A method for detecting disease-characteristic polypeptide markers based on endogenous polypeptides

Abstract

The present invention relates to a detection method for disease characteristic polypeptide markers based on endogenous polypeptides, and belongs to the field of biomedicine technology. The present invention separates and enriches endogenous peptides from samples by using magnetic copper ion mesoporous dopamine material as a solid phase adsorbent; MALDI-TOF / TOFMS analysis is performed on the separated and enriched products; peak extraction and normalization are performed on the mass spectrum, and orthogonal partial least squares discriminant analysis and heat map analysis are performed to obtain characteristic polypeptide markers for detecting diseases; nano-LC-MS / MS analysis is then performed on the separated and enriched products; the mass-to-charge ratio of the characteristic polypeptide marker is matched with the polypeptide identified by nano-LC-MS / MS analysis to determine the amino acid sequence of the characteristic polypeptide marker. The method provided by the present invention has high sensitivity, low cost, high throughput, and low sample consumption, and has broad application prospects in large-scale population screening, disease diagnosis, and prognosis evaluation.

Description

Technical Field

[0001] The present invention relates to a method for detecting disease characteristic polypeptide markers based on endogenous polypeptides, and belongs to the technical field of biomedicine. Background Art

[0002] Omics-driven disease diagnosis and monitoring are new approaches to clinical diagnosis that have attracted widespread attention over the past few decades. However, sequencing-based genomics and transcriptomics disease detection is costly, while LC-MS-based proteomics and metabolomics disease detection is time-consuming. Neither approach is optimal for personalized disease monitoring in the era of precision medicine. Generally speaking, rapid testing of large-scale clinical samples is an important prerequisite for screening specific molecular features in precision medicine. In this regard, matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) has attracted much attention due to its rapid and high-throughput detection capabilities.

[0003] The peptidome, a low-molecular-weight protein group, is widely distributed in serum and closely correlates with the state of an organism. Numerous studies have shown that peptides are associated with the onset and progression of disease, and some studies have suggested that peptides can serve as prospective disease biomarkers. For example, serum amyloid A-2, serum amyloid A-1, and C-reactive protein can monitor COVID-19. Fibrinogen α chain (605-629, m / z 2660), α-trypsin inhibitor heavy chain H4 (347-356, m / z 1061), and apolipoprotein A-II (43-52, m / z 1041) can serve as serum peptide biomarkers for breast cancer. Recently, MALDI-TOF MS has been initially applied to disease diagnosis based on endogenous peptide analysis, successfully enabling the classification of healthy individuals from diseased patients. However, due to the low abundance of endogenous peptides and the complexity of actual samples, current work has not yet achieved comprehensive disease monitoring. Therefore, it is necessary to develop functionalized nanomaterials to enrich endogenous peptides and improve detection sensitivity.

[0004] Among the many functionalized nanomaterials, copper ion materials can achieve comprehensive enrichment of peptides due to their excellent coordination ability with the carboxyl and amino groups of peptides. Dopamine, which has a large number of catechol functional groups, can be polymerized under mild conditions and adhere to the surface of almost any material. In addition, polydopamine can be adjusted to a highly open pore structure through a soft template method, and has the ability to fully utilize its own functional groups for ion chelation, thereby fixing a large number of copper ions on the material surface. Therefore, it is necessary to combine the material advantages of copper ions and mesoporous polydopamine to prepare a probe with excellent peptide coordination ability to facilitate the efficient identification of endogenous peptides.

[0005] Membranous nephropathy (MN) is an autoimmune glomerular disease and the most common pathological form of nephrotic syndrome. Without precise diagnosis and treatment, most patients with MN will develop chronic kidney disease or end-stage renal disease, and may also develop other diseases such as systemic lupus erythematosus (SLE) and cancer, leading to a high mortality rate. Currently, antiphospholipase A2 receptor (PLA2R) and thrombospondin type I containing 7A domain (THSD7A) are commonly used diagnostic strategies for MN, however, their sensitivities are only 70% and 3%, respectively. MN and other primary nephrotic syndrome subtypes, such as minimal change disease, share similar clinical manifestations and differ in treatment strategies. Clinically, these two subtypes are differentiated by the highly invasive and equipment-intensive renal biopsy. Regarding clinical prognosis, prognostic assessment based on anti-PLA2R1 antibody levels remains controversial and challenging, as the drugs used are expensive and potentially toxic. Therefore, it is necessary to develop an advanced method with high sensitivity and low damage to achieve comprehensive individualized monitoring of membranous nephropathy, including accurate diagnosis and prognosis monitoring. Summary of the Invention

[0006] The purpose of the present invention is to solve the technical problem of how to provide a method for detecting disease characteristic polypeptide markers based on endogenous polypeptides.

[0007] The present invention provides a method for detecting disease characteristic polypeptide markers based on endogenous polypeptides. The method comprises the following steps: using a magnetic copper ion mesoporous dopamine material as a solid phase adsorbent to separate and enrich endogenous peptides from a sample; performing MALDI-TOF / TOF MS analysis on the separated and enriched product; performing peak extraction and normalization on the MALDI-TOF / TOF MS mass spectrum, and performing orthogonal partial least squares discriminant analysis and heat map analysis to obtain characteristic polypeptide markers for detecting the disease; then performing nano-LC-MS / MS analysis on the separated and enriched product; and matching the mass-to-charge ratio of the characteristic polypeptide marker obtained by the MALDI-TOF / TOF MS analysis with a polypeptide identified by the nano-LC-MS / MS analysis to determine the amino acid sequence of the characteristic polypeptide marker.

[0008] Preferably, the steps include:

[0009] Step 1: Separate and enrich the sample using a magnetic copper ion mesoporous dopamine material; prepare the magnetic copper ion mesoporous dopamine material and a loading buffer into a material dispersion, add the material dispersion and the target sample into the loading buffer, wash with the loading buffer, and add an elution buffer to obtain an eluate;

[0010] Step 2: The eluate obtained in step 1 is mixed with the matrix target and analyzed by MALDI-TOF / TOF MS;

[0011] Step 3: Peak extraction and normalization are performed on the mass spectrum of MALDI-TOF / TOF MS obtained in step 2, and orthogonal partial least squares discriminant analysis and heat map analysis are performed to select characteristic peptide markers;

[0012] Step 4: The eluate obtained in step 1 is lyophilized and subjected to nano-LC-MS / MS analysis to identify the peptides;

[0013] Step 5: Match the mass-to-charge ratio of the characteristic polypeptide marker obtained in step 3 with the polypeptide identified by nano-LC-MS / MS analysis in step 4 to determine the amino acid sequence of the characteristic polypeptide marker.

[0014] Preferably, the synthesis steps of the magnetic copper ion mesoporous dopamine material used in step 1 are as follows:

[0015] Step 1.1: Dissolve ferric chloride hexahydrate in ethylene glycol. After the solution becomes clear and transparent, add anhydrous sodium acetate. After thorough stirring and ultrasonication, transfer the mixture to a reactor and heat at 100-450°C for 10-20 hours. After the reaction is complete, cool the reactor to room temperature, thoroughly wash the resulting product with deionized water and anhydrous ethanol, and vacuum dry at 40-75°C. The mass-to-volume ratio of ferric chloride hexahydrate, ethylene glycol, and anhydrous sodium acetate is 1.35 g:75 mL:3.6 g.

[0016] Step 1.2: The product obtained in step 1.1 and Pluronic F127 are uniformly dispersed in a mixed solution of ethanol and deionized water. After ultrasonic homogenization, 1,3,5-trimethylbenzene, dopamine hydrochloride, and concentrated ammonia water are respectively injected into the solution and stirred at room temperature for 2-3 hours. After the reaction, the resulting product is thoroughly washed with water and anhydrous ethanol, refluxed with acetone at 60-90°C for 24 hours, and vacuum dried at 40-75°C. The mass volume ratio of Pluronic F127, ethanol, deionized water, 1,3,5-trimethylbenzene, dopamine hydrochloride, and concentrated ammonia water is 200 mg:10 mL:10 mL:0.2 mL:120 mg:0.16 mL;

[0017] Step 1.3: The product obtained in step 1.2 was evenly dispersed in 0.1 mol / L copper sulfate solution. After ultrasonic homogenization, the mixture was stirred at room temperature for 2-3 hours. The product was thoroughly washed with deionized water and anhydrous ethanol, and dried in vacuum at 40-75°C.

[0018] Preferably, the sample comprises serum or urine.

[0019] The present invention provides the application of the above-mentioned method for detecting disease characteristic polypeptide markers based on endogenous polypeptides in non-diagnostic methods and non-therapeutic methods.

[0020] Preferably, the above application includes application in preparing a disease detection and disease prognosis monitoring kit.

[0021] The present invention provides a kit for detecting or monitoring the prognosis of membranous nephropathy, which includes reagents for detecting 12 characteristic polypeptide markers; the amino acid sequences of the 12 characteristic polypeptide markers are shown in SEQ ID NOs: 1-12;

[0022] SEQ ID NO: 1: YVKVTSIQDWVQKTIAEN;

[0023] SEQ ID NO: 2: SSYSKQFTSSTSYNRGDSTFESKSY;

[0024] SEQ ID NO: 3: SEAEDASLLSFMQGYMKHAT;

[0025] SEQ ID NO: 4: DAHKSEVAHRFKDLGEENFKALVLIAF;

[0026] SEQ ID NO: 5: WDLDPEVRPTSAVAA;

[0027] SEQ ID NO: 6: DAHKSEVAHRFKDLGEENFKALVLIAFAQY;

[0028] SEQ ID NO: 7: EEAGARVQQNVPSGTTDTGD;

[0029] SEQ ID NO: 8: SLEDKTERELLESYIDG;

[0030] SEQ ID NO: 9: SPMYSIITPNILRLESEE;

[0031] SEQ ID NO: 10: IAALLSPYSYSTTAVVTNPKE;

[0032] SEQ ID NO: 11: SSSYSKQFTSSTSYNRGDSTFESKSYKMADEAGSEADHEGTHST;

[0033] SEQ ID NO: 12: TFPGFFSPMLGEFVSETESRGSESGIFTNTKESSSHHPGIAEFPSRG.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] 1. The present invention proposes to fix metal ions in the pores of magnetic mesoporous dopamine. Magnetic mesoporous dopamine has a large specific surface area and good magnetic responsiveness, and can fully utilize the catechol functional group to chelate a large number of metal ions. Therefore, the method of the present invention can more sensitively separate and enrich endogenous peptides.

[0036] 2. The copper ions of the magnetic copper ion mesoporous dopamine material have excellent coordination effects with endogenous peptides. Therefore, the method of the present invention shows good enrichment ability for endogenous peptides in complex biological samples.

[0037] 3. The method of the present invention can analyze the expression differences of endogenous peptides between healthy people and disease patients through machine learning algorithms, thereby screening characteristic peptide markers. Combined with nano-LC MS / MS, it can identify endogenous peptides on a large scale and deeply analyze biological functions.

[0038] In summary, the magnetic copper ion mesoporous dopamine material prepared by the present invention has a large amount of copper ions and excellent magnetic responsiveness, and can be successfully used to specifically separate and enrich endogenous peptides in the serum or urine of patients with membranous nephropathy, minimal change disease patients, healthy people, partial remission and complete remission patients, and screened out 12 characteristic endogenous peptides as potential membranous nephropathy markers, successfully realizing the preliminary diagnosis and disease classification of membranous nephropathy patients, which indicates that it has great application prospects in large-scale population screening, disease diagnosis and prognosis monitoring. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a scanning electron microscope photograph of the magnetic copper ion mesoporous dopamine material of the embodiment;

[0040] Figure 2 This is a transmission electron microscope photograph of the magnetic copper ion mesoporous dopamine material of the embodiment;

[0041] Figure 3 This is an elemental analysis diagram of the magnetic copper ion mesoporous dopamine material of the embodiment;

[0042] Figure 4 This is a representative mass spectrogram of the magnetic copper ion mesoporous dopamine material enriched with serum endogenous peptides from membranous nephropathy patients, healthy people, partial remission patients, complete remission patients, and minimal change disease patients.

[0043] Figure 5 : is a heat map of 2800 polypeptides based on all data in the embodiment.

[0044] Figure 6 3D scatter plot based on the training set of the embodiment.

[0045] Figure 7 3D scatter plot based on the validation set of the embodiment.

[0046] Figure 8 12 characteristic polypeptides based on all data in the embodiment. DETAILED DESCRIPTION

[0047] In order to make the present invention more clearly understood, preferred embodiments are described in detail below with reference to the accompanying drawings:

[0048] The purpose of the present invention is to provide a method for detecting disease characteristic polypeptide markers based on endogenous polypeptides; the specific steps are as follows:

[0049] Step 1: Separation and enrichment using magnetic copper ion mesoporous dopamine material: Mix the magnetic copper ion mesoporous dopamine material with the loading buffer at a weight-to-volume ratio of 10 g:1 L to prepare a material dispersion, take 20 μL of the material dispersion and 2 μL of the target sample and add them to 200 μL of the loading buffer, incubate at 37°C for 30 minutes, wash with the loading buffer, add 10 μL of the elution buffer, incubate at 37°C for 30 minutes, and obtain the eluate;

[0050] The loading buffer is a buffer solution containing acetonitrile, water, and trifluoroacetic acid in a volume ratio of 90:9:1;

[0051] The elution buffer was 0.4 mol L -1 of ammonia;

[0052] Step 2: Mix 1 μL of the eluate from step 1 with 1 μL of the matrix target and dry it naturally before performing MALDI-TOF / TOF MS analysis. The matrix is ​​a 20 mg mL solution containing acetonitrile, water, and trifluoroacetic acid in a volume ratio of 50:49.9:0.1. -1 2,5-Dihydroxybenzoic acid buffer solution;

[0053] Step 3: Peak extraction and normalization are performed on the mass spectrum of MALDI-TOF / TOF MS obtained in step 2, and orthogonal partial least squares discriminant analysis and heat map analysis are performed to select characteristic peptide markers;

[0054] Step 4: The eluate obtained in step 1 is lyophilized and subjected to nano-LC-MS / MS analysis to identify the peptides;

[0055] Step 5: Match the mass-to-charge ratio of the characteristic polypeptide marker obtained in step 3 with the polypeptide identified by nano-LC-MS / MS analysis in step 4 to determine the amino acid sequence of the characteristic polypeptide marker.

[0056] In the present invention, the synthesis steps of the magnetic copper ion mesoporous dopamine material used in the above step 1 are as follows:

[0057] Step 1.1: Dissolve ferric chloride hexahydrate in ethylene glycol. Add anhydrous sodium acetate until the solution becomes clear and transparent. After thorough stirring and ultrasonication, transfer the mixture to a reactor and heat at 100-450°C for 10-20 hours. After the reaction is complete, cool the reactor to room temperature, thoroughly wash the resulting product with deionized water and anhydrous ethanol, and dry it in vacuo at 40-75°C.

[0058] The mass volume ratio of ferric chloride hexahydrate, ethylene glycol and anhydrous sodium acetate is 1.35 g:75 mL:3.6 g;

[0059] Step 1.2: The product obtained in step 1.1 and Pluronic F127 are uniformly dispersed in a mixed solution of ethanol and deionized water. After ultrasonic homogenization, 1,3,5-trimethylbenzene, dopamine hydrochloride, and concentrated ammonia water are respectively injected into the solution and stirred at room temperature for 2-3 hours. After the reaction, the product is thoroughly washed with water and anhydrous ethanol, refluxed with acetone at 60-90°C for 24 hours, and then vacuum dried at 40-75°C.

[0060] The mass volume ratio of Pluronic F127, ethanol, deionized water, 1,3,5-trimethylbenzene, dopamine hydrochloride, and concentrated ammonia is 200 mg:10 mL:10 mL:0.2 mL:120 mg:0.16 mL;

[0061] Step 1.3: The product obtained in step 1.2 was uniformly dispersed in 0.1 mol L -1 After ultrasonic homogenization in copper sulfate solution, stir at room temperature for 2-3 hours, wash the obtained product thoroughly with deionized water and anhydrous ethanol, and dry it in vacuum at 40-75°C.

[0062] In the present invention, the specific conditions for the MALDI-TOF / TOF MS analysis in step 2 are as follows: using a Bruker UltrafleXtreme MALDI-TOF / TOF mass spectrometer, a 355 nm Nd:YAG laser light source, a laser frequency of 2000 Hz, an acceleration voltage of 20 kV, wherein the voltage at ion source 1 is 20 kV, and the voltage at ion source 2 is 17.6 kV; the acquisition mode is reflectron positive ion mode, and the acquisition range is m / z 700-5000 Da; mass spectrometry data are obtained from Flexcontrol 3.4 and exported in Flexanalysis 3.4.

[0063] In the present invention, the specific conditions for nano-LC-MS / MS analysis in step 4 are as follows: using an EASY-nLC 1000 liquid chromatograph (Thermo Fisher Scientific) coupled with an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific), the A phase and the B phase of the liquid chromatography are water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid, respectively. The eluate obtained in step 1 is lyophilized and redissolved in the A phase, and a linear gradient of 5% A to 30% B in 50 min is applied to the analytical column (Thermo Scientific Acclaim PepMap C18, 75 μm × 25 cm); the electrospray voltage was 2.3 kV, and the parent ion scan range for the primary spectrum was m / z = 350-1600, with a resolution of 60,000 (m / z = 200, and the secondary spectrum was obtained by high-energy collisional dissociation with a resolution of 15,000, m / z = 200; the high-energy collisional dissociation mode was selected to sequentially fragment parent ions with charges of +2, +3, and +4; the normalized collision energy was 28%;

[0064] Tandem mass spectra were extracted by Proteome Discoverer (Thermo Fisher Scientific, version 2.4.0.305) and database searching was performed using the Uniprot-SwissProt database (taxonomy: Homo sapiens, 20,386 entries) with a precursor ion mass tolerance of 10 ppm and a fragment ion mass tolerance of 0.020 Da.

[0065] In the present invention, the conditions for selecting the characteristic polypeptide marker in step 3 are VIP value>1, P value<0.05 and FC value>2 or <0.5.

[0066] In the present invention, the polypeptide matching condition in step 5 is that the mass-to-charge ratio of the characteristic polypeptide marker selected by MALDI-TOF / TOF MS analysis and the mass-to-charge ratio of the polypeptide identified by nano-LC-MS / MS analysis are consistent within 100 ppm.

[0067] In the present invention, in step 5, the mass-to-charge ratio of the MALDI-TOF / TOF MS characteristic polypeptide marker in step 3 is matched with the polypeptide identified by nano-LC-MS / MS in step 4 to determine the amino acid sequences of 12 characteristic polypeptide markers (m / z value of 708.38 corresponds to YVKVTSIQDWVQKTIAEN, m / z value of 712.35 corresponds to SSYSKQFTSSTSYNRGDSTFESKSY, m / z value of 739.21 corresponds to SEAEDASLLSFMQGYMKHAT, m / z value of 771.99 corresponds to DAHKSEVAHRFKDLGEENFKALVLIAF, m / z value of 813.88 corresponds to WDLDPEVRPTSAVAA, m / z value of 862.32 corresponds to DAHKSEVAHRFKDLGEENF KALVLIAFAQY, m / z value of 966.18 corresponds to EEAGARVQQNVPSGTDTGD, m / z value of 998.99 corresponds to SLEDKTERELLESYIDG, m / z value of 1046.24 corresponds to SPMYSIITPNILRLESEE, m / z value of 1113.31 corresponds to IAALLSPYSYSTTAVVTNPKE, m / z value of 1197.30 corresponds to SSSYSKQFTSSTSYNRGDSTFESKSYKMADEAGSEADHEGTHST, m / z value of 1266.32 corresponds to TFPGFFSPMLGEFVSETESRGSESGIFTNTKESSSHHPGIAEFPSRG.

[0068] In the present invention, the target sample in step 1 is a body fluid such as serum or urine.

[0069] Example 1

[0070] 1. Synthesis of magnetic copper ion mesoporous dopamine materials.

[0071] Step 1.1: Dissolve 1.35 g of FeCl₃·6H₂O in 75 mL of ethylene glycol under magnetic stirring until the solid is completely dissolved. Then, add 3.6 g of sodium acetate. After thorough stirring and sonication, transfer the mixture to a hydrothermal reactor and heat at 200°C for 16 hours. After cooling, wash the product three times with deionized water and ethanol, respectively, and dry it in a vacuum oven at 50°C.

[0072] Step 1.2: 50 mg of the product obtained in step 1.1 and 200 mg of Pluronic F127 (Sigma-Aldrich) were uniformly dispersed in a mixed solution of 10 mL of ethanol and 10 mL of deionized water. The mixture was then ultrasonicated until uniform. 0.2 mL of 1,3,5-trimethylbenzene, 120 mg of dopamine hydrochloride, and 0.16 mL of concentrated ammonia were injected into the solution respectively. The mixture was stirred at room temperature for 2 hours. After the reaction, the product was thoroughly washed with water and anhydrous ethanol, refluxed with 200 mL of acetone at 80°C for 48 hours, and dried in vacuo at 50°C.

[0073] Step 1.3: Disperse 50 mg of the product obtained in step 1.2 uniformly in 100 mL of 0.1 mol L -1 In copper sulfate solution, after ultrasonic homogenization, stir at room temperature for 2-3 hours, wash the obtained product thoroughly with deionized water and anhydrous ethanol, and vacuum dry at 50°C to obtain magnetic copper ion mesoporous dopamine material, named Cu(II)-FPA.

[0074] Scanning electron microscope images of magnetic copper ion mesoporous dopamine materials Figure 1 ; Transmission electron microscopy images of magnetic copper ion mesoporous dopamine materials Figure 2 ; Elemental analysis of magnetic copper ion mesoporous dopamine material Figure 3 .

[0075] from Figures 1 to 3 It can be seen that the magnetic copper ion mesoporous dopamine material presents a uniform spherical morphology and core-shell structure, and contains a large amount of copper ions.

[0076] 2. Magnetic copper ion mesoporous dopamine material as a solid phase adsorbent for the separation and enrichment of endogenous peptides:

[0077] The magnetic copper ion mesoporous dopamine material obtained above was used as a solid phase adsorbent to separate and enrich endogenous peptides in the sera of 124 membranous nephropathy patients, 171 healthy people, 53 partial remission patients, 24 complete remission patients, and 34 minimal change disease patients.

[0078] Step 2.1: All serum samples were divided into training and validation sets, corresponding to sera from 98 patients with membranous nephropathy, 131 healthy subjects, 42 patients with partial remission, 19 patients with complete remission, and 27 patients with minimal change disease; and sera from 26 patients with membranous nephropathy, 40 healthy subjects, 11 patients with partial remission, 5 patients with complete remission, and 7 patients with minimal change disease, respectively.

[0079] Step 2.2: 2 mg of magnetic copper ion mesoporous dopamine material was mixed with 200 μL of loading buffer (acetonitrile / water / trifluoroacetic acid volume ratio = 90 / 9 / 1) to prepare a material dispersion. 20 μL of the material dispersion and 2 μL of serum were added to 200 μL of loading buffer and incubated at 37°C for 30 minutes. The material was washed with loading buffer and 10 μL of elution buffer (0.4 mol L -1 and eluted at 37°C for 30 minutes to obtain an eluate.

[0080] Step 2.3: Mass spectrometry analysis: Take 1 μL of the above eluate and mix it with 1 μL of 20 mg mL -1 2,5-Dihydroxybenzoic acid (DHB) matrix (acetonitrile / water / trifluoroacetic acid volume ratio = 50 / 49.9 / 0.1) was spotted and dried naturally before MALDI-TOF / TOF MS analysis using a Bruker UltrafleXtreme MALDI-TOF / TOF mass spectrometer with a 355 nm Nd:YAG laser source, a laser frequency of 2000 Hz, an accelerating voltage of 20 kV (20 kV at ion source 1, 17.6 kV at ion source 2), and a reflectron cation acquisition mode with an acquisition range of m / z 700-5000 Da. Mass spectral data were acquired using Flexcontrol 3.4 and exported to Flexanalysis 3.4. The mass spectrum is shown in Figure 2. Figure 4 shown.

[0081] Analysis results: Figure 4 It can be seen that the present material can capture abundant serum endogenous peptides with a mass-to-charge ratio ranging from 700 to 5000 Daltons.

[0082] 3. Data processing to obtain characteristic peptide markers:

[0083] The obtained serum endogenous peptide mass spectra were peak extracted and normalized, and the training set was subjected to orthogonal partial least squares discriminant analysis using Metaboanalyst 5.0 and SIMCA. The VIP value, P value, and FC value of each peptide were calculated to screen characteristic peptide markers.

[0084] Step 3.1: Peak extraction and normalization of the serum endogenous peptide mass spectra were performed using the R packages MALDIquant, MALDIquantForeign, and limma, resulting in 2800 peptide peaks. A heat map was created for the entire data set based on all peptides.

[0085] Step 3.2: Use SIMCA to perform orthogonal partial least squares discriminant analysis on the training sets of healthy subjects, patients with membranous nephropathy, patients with minimal change disease, patients with partial remission, and patients with complete remission, and draw a three-dimensional scatter plot.

[0086] Step 3.3: Orthogonal partial least squares discriminant analysis was performed using SIMCA on the training sets of healthy subjects and patients with primary nephrotic syndrome, patients with membranous nephropathy and patients with minimal change disease, healthy subjects and patients in remission, patients with membranous nephropathy and patients in remission, and patients with partial remission and complete remission. VIP values, P values, and FC values ​​were calculated for each peptide. Based on the conditions of VIP values ​​> 1, P values ​​< 0.05, and FC values ​​> 2 or < 0.5, the intersection of the five groups was taken to screen out 12 characteristic peptide markers. Orthogonal partial least squares discriminant analysis was performed on the validation set based on the characteristic peptide markers, and a three-dimensional scatter plot was drawn.

[0087] Step 3.4: Draw a heat map for all data based on the characteristic peptide markers.

[0088] Analysis results: Figure 5 It can be seen that there are differences in endogenous peptides among the five groups of healthy people, membranous nephropathy patients, minimal change disease patients, partial remission patients and complete remission patients. Figure 6 It can be seen that the training set based on 2800 peptide peaks has a better discrimination effect. Figure 7 It can be seen that the 12 characteristic peptide markers have excellent discrimination effect on the validation set. Figure 8 It can be seen that the 12 characteristic polypeptide markers play an important role in the comprehensive monitoring of patients with membranous nephropathy.

[0089] 4. Identification of polypeptide sequences:

[0090] The eluate obtained in step 2.2 was subjected to nano-LC-MS / MS analysis to identify the peptide sequence.

[0091] Step 4.1: Using an EASY-nLC 1000 liquid chromatograph (Thermo Fisher Scientific) coupled to an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific), the liquid chromatography phase A and phase B were water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid, respectively. The eluate obtained in step 2.2 was lyophilized and redissolved in phase A. A linear gradient of 5% A to 30% B in 50 min was applied to the analytical column (Thermo Scientific Acclaim PepMap C18, 75 μm × 25 cm); the electrospray voltage was 2.3 kV, and the parent ion scan range for the primary spectrum was m / z = 350-1600, with a resolution of 60,000 (m / z = 200, and the secondary spectrum was obtained by high-energy collisional dissociation with a resolution of 15,000, m / z = 200; the high-energy collisional dissociation mode was selected to sequentially fragment parent ions with charges of +2, +3, and +4; the normalized collision energy was 28%;

[0092] Step 4.2: Tandem mass spectra were extracted by Proteome Discoverer (Thermo Fisher Scientific, version 2.4.0.305) and database search was performed using the Uniprot-SwissProt database (taxonomy: Homo sapiens, 20,386 entries) with a precursor ion mass tolerance of 10 ppm and a fragment ion mass tolerance of 0.020 Da.

[0093] Step 4.3: Match the mass-to-charge ratios of the characteristic peptide markers obtained in step 3.3 with the peptides identified by nano-LC-MS / MS, and determine the amino acid sequences of the 12 characteristic peptide markers based on the principle of consistency within 100 ppm.

[0094] Analysis results: The amino acid sequences of the 12 characteristic polypeptide markers are as follows:

[0095] The m / z value is 708.38, corresponding to YVKVTSIQDWVQKTIAEN,

[0096] The m / z value is 712.35, corresponding to SSYSKQFTSSTSYNRGDSTFESKSY,

[0097] The m / z value is 739.21, corresponding to SEAEDASLLSFMQGYMKHAT.

[0098] The m / z value is 771.99, corresponding to DAHKSEVAHRFKDLGEENFKALVLIAF,

[0099] The m / z value is 813.88 corresponding to WDLDPEVRPTSAVAA,

[0100] The m / z value is 862.32, corresponding to DAHKSEVAHRFKDLGEENFKALVLIAFAQY,

[0101] The m / z value is 966.18 corresponding to EEAGARVQQNVPSGTDTGD,

[0102] The m / z value is 998.99 corresponding to SLEDKTERELLESYIDG,

[0103] The m / z value is 1046.24, corresponding to SPMYSIITPNILRLESEE,

[0104] The m / z value is 1113.31, corresponding to IAALLSPYSYSTTAVVTNPKE.

[0105] The m / z value is 1197.30, corresponding to SSSYSKQFTSSTSYNRGDSTFESKSYKMADEAGSEADHEGTHST,

[0106] The m / z value is 1266.32, corresponding to TFPGFFSPMLGEFVSETESRGSESGIFTNTKESSSHHPGIAEFPSRG.

[0107] Example 2:

[0108] The magnetic copper ion mesoporous dopamine material obtained in Example 1 was used as a solid phase adsorbent to separate and enrich endogenous peptides in the sera of 124 membranous nephropathy patients, 171 healthy people, 53 partial remission patients, 24 complete remission patients, and 34 minimal change disease patients.

[0109] (1) All urine samples were divided into a training set and a validation set, corresponding to urine samples from 98 patients with membranous nephropathy, 131 healthy subjects, 42 patients with partial remission, 19 patients with complete remission, and 27 patients with minimal change disease; and urine samples from 26 patients with membranous nephropathy, 40 healthy subjects, 11 patients with partial remission, 5 patients with complete remission, and 7 patients with minimal change disease, respectively.

[0110] (2) The urine sample was centrifuged at 4000 rpm for 20 minutes at 4°C, and the supernatant was stored at -80°C. 2 mg of the magnetic copper ion mesoporous dopamine material obtained in Example 1 was mixed with 200 μL of loading buffer (acetonitrile / water / trifluoroacetic acid volume ratio = 90 / 9 / 1) to prepare a material dispersion. 20 μL of the material dispersion and 2 μL of urine were added to 200 μL of loading buffer, incubated at 37°C for 30 minutes, washed with loading buffer, and 10 μL of elution buffer (0.4 mol L-1 ammonia water) was added. The mixture was eluted at 37°C for 30 minutes to obtain an eluate.

[0111] (3) Mass spectrometry analysis: 1 μL of the eluate from step (2) was mixed with 1 μL of 20 mg mL-1,2,5-dihydroxybenzoic acid (DHB) matrix (acetonitrile / water / trifluoroacetic acid volume ratio = 50 / 49.9 / 0.1) and the target was exposed to air drying before MALDI-TOF / TOF MS analysis. A Bruker UltrafleXtreme MALDI-TOF / TOF mass spectrometer was used, using a 355 nm Nd:YAG laser source with a laser frequency of 2000 Hz and an acceleration voltage of 20 kV (20 kV at ion source 1 and 17.6 kV at ion source 2). The acquisition mode was reflectron cation mode, and the acquisition range was m / z 700-5000 Da. Mass spectrometry data were obtained from Flexcontrol 3.4 and exported in Flexanalysis 3.4.

[0112] The other conditions are the same as those in Example 1, except for the step (2) of obtaining the eluate.

[0113] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form or substance. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the scope of protection of the present invention. Any equivalent changes, modifications and evolutions made by technicians familiar with this profession without departing from the spirit and scope of the present invention by using the technical content disclosed above are all equivalent embodiments of the present invention; at the same time, any equivalent changes, modifications and evolutions made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A kit for detecting or monitoring prognosis of membranous nephropathy, characterized in that: It comprises reagents for detecting 12 characteristic polypeptide markers; the amino acid sequences of the 12 characteristic polypeptide markers are shown as SEQ ID NOs: 1-12.

Images