Kit and method for detecting pepsin based on polypeptide

Abstract

The invention discloses a kit and a method for detecting pepsin based on polypeptide, and belongs to the technical field of proteomics. The amino acid sequence of the polypeptide is as shown in SEQ ID No. 1. The polypeptide is obtained based on enzymolysis of pepsinogen PGA4 and screening, the polypeptide and isotope labeled polypeptide are utilized to establish a standard curve, a biological sample is further subjected to enzymolysis to obtain a peptide fragment sample, and pepsin in the biological sample can be quantified by detecting the content of the polypeptide in the peptide fragment sample. By utilizing the method disclosed by the invention, the pepsin in the biological sample can be rapidly quantified, so that pathological reflux and physiological reflux are diagnosed and distinguished, and the accuracy rate is high.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of proteomics, and particularly relates to a kit and method for detecting pepsin based on polypeptides. BACKGROUND

[0002] Salivary pepsin detection is a non-invasive examination, which assists in diagnosing gastroesophageal reflux disease (GERD) or laryngopharyngeal reflux (LPR) by analyzing the concentration of pepsin in saliva. The core principle is that pepsin is secreted by the stomach, and if it refluxes into the esophagus or throat, it may enter the saliva; the detection of the enzyme indicates the presence of abnormal reflux, which is further combined with symptoms and other examinations for comprehensive judgment.

[0003] At present, the methods for detecting salivary pepsin mainly include immunochromatography (colloidal gold method), enzyme-linked immunosorbent assay (ELISA) and new probe technology.

[0004] However, laryngopharyngeal reflux is intermittent, the concentration of pepsin in saliva fluctuates greatly, the existing technology has a low detection rate for samples of <10 ng / mL, and salivary mucin, food residues and the like easily lead to false negatives / positives, which is particularly obvious in the colloidal gold method. SUMMARY

[0005] In order to solve at least one of the above technical problems, the technical scheme adopted by the present application is as follows.

[0006] The first aspect of the present application provides an application of a polypeptide in the preparation of a kit for detecting pepsin in a biological sample, wherein the amino acid sequence of the polypeptide is shown in SEQ ID No. 1.

[0007] In some embodiments of the present application, the polypeptide is labeled with or without isotopes.

[0008] In some embodiments of the present application, the polypeptide labeled with isotopes is used as an internal standard, and the polypeptide not labeled with isotopes is used as a standard for making a standard curve, so as to perform quantitative detection.

[0009] In some embodiments of the present application, the isotope includes 13 C and / or 15 N.

[0010] In some embodiments of the present application, the labeling site of the isotope is lysine.

[0011] In some embodiments of the present application, the biological sample is saliva.

[0012] The second aspect of the present application provides a kit for detecting pepsin in a biological sample, comprising a polypeptide with an amino acid sequence as shown in SEQ ID No. 1, and an isotopically labeled polypeptide.

[0013] In some embodiments of the present application, the kit is based on LC-MS / MS detection, and further comprises a mobile phase reagent.

[0014] In some embodiments of the present application, the mobile phase comprises liquid A and liquid B, liquid A is 0.1% formic acid aqueous solution, and liquid B is acetonitrile solution containing 0.1% formic acid.

[0015] In some embodiments of the present application, the volume concentration of the acetonitrile solution is 80% to 90%.

[0016] The third aspect of the present application provides a method for detecting pepsin in a biological sample based on a polypeptide, wherein the amino acid sequence of the polypeptide is as shown in SEQ ID No. 1, and the method comprises the following steps: S1, protein extraction: extracting a protein sample from a biological sample; S2, proteolysis: using a protease to digest the protein sample to obtain a peptide sample; S3, peptide desalination: removing impurities in the peptide sample by chromatography; S4, mixing a part of the peptide sample after removing impurities with an isotopically labeled polypeptide, performing LC-MS / MS analysis, using a standard curve prepared based on the polypeptide and the isotopically labeled polypeptide to obtain the concentration of the polypeptide in the peptide sample, thereby obtaining the concentration of pepsin in the biological sample.

[0017] In the above method, protein extraction and proteolysis are critical.

[0018] For different types of biological samples, the processing method of protein extraction is different, as follows: For cell samples, a lysis solution containing enzyme inhibitors is usually added in a low temperature environment, followed by ultrasonic or homogenization treatment to make it homogeneous.

[0019] For urine samples, proteins are extracted through solvent precipitation, ultrafiltration, centrifugation, dialysis and freeze-drying, which are familiar to those skilled in the art.

[0020] For blood samples, plasma or serum is usually collected by centrifugation.

[0021] For tissue samples, pre-cooled PBS is usually used for flushing to remove blood and fat. After washing, tissue disruption is performed by liquid nitrogen grinding or homogenization, followed by the addition of RIPA lysis buffer with ultrasonic-assisted lysis. After lysis, unlysed tissue components and other impurities such as connective tissue can be removed by centrifugation to obtain the supernatant.

[0022] Enzymolysis refers to the decomposition of proteins into peptide segments under the action of proteases. Before enzymolysis, a reducing agent (including but not limited to DTT and TCEP) is usually added first to open the disulfide bond, and an alkylating agent such as iodoacetamide (IAA) or chloroacetamide (CAA) is used to block the free thiol group, thereby further destroying the protein secondary structure and improving the enzymolysis efficiency.

[0023] In some embodiments of the present application, the protease is selected from one of chymotrypsin, trypsin, endoproteinase Lys-C, endoproteinase Glu-C and endoproteinase Asp-N. In some specific embodiments of the present application, the protease is trypsin.

[0024] In some embodiments of the present application, the mobile phase for LC-MS / MS analysis includes A liquid and B liquid, the A liquid is 0.1% formic acid aqueous solution, and the B liquid is acetonitrile solution containing 0.1% formic acid.

[0025] In some embodiments of the present application, the relevant liquid gradient is: 0~1min, B liquid maintains at 5%; 1~6min, B liquid linearly changes from 5% to 99%; 6~8min, B liquid maintains at 99%; 8~8.1min, B liquid linearly changes from 99% to 5%; 8.1~10min, B liquid maintains at 5%.

[0026] In some embodiments of the present application, the preparation method of the standard curve is: Different masses of the polypeptide are dissolved in 50% methanol aqueous solution to obtain standard solution with gradient concentration (for example, 0.95 pmol / mL, 1.90 pmol / mL, 4.74 pmol / mL, 9.48 pmol / mL, 18.96 pmol / mL, 47.41 pmol / mL, 94.82 pmol / mL, 474.12 pmol / mL, 948.24 pmol / mL), and the same mass of isotope-labeled polypeptide is added, and placed in a 4°C automatic sampler, with a column temperature of 40°C, a flow rate of 300 µL / min, and a sample injection amount of 5 µL.

[0027] The relevant liquid phase gradient is as follows: 0-1 min, B liquid is maintained at 5%; 1-6 min, B liquid is linearly changed from 5% to 99%; 6-8 min, B liquid is maintained at 99%; 8-8.1 min, B liquid is linearly changed from 99% to 5%; 8.1-10 min, B liquid is maintained at 5%.

[0028] Mass spectrometry is performed by using a QTRAP5500 mass spectrometer (AB SCIEX) in a positive ion mode. The 5500 QTRAP ESI source parameters are as follows: Positive ion mode: Source Temperature (source temperature): 500℃, Ion Source Gas1 (GAS1, spray gas): 40, Ion Source Gas2 (GAS2, heating gas): 50, Curtain Gas (CUR, gas curtain gas): 35, Ion Spray Voltage Floating (ISVF, ion spray voltage floating): 5500V. The multiple reaction monitoring (MRM) mode is used to detect the ion pair to be detected.

[0029] The standard curve is drawn by using the concentration of the polypeptide and the ratio of the peak area of the polypeptide to the peak area of the isotopically labeled polypeptide.

[0030] In the present application, the amount of isotopically labeled polypeptide added is consistent when preparing the standard curve and in the detection of biological samples.

[0031] In the detection of biological samples, by obtaining the ratio of the peak area of the polypeptide to the peak area of the isotopically labeled polypeptide, the concentration of the polypeptide in the peptide segment sample on the machine can be obtained by using the standard curve. By using the concentration, the concentration of pepsin in the biological sample can be obtained: Pepsin content in the biological sample = [concentration of the polypeptide * volume on the machine] / (peptide segment sampling ratio * original biological sample volume) Peptide segment sampling ratio = on-machine peptide segment mass / total mass of sample peptide segments Compared with the prior art, the present application has the following beneficial effects: The polypeptide in the present application is based on the enzyme digestion of pepsinogen PGA4 and is screened, and is targeted, the standard curve is established by using the polypeptide and the isotopically labeled polypeptide, and the peptide segment sample is further obtained by enzyme digestion of the biological sample, and the content of the polypeptide in the peptide segment sample is detected, so that the pepsin in the biological sample can be quantified. By using the method of the present application, the pepsin in the biological sample can be quickly quantified, and the accuracy is high.

[0032] By using the kit and the method of the present application, the concentration of pepsin is quantitatively determined when detecting pepsin, so as to distinguish physiological and pathological reflux.

[0033] The kit and method of the present application have high detection rate and sensitivity, and are simple to operate, and are suitable for further promotion in clinic.

[0034] It should be understood that the matters described in this section are not intended to identify key or important features of the embodiments of the present application, nor are they used to limit the scope of the present application. Other features of the present application will become apparent from the following description. BRIEF DESCRIPTION OF DRAWINGS

[0035] The above and other objects, features and advantages of the example embodiments of the present application will be more apparent from the following detailed description read in conjunction with the accompanying drawings, in which several embodiments of the present application are shown by way of example, and in which: Figure 1 A secondary mass spectrum matching diagram of the target peptide segment screened in Example 1 of the present application is shown, in which b2 represents a fragment ion (daughter ion) composed of 2 amino acid residues from the N-terminal of the target peptide segment, i.e., the b2 daughter ion is composed of 2 amino acid residues A and N, b2-NH3 represents the ion after the b2 daughter ion loses one molecule of ammonia, b3 to b8 and b3-NH3 to b8-NH3 are the same, y2 represents a fragment ion (daughter ion) composed of 2 amino acid residues from the C-terminal of the target peptide segment, i.e., the y2 daughter ion is composed of 2 amino acid residues V and A, y3, y4 are the same, and so on; Figure 2 A standard curve constructed in Example 2 of the present application is shown. DETAILED DESCRIPTION

[0036] Unless otherwise stated, implied from context, or according to the practice in the art, all parts and percentages in the present application are based on weight, and the test and characterization methods used are contemporary with the filing date of the present application. The contents of any patents, patent applications, or publications mentioned in the present application are hereby incorporated by reference in their entirety, and equivalent homologous patents in other countries are also incorporated by reference, especially the definitions of relevant terms disclosed in these documents. If the definition of a specific term disclosed in the prior art is inconsistent with any definition provided in the present application, the definition provided in the present application shall prevail.

[0037] In order to make the technical problems, technical solutions and beneficial effects solved by the present application more clear and explicit, the present application is further described in detail below in combination with examples.

[0038] The following examples are used to illustrate preferred embodiments of this application. Those skilled in the art will understand that the techniques disclosed in the examples represent technologies discovered by the inventors that can be used to implement this application, and therefore can be considered preferred embodiments of this application. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed herein, still yielding the same or similar results, without departing from the spirit or scope of this application.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains, and all materials cited herein and referenced by them are incorporated herein by reference.

[0040] Those skilled in the art will recognize, or can learn through routine experimentation, many equivalents of the specific embodiments of the invention described herein. These equivalents will be included in the claims.

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0042] Example 1: Acquisition and Screening of Peptides 1. Enzymatic hydrolysis of pepsinogen standards and desalting of peptides Take an appropriate amount of pepsinogen PGA4 standard (Pepsin (PGA4)(NM 001079808) Human Recombinant Protein, product code: TP319574, brand: origene), add DTT to a final concentration of 100 mM, incubate at 37°C for 2.5 h, and cool to room temperature. Add IAA to a final concentration of 50 mM, incubate in the dark for 30 min. Add 5 volumes of 100 mM NH4HCO3, and precipitate the protein using TCA / acetone. Reconstitute the protein precipitate with 8 M urea, dilute 10-fold with 50 mM NH4HCO3, and then add trypsin at a 1:50 ratio, and enzymatically hydrolyze at 37°C for 18 h. Desalt the hydrolyzed peptides using an SPE C18 column (WatersWAT051910) and lyophilize.

[0043] 2. LC-MS / MS An appropriate amount of peptide was taken and separated by chromatography using an Easy nLC 1200 chromatography system (Thermo Scientific) with a flow rate of nanoliters.

[0044] Buffer: A is 0.1% formic acid in water, B is 0.1% formic acid in acetonitrile (85% acetonitrile by volume).

[0045] The column was equilibrated with 95% A, and the sample was injected and separated by a gradient on a column (75 µm x 200 mm, 3 µm, C18, Dr. Maisch GmbH) at a flow rate of 300 nL / min.

[0046] The gradient for liquid separation was as follows: 0-2 min, B linearly from 2% to 8%; 2-45 min, B linearly from 8% to 28%; 45-50 min, B linearly from 28% to 40%; 50-52 min, B linearly from 40% to 100%; 52-60 min, B maintained at 100%.

[0047] After the separation of the peptide fragments, DDA (data-dependent acquisition) mass spectrometry was performed using a Q-Exactive HF-X mass spectrometer (Thermo Scientific). The analysis time was 60 min, and the detection mode was positive ion, the parent ion scanning range was 350-1800 m / z, the first mass resolution was 60,000 @ m / z 200, the AGC target (automatic gain control target) was 3e6, and the first Maximum IT (maximum injection time) was 50 ms.

[0048] The secondary mass spectrometry analysis of the peptide fragments was collected according to the following method: after each full scan (full scan), the secondary mass spectrum of 20 highest intensity parent ions (MS2 scan) was collected, the secondary mass resolution was 45,000 @ m / z 200, the AGC target was 1e5, the secondary Maximum IT was 50 ms, the MS2 Activation Type (secondary mass spectrum activation type) was HCD (high-energy collision-induced dissociation), the Isolation window (isolation window) was 1.2 m / z, and the Normalized collision energy (normalized collision energy) was 32.

[0049] 3. Database retrieval The final LC-MS / MS raw RAW file was imported into the MaxQuant software (version number 2.0.1.0) database retrieval, and the database used was: BP20231405-Traget.fasta.

[0050] A total of 9 pepsinogen peptides were obtained, and the relevant information is shown in Table 1.

[0051] Table 1: Pepsinogen peptide information

[0052] Among them, after multiple tests, the peptide segment shown in SEQ ID No. 1 is ionized well and easy to be detected by mass spectrometry. In addition, the peptide segment has no enzyme leakage site, low peptide segment matching error rate (<0.001), high peptide segment score (125.25), and high mass spectrometry intensity value (1012900000), so it is selected as the target peptide segment. The secondary mass spectrometry matching diagram thereof is shown in Figure 1

[0053] Example 2: Establishing a standard curve using a target peptide segment as a standard 1. Peptide segment synthesis ANNQVGLAPVA is the target peptide segment screened in Example 1. In this embodiment, two target peptide segments, one non-heavy labeled peptide segment (light labeled peptide) and one heavy labeled peptide segment (heavy labeled peptide), are synthesized, which are: ANNQVG-L-APVA and ANNQVG-L (C6) -APVA, i.e. the light labeled peptide is a polypeptide without isotopic labeling, and the heavy labeled peptide is labeled on lysine using C6 and N. 13 C6 15 N. 13 C and 15 N.

[0054] 2. Ion pair mass spectrometry condition optimization The two synthesized peptide segments are prepared into a standard solution with a concentration of 9.48 nmol / mL. Using a QTRAP5500 mass spectrometer, the parent ion and daughter ion of the two synthesized peptides are optimized in positive ion mode for declustering potential and collision energy parameters, and the optimal mass spectrometry on-machine conditions are finally selected for standard curve mass spectrometry on-machine.

[0055] The selected target peptide segment is tested for ion pair optimization. The specific ion pair selection and results are shown in Table 2.

[0056] Table 2: Target peptide segment ion pair optimization

[0057] The final ion pair optimization results are shown in Table 2 (rows 4 and 7 in bold). The standard curve is finally established under this mass spectrometry condition.

[0058] 3. Standard curve establishment ​Shimadzu Nexera X2 A8-30AD high performance liquid chromatography was used. The chromatographic column was Waters Acquity UPLC BEH C18 column (1.7 µm, 2.1 × 100 mm Column). The mobile phase was A liquid of 0.1% formic acid aqueous solution, B liquid of acetonitrile solution containing 0.1% formic acid (the volume concentration of acetonitrile was 85%).

[0059] Different mass light standard peptides were dissolved in 50% methanol aqueous solution to obtain standard solution with concentrations of 0.95 pmol / mL, 1.90 pmol / mL, 4.74 pmol / mL, 9.48 pmol / mL, 18.96 pmol / mL, 47.41 pmol / mL, 94.82 pmol / mL, 474.12 pmol / mL, and 948.24 pmol / mL, and the same mass of heavy standard peptides was added (the final concentration was 470.99 pmol / mL), and placed in a 4°C automatic sampler, the column temperature was 40°C, the flow rate was 300 µL / min, and the injection volume was 5 µL.

[0060] The relevant liquid gradient was as follows: 0~1 min, B liquid maintained at 5%; 1~6 min, B liquid linearly changed from 5% to 99%; 6~8 min, B liquid maintained at 99%; 8~8.1 min, B liquid linearly changed from 99% to 5%; 8.1~10 min, B liquid maintained at 5%.

[0061] Mass spectrometry was performed by QTRAP5500 mass spectrometer (AB SCIEX) in positive ion mode. The 5500 QTRAP ESI source parameters were as follows: Positive ion mode: Source Temperature (source temperature): 500°C, Ion Source Gas1 (GAS1, spray gas): 40, Ion Source Gas2 (GAS2, heating gas): 50, Curtain Gas (CUR, gas curtain gas): 35, Ion Spray Voltage Floating (ISVF, ion spray voltage floating): 5500V. The multiple reaction monitoring (MRM) mode was used to detect the ion pairs to be detected.

[0062] The detection of standard solutions with different concentrations is shown in Table 3: Table 3: Establishment of standard curve

[0063] Wherein, Ratio represents the ratio of the peak area of light standard peptide to the peak area of heavy standard peptide. The standard curve was drawn according to the concentration of standard solution and Ratio, as Figure 2The equation of the standard curve is y = a - b * x, where y represents Ratio; x represents the concentration of the standard solution; a represents the standard curve intercept, with a value of -0.00496 ± 0.0085; b represents the standard curve slope, with a value of 0.00218 ± 2.39353E-05. The Pearson correlation coefficient is 0.99958, the correlation R 2 is 0.99916, and the adjusted R 2 is 0.99904.

[0064] Quantitative detection of actual samples Three saliva samples were obtained, designated as PGA4-1, PGA4-2, and PGA4-3, respectively.

[0065] For each original saliva sample, 100 µL was taken and lyophilized, and then 50 µL of UA lysis solution was added, transferred to an EP tube, homogenized using a homogenizer, incubated at 37°C for 30 min, and subjected to protein quantification using the BCA method.

[0066] All proteins were taken and added with an appropriate amount of 1M DTT to a final concentration of 100mM, incubated at 37°C for 30 min, and cooled to room temperature. Then an appropriate amount of IAA was added to each sample to a final concentration of 50mM, and incubated at room temperature in the dark for 30 min. 50mM NH4HCO3 was added to each sample to dilute 6 times, then 2µg Trypsin was added, shaken at 600rpm for 1 min, and incubated at 37°C for 20h. After enzymolysis, the sample was acidified with an appropriate amount of TFA to terminate the enzymolysis. Then C18 Cartridge was used for desalting treatment of the enzymolyzed peptides, and finally OD280 was used for peptide quantification. The protein and peptide quantification information of each sample is shown in Table 4.

[0067] Table 4: Protein and peptide quantification information of each sample

[0068] The peptides of the three samples were subjected to LC-MS / MS analysis using Shimadzu Nexera X2 A8-30AD high performance liquid chromatography. The chromatographic column was Waters Acquity UPLC BEH C18 column (1.7µm, 2.1×100mm Column).

[0069] The mobile phase was A liquid (0.1% formic acid aqueous solution) and B liquid (0.1% formic acid acetonitrile solution).

[0070] Each sample of the same quality (1.875 μg) of peptide segment was reconstituted with 10 μL of 50% methanol aqueous solution, 10 μL of heavy peptide (final concentration 470.99 pmol / mL) was added, and the sample was placed in a 4°C automatic injector, the column temperature was 40°C, the flow rate was 300 μL / min, and the injection volume was 15 μL.

[0071] The relevant liquid gradient was as follows: 0~1 min, B liquid maintained at 5%; 1~6 min, B liquid linearly changed from 5% to 99%; 6~8 min, B liquid maintained at 99%; 8~8.1 min, B liquid linearly changed from 99% to 5%; 8.1~10 min, B liquid maintained at 5%.

[0072] Mass spectrometry was performed by using a QTRAP5500 mass spectrometer (AB SCIEX) in positive ion mode. The 5500 QTRAP ESI source parameters were as follows: Positive ion mode: Source Temperature (source temperature): 500°C, Ion Source Gas1 (GAS1, spray gas): 40, Ion Source Gas2 (GAS2, heating gas): 50, Curtain Gas (CUR, gas curtain gas): 35, Ion Spray Voltage Floating (ISVF, ion spray voltage floating): 5500V. The multiple reaction monitoring (MRM) mode was used to detect the ion pairs to be detected.

[0073] The peak area ratio of the target peptide to the heavy peptide in each sample was substituted into the standard curve obtained in Example 2 to calculate the loading concentration of the target peptide in each sample, and then the content of the target PGA4 protein in the initial sample was finally obtained according to the reconstitution volume.

[0074] In this example, the target peptide quantitative values of each saliva sample are shown in Table 5.

[0075] Table 5: Absolute quantification results of saliva samples

[0076] Saliva PGA4 protein content = [target peptide concentration * injection volume (15 μL)] / (peptide segment sampling ratio * original saliva volume) Peptide segment sampling ratio = injection peptide segment mass (1.875 μg) / total sample peptide segment mass Example 4: Application of pepsin quantitative detection Ten clinical symptomless physiological reflux patient saliva and 10 pathological reflux patient saliva were obtained from the Second Affiliated Hospital of Chongqing Medical University Infection Department, and based on the method of Example 2, the quantitative detection results of pepsin were obtained. The pepsin quantitative concentration of the detection sample was not greater than 50 ng / mL, which was determined as physiological reflux. The pepsin quantitative concentration of the detection sample was greater than 50 ng / mL, which was determined as pathological reflux.

[0077] The pepsin semi-quantitative detection test strip (prepared according to the method disclosed in the Chinese invention patent publication CN113049825A “Semi-quantitative pepsin detection product”) was used for detection as a comparison. The pepsin semi-quantitative concentration of the detection sample was 0 ng / mL, less than 5 ng / mL, 5 ng / mL, 10 ng / mL or 25 ng / mL, which was determined as physiological reflux. The pepsin semi-quantitative concentration of the detection sample was 50 ng / mL, 100 ng / mL, 200 ng / mL or greater than 200 ng / mL, which was determined as pathological reflux.

[0078] The detection comparison results are shown in Table 6.

[0079] Table 6: Clinical sample detection results

[0080] As can be seen from Table 6, based on the method of Example 2, the quantitative concentration of pepsin in the saliva sample can be accurately obtained compared with semi-quantitative detection.

[0081] In addition, it should be understood that, after reading the above teachings of the present application, those skilled in the art can make various modifications or changes to the present application, and these equivalent forms also fall within the scope defined by the claims of the present application.

Claims

1. Use of a polypeptide for the preparation of a kit for the detection of pepsin in a biological sample, characterized in that, The amino acid sequence of the polypeptide is shown as SEQ ID No.

1.

2. Use according to claim 1, characterized in that, The polypeptide is labeled with or without isotope.

3. Use according to claim 2, characterized in that, The polypeptide labeled with isotope is used as an internal standard, and the polypeptide not labeled with isotope is used as a standard sample to prepare a standard curve, so as to perform quantitative detection.

4. Use according to claim 2 or 3, characterized in that, The isotopes include 13 C and / or 15 N.

5. Use according to claim 4, characterized in that, The labeling site of the isotope is lysine.

6. A kit for detecting pepsin in a biological sample, characterized by, The polypeptide comprising the amino acid sequence shown as SEQ ID No. 1 also comprises the isotope-labeled polypeptide.

7. The kit of claim 6, wherein The kit is based on LC-MS / MS detection and further comprises mobile phase reagents.

8. A method for detecting pepsin in a biological sample based on a polypeptide, characterized by, The amino acid sequence of the polypeptide is shown as SEQ ID No. 1, and the method comprises the following steps: S1, protein extraction: extracting a protein sample of a biological sample; S2, protein digestion: using a protease to digest the protein sample to obtain a peptide sample; S3, peptide desalting: removing impurities in the peptide sample by chromatography; S4, mixing part of the peptide sample after removing impurities with the isotope-labeled polypeptide to perform LC-MS / MS analysis, using a standard curve prepared based on the polypeptide and the isotope-labeled polypeptide to obtain the concentration of the polypeptide in the peptide sample, so as to obtain the concentration of pepsin in the biological sample.

9. The method of claim 8, wherein, The mobile phase for LC-MS / MS analysis comprises A liquid and B liquid, the A liquid is 0.1% formic acid aqueous solution, and the B liquid is acetonitrile solution containing 0.1% formic acid.

10. The method of claim 9, wherein, The related liquid gradient is as follows: 0~1min, B liquid maintains at 5%; 1~6min, B liquid linearly changes from 5% to 99%; 6~8min, B liquid maintains at 99%; 8~8.1min, B liquid linearly changes from 99% to 5%; 8.1~10min, B liquid maintains at 5%.

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