection limit (S / N = 6.1) was 0.1 ng / mL, and the quantification limit (S / N = 28.9) was 20 ng / mL. (6) Spike recovery test Weigh 0.15 g of defatted powder of Sandworm, and weigh 9 portions in parallel and place them in conical flasks. Add the standard peptide solution prepared at 1.0 mg / mL under 1.4.4 of this chapter at a ratio of 50%, 100% and 150% of the PSC-1 and PSC-2 contents in the samples. Prepare the Sandworm solution according to 1.4.3 of this chapter and inject it into LC-MS for analysis and determination. The average recovery rate of Sandworm peptides is between 95.77% and 104.37%, and the RSD% is less than 5%, indicating that the recovery rate of this method is good.

[0025] Table 2 Recovery results of peptide components from Treptospira spp.

[0026] Systematic methodological studies have shown that the linear relationships of the Treptospira peptides are all greater than 0.99, indicating a good linear relationship. The repeatability, stability, and instrument precision of this method all meet the requirements of relevant methodologies. The spiked recovery rate of this method is between 95% and 105%, indicating that this method can be used for sample content determination.

[0027] like Figure 1 It is the detection spectrum of PSC-1 in the content determination method of the Treptospira polypeptide of the present invention; Figure 2 is the detection spectrum of PSC-2; Figure 3 is the linear graph of LIRF content determination; Figure 4 This is the linear graph of LEI content determination.

[0028] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations that come within the meaning and range of equivalents of the claims be embraced therein.

[0029] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A method for determining the content of a Treptospira polypeptide, characterized in that: The following steps are involved: First, mass spectrometry was used to determine the characteristic peptide sequences of the worm, LIRF and LEI, which were named PSC-1 and PSC-2. (1) Preparation of the Sandworm Solution: Weigh the defatted Sandworms into a conical flask, add water and shake well, adjust the pH and add pepsin, heat in a 37°C water bath for enzymatic hydrolysis, then inactivate in a boiling water bath, cool to room temperature, centrifuge and collect the supernatant to obtain the Sandworm Solution; (2) Preparation of reference solution: Weigh the reference substances PSC-1 and PSC-2 of the Treptilophora spp. peptide reference substances respectively, dissolve them in ultrapure water to obtain the reference solution stock solution; (3) Establishment of a standard curve: Prepare the mother solution of the reference substance in step (2) into different mass concentrations and inject it into the LC-MS, export the mass spectrometry data, and use Skyline software for data analysis. Draw a standard curve with mass concentration as the horizontal axis and peak area as the vertical axis; (4) Content determination: According to the method and detection conditions of step (3), the solution of the crawling sandworm in step (1) was prepared and injected into the LC-MS, the mass spectrometry data was exported and imported into the Skyline software to calculate the content of PSC-1 and PSC-2 in the crawling sandworm solution.

2. the content assay method of Treptospira polypeptide according to claim 1, is characterized in that: In the step (1), 0.15 g of defatted sandworms were weighed and placed in a conical flask, 9 mL of water was added and shaken, the pH was adjusted to 2, protease was added so that the enzyme activity was 450 U / mL, and the mixture was enzymatically hydrolyzed in a water bath at 37°C for 60 min, then taken out, and pepsin was inactivated in a boiling water bath for 10 min. After standing at room temperature, the mixture was centrifuged at 8000 rpm for 5 min, 100 μL of the supernatant was taken out and added to 900 μL of methanol, and the mixture was centrifuged at 4°C and 12000 rpm for 15 min to precipitate large proteins, and then the mixture was taken out and filtered through a 0.22 μm organic filter membrane to obtain a sandworm solution.

3. the content assay method of Treptospira polypeptide according to claim 1, is characterized in that: In steps (2) and (3), 2 mg of PSC-1 and PSC-2 reference substances were weighed, respectively, dissolved in 2 mL of ultrapure water, ultrasonically shaken, and mixed to prepare a 1 mg / mL reference substance solution stock solution; the reference substance solution stock solution was further diluted to 20, 40, 80, 100, 200, 400, 800, and 1000 ng / mL.

4. the content assay method of Treptospira polypeptide according to claim 1, is characterized in that: In step (3) and step (4), the detection conditions include: Chromatographic conditions: Thermo Kinetex C18 column (2.1 × 150 mm, 2.6 μm); mobile phase A: 0.1% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile; flow rate, 0.2 mL / min; gradient elution: 0-0.5 min, 8% B, 1-8 min, 8%-70% B, 8-9.5 min, 70%-8% B, 9.5-10 min, 8% B; column temperature, 25°C; injection volume, 1 μL; Mass spectrometry conditions: Positive ion multiple reaction monitoring (MRM) mode was used: curtain gas 40 psi; ionization voltage 5500 V; ion source temperature 500°C; nebulizer gas 50 psi; auxiliary heater gas 50 psi. The reaction parameters for the Treptospira peptide are as follows: 。 5. The method for determining the content of the Treptospira polypeptide according to claim 1, wherein In step (3) and step (4), the parameters of Skyline are set as follows: the mass spectrometry parameters Molecule List Name, Precursor Name, Precursor m / z, Precursor charge, Precursor RT, Precursor CE, Product m / z, and Product Charge of PSC-1 and PSC-2 are imported into Skyline software to establish a qualitative analysis method, and the mass spectrometry data in .wiff format are imported to establish a quantitative analysis method using a standard curve.

6. The method for determining the content of the Treptospira polypeptide according to claim 1, wherein The ion pair information of the LIRF is 548.2→401.2; the ion pair information of the LEI is 374.2→243.3.

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