Method for detecting membranous nephropathy target antigen

Abstract

The invention discloses a method for detecting a membranous nephropathy target antigen, which comprises the following steps: adding a protein extracting solution into a glomerular tissue to be detected, and incubating; adding a mixed enzyme of a LysC enzyme and a Trypsin enzyme, and incubating; adding a dithiothreitol solution, and incubating; adding an iodo-acetamide solution, and incubating at room temperature in a dark place; adding a precipitating agent, performing ultrasonic treatment and centrifugation, taking supernate, loading the supernate to a C18 small column, eluting the small column, and collecting eluent; freezing the eluent, blow-drying the eluent, and redissolving the eluent with a loading solution to obtain a test solution; and carrying out liquid chromatography-tandem mass spectrometry detection on the test solution. By adopting the detection method disclosed by the invention, the target antigen of membranous nephropathy can be accurately detected when the sampling volume is obviously reduced, so that the sampling difficulty is greatly reduced, and the application of the laser microdissection and liquid chromatography-mass spectrometry combined technology in the detection of the target antigen of membranous nephropathy is greatly promoted. The method is simple in process and short in detection time.

Description

Technical Field

[0001] This invention belongs to the field of disease detection technology. More specifically, this invention relates to a method for detecting target antigens in membranous nephropathy based on laser microdissection combined with liquid chromatography-mass spectrometry (LMD-LC / MS). Background Technology

[0002] Membranous nephropathy (MN) is a glomerular disease caused by the deposition of immune complexes along the subepithelial region of the glomerular basement membrane. Identification and subtyping of membranous nephropathy target antigens (MNAs) are crucial for the accurate diagnosis of MN, and understanding these target antigens is a core prerequisite for understanding its pathogenesis and guiding individualized treatment. Currently, at least 14 MNAs have been identified, and new target antigens are being discovered.

[0003] Currently, the mainstream (traditional) method for identifying membranous nephropathy (MNA) in clinical practice is immunohistochemistry / immunofluorescence (IHC / IF). Routine pathological examination can detect two antigens, PLA2R and THSD7A. A few testing institutions can also detect NELL1, EXT1, EXT2, Sema3B, and PCDH7 antigens. However, due to the limited availability of commercially available antibodies, most antigens cannot be detected using this traditional method. Furthermore, the detection of membranous nephropathy using existing proteomics methods often takes 20-24 hours, making the process cumbersome and time-consuming.

[0004] The rise of laser microdissection combined with liquid chromatography-mass spectrometry (LMD-LC / MS) has enabled the depth of disease diagnosis to extend from the tissue and cellular level to the molecular level. However, there are very few reports on the application of LMD-LC / MS in the detection of target antigens in membranous nephropathy, and standardized methodological guidance is lacking. Furthermore, in the few existing reports on the use of this method for detecting target antigens in membranous nephropathy, the required tissue volume is relatively large (250,000 μm). 2 *10 μm~550000 μm 2 *10 μm). Because the kidney is a vital solid organ in the human body, obtaining kidney tissue samples from pediatric patients is extremely difficult, and obtaining too much sample can increase the burden on kidney function and even cause irreversible damage to kidney tissue. Therefore, the high sample volume required has hindered the research progress of target antigen detection in membranous nephropathy. Summary of the Invention

[0005] Based on this, the purpose of the present invention is to provide a method for detecting target antigens of membranous nephropathy based on laser microdissection combined with liquid chromatography-mass spectrometry (LMD-LC / MS). When using this method to detect target antigens of membranous nephropathy, the required tissue volume is small and the detection efficiency is high.

[0006] The technical solutions for achieving the above-mentioned objectives include the following.

[0007] This invention provides a method for detecting target antigens in membranous nephropathy, comprising the following steps:

[0008] (1) Add protein extraction solution to the glomerular tissue to be tested and incubate at 90℃~110℃ for 40 min~50 min; the protein extraction solution includes 10.8 mmol / L~12.0 mmol / L sodium deoxycholate, 18.0 mmol / L~22.0 mmol / L tris(hydroxymethyl)aminomethane and 1.8 mmol / L~2.2 mmol / L ethylenediaminetetraacetic acid;

[0009] (2) Add a mixture of LysC enzyme and Trypsin enzyme and incubate at 36℃~38℃ for 3 h~8 h;

[0010] (3) Add dithiothreitol solution and incubate at 36℃~38℃ for 20 to 40 minutes;

[0011] (4) Add iodoacetamide solution and incubate at room temperature in the dark for 20 to 40 minutes;

[0012] (5) Add precipitant, sonicate, centrifuge, and load the supernatant onto C. 18 Small column, elute the small column, and collect the eluent;

[0013] (6) The eluent is frozen and dried, and then reconstituted with the sample solution to obtain the test solution;

[0014] (7) The test solution was detected by liquid chromatography-tandem mass spectrometry.

[0015] The inventors of this invention discovered, while exploring methods for detecting target antigens in membranous nephropathy, that when the sample volume of the glomerular tissue to be tested is significantly reduced, the target antigens of membranous nephropathy can still be accurately detected by using appropriate test solution preparation methods and optimized liquid chromatography-tandem mass spectrometry conditions. This greatly reduces the difficulty of sampling and significantly promotes the application of laser microdissection combined with liquid chromatography-mass spectrometry (LMD-LC / MS) in the detection of target antigens in membranous nephropathy.

[0016] In addition, the detection method of the present invention has a simple process and a short detection time. Attached Figure Description

[0017] Figure 1 This is a chromatogram of the test solution prepared when the enzyme dosage was 0.5 μg in Example 6 of the present invention.

[0018] Figure 2 This is a chromatogram of the test solution prepared when the enzyme dosage was 0.05 μg in Example 6 of the present invention.

[0019] Figure 3This is a chromatogram of the test solution prepared when the enzyme dosage was 0.025 μg in Example 6 of the present invention.

[0020] Figure 4 This is a chromatogram obtained by using elution process A in Example 12 of the present invention.

[0021] Figure 5 This is a chromatogram obtained by elution process B in Example 12 of the present invention.

[0022] Figure 6 This is a chromatogram obtained by using elution process C in Example 12 of the present invention. Detailed Implementation

[0023] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0024] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0025] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the examples are commercially available products.

[0026] In some embodiments of the present invention, a method for detecting target antigens of membranous nephropathy is disclosed, comprising the following steps:

[0027] (1) Add protein extraction solution to the glomerular tissue to be tested and incubate at 90℃~110℃ for 40 min~50 min; the protein extraction solution includes 10.8 mmol / L~12.0 mmol / L sodium deoxycholate, 18.0 mmol / L~22.0 mmol / L tris(hydroxymethyl)aminomethane and 1.8 mmol / L~2.2 mmol / L ethylenediaminetetraacetic acid;

[0028] (2) Add a mixture of LysC enzyme and Trypsin enzyme and incubate at 36℃~38℃ for 3 h~8 h;

[0029] (3) Add dithiothreitol solution and incubate at 36℃~38℃ for 20 to 40 minutes;

[0030] (4) Add iodoacetamide solution and incubate at room temperature in the dark for 20 to 40 minutes;

[0031] (5) Add precipitant, sonicate, centrifuge, and load the supernatant onto C. 18 Small column, elute the small column, and collect the eluent;

[0032] (6) The eluent is frozen and dried, and then reconstituted with the sample solution to obtain the test solution;

[0033] (7) The test solution was detected by liquid chromatography-tandem mass spectrometry.

[0034] In one embodiment, the protein extract in step (1) comprises 11.0 mmol / L-12.0 mmol / L sodium deoxycholate, 19.0 mmol / L-21.0 mmol / L tris(hydroxymethyl)aminomethane and 1.9 mmol / L-2.1 mmol / L ethylenediaminetetraacetic acid.

[0035] In one embodiment, the protein extraction solution in step (1) comprises 11.5 mmol / L-12.0 mmol / L sodium deoxycholate, 20.0 mmol / L-21.0 mmol / L tris(hydroxymethyl)aminomethane, and 2.0 mmol / L-2.1 mmol / L ethylenediaminetetraacetic acid. Using this protein extraction solution results in high protein extraction efficiency and a large number of proteins and peptides obtained.

[0036] In one embodiment, the glomerular tissue to be tested is obtained in step (1) using a laser microdissection instrument.

[0037] In one embodiment, the volume of the glomerular tissue in step (1) is 250,000 μm. 2 ~300000 μm 2 (7 μm~8 μm). This invention achieves accurate detection of target antigens even with small tissue dosage.

[0038] In one embodiment, the volume of the glomerular tissue in step (1) is 280,000 μm. 2 ~300000 μm 2 (7 μm~8 μm).

[0039] In one embodiment, the ratio of glomerular tissue to protein extract in step (1) is (250000 μm) 2 ~300000 μm 2 )*(7 μm~8 μm): 50 μL.

[0040] In one embodiment, the incubation temperature in step (1) is 95°C to 105°C, and the incubation time is 44 min to 46 min.

[0041] In one embodiment, the ratio of LysC enzyme to Trypsin enzyme in the mixed enzyme in step (2) is 0.8~1.2:1.

[0042] In one embodiment, the ratio of the mixed enzyme in step (2) to the glomerular tissue in step (1) is 0.02 μg~0.04 μg: (250000 μm 2 ~300000 μm 2 (7 μm~8 μm).

[0043] In one embodiment, the ratio of the mixed enzyme in step (2) to the glomerular tissue in step (1) is 0.025 μg to 0.030 μg: (250000 μm 2 ~300000 μm 2 (7 μm~8 μm).

[0044] In one embodiment, the incubation time in step (2) is 3 h to 4 h. An enzyme reaction time of 3 h to 4 h can find a balance between specificity and efficiency, improving the yield and detectability of the target peptide.

[0045] In one embodiment, the concentration of the dithiothreitol solution in step (3) is 4 mmol / L to 6 mmol / L.

[0046] In one embodiment, the concentration of the iodoacetamide solution in step (4) is 14 mmol / L to 16 mmol / L.

[0047] In one embodiment, the precipitant in step (5) is a 3.5%~4.5% aqueous solution of trifluoroacetic acid or a 3.5%~4.5% aqueous solution of formic acid.

[0048] In one embodiment, the precipitant in step (5) is a 3.8%~4.2% aqueous solution of trifluoroacetic acid. Using an aqueous solution of trifluoroacetic acid as the precipitant shortens the vacuum concentration and drying time while achieving sodium deoxycholate precipitation.

[0049] In one embodiment, the ratio of the precipitant in step (5) to the protein extract in step (1) is 4 μL to 6 μL: 50 μL.

[0050] In one embodiment, the ratio of the precipitant in step (5) to the protein extract in step (1) is 4.5 μL to 5.5 μL: 50 μL.

[0051] In one embodiment, the solvent used for elution in step (5) is a mixture of acetonitrile, water and trifluoroacetic acid in a volume ratio of 55~65:35~45:0.08~0.12.

[0052] In one embodiment, the freezing temperature in step (6) is -82°C to -78°C, and the freezing time is 15 min to 25 min.

[0053] In one embodiment, the loading solution in step (6) is a 0.08%~0.12% aqueous solution of trifluoroacetic acid.

[0054] In one embodiment, the chromatographic conditions for liquid chromatography in step (7) include:

[0055] Mobile phase A: 0.08%~0.12% formic acid aqueous solution; Mobile phase B: 75%~85% acetonitrile solution containing 0.08%~0.12% formic acid; Injection volume: 14 μL~16 μL;

[0056] The liquid phase gradient program for the sample loading pump was as follows: 0 → 5 min, flow rate 5.000 μL / min; 5 min → 6 min, flow rate 5.000 μL / min → 1.000 μL / min; 6 min → 45 min, flow rate 1.000 μL / min; 45 min → 46 min, flow rate 1.000 μL / min → 5.000 μL / min; 46 min → 50 min, flow rate 5.000 μL / min; 0 → 50 min, 100% mobile phase A;

[0057] The liquid phase gradient program for the nano-pump is as follows: 0 → 5 min, 90% mobile phase A; 5 min → 35 min, 90% mobile phase A → 55% mobile phase A; 35 min → 40 min, 55% mobile phase A → 10% mobile phase A; 40 min → 45 min, 10% mobile phase A; 45 min → 46 min, 10% mobile phase A → 90% mobile phase A; 46 min → 50 min, 90% mobile phase A; 0 → 50 min, flow rate 0.3 μL / min.

[0058] In one implementation, the mass spectrometry parameters in step (7) include:

[0059] Resolution: 70000 (MS1), 17500 (MS2);

[0060] MS1 scan range: 300 m / z - 2000 m / z;

[0061] Collision energy: 30% HCD;

[0062] Isolation window: 2.0 m / z (DDA);

[0063] Maximum injection time: 50 ms (MS1) / 200 ms (MS2).

[0064] The target antigens for membranous nephropathy include PLA2R, THSD7A, NELL1, SEMA3B, VASN, HTRA1, NTNG1, EXT1, EXT2, NCAM1, CNTN1, FAT1, NDNF, and / or PCSK6.

[0065] In the following embodiments of the present invention, the solvents used, such as water, acetonitrile, and trifluoroacetic acid, were all LC / MS grade. Sodium deoxycholate, tris(hydroxymethyl)aminomethane, ethylenediaminetetraacetic acid, and trifluoroacetic acid were purchased from Sigma-Aldrich; trypsin, water, a mixture of LysC enzyme and Trypsin enzyme for LC / MS, were purchased from Thermo Fisher; methanol and acetonitrile were purchased from Merck Millipore. A Thermo Q-Exactive LC-MS system was used.

[0066] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0067] Example 1: Method for detecting target antigens in membranous nephropathy

[0068] Includes the following steps:

[0069] 1. Preparation of the test solution

[0070] (1) A 7 μm thick kidney tissue was cut and adhered to the membrane;

[0071] (2) A laser microdissection instrument was used to obtain 300,000 μm samples under a microscope. 2 The glomeruli (approximately 10-12 glomeruli) were collected in a 0.6 mL EP tube;

[0072] (3) Add 50 μL of protein extraction buffer (sodium deoxycholate concentration of 12.0 mmol / L, tris(hydroxymethyl)aminomethane concentration of 20.0 mmol / L, ethylenediaminetetraacetic acid concentration of 2.0 mmol / L, pH 8.0), mix well and centrifuge;

[0073] (4) Place in a 100℃ constant temperature mixer and incubate for 45 min;

[0074] (5) Add 0.025 μg of mixed enzyme (LysC enzyme + Trypsin enzyme, weight ratio 1:1) and incubate at 37℃ for 3~4h.

[0075] (6) Add 1.5 μL of 5 mmol / L dithiothreitol solution, vortex, and incubate at 37°C for 30 minutes;

[0076] (7) Add 1.5 μL of 15 mmol / L iodoacetamide solution, vortex, and incubate at room temperature in the dark for 30 minutes;

[0077] (8) Add 5 μL of precipitant (4% trifluoroacetic acid aqueous solution), sonicate in water bath for 10 min, centrifuge for 5 min, and obtain the supernatant;

[0078] (9) Load the supernatant onto Ziptip C 18 The target analyte was eluted into another clean 1.5 mL centrifuge tube using 50 μL of elution buffer (water, acetonitrile, trifluoroacetic acid, volume ratio 40:60:0.1).

[0079] (10) Vacuum concentration and drying;

[0080] (11) Add 20 μL of sample solution (0.1% trifluoroacetic acid aqueous solution), vortex for 30 s to dissolve and mix, centrifuge for 5 min, and completely transfer to the sample bottle to obtain the test solution.

[0081] 2. Perform liquid chromatography-mass spectrometry (LC-MS) detection on the test solution.

[0082] (1) Chromatographic conditions include:

[0083] Mobile phase A: 0.1% formic acid aqueous solution

[0084] Mobile phase B: 80% acetonitrile aqueous solution containing 0.1% formic acid.

[0085] Injection volume: 15 μL

[0086] Gradient elution: The liquid phase gradient of the loading pump is shown in Table 1, and the liquid phase gradient of the nano-pump is shown in Table 2.

[0087] Table 1

[0088]

[0089] Table 2

[0090]

[0091] (2) Mass spectrometry detection

[0092] The mass spectrometry parameters are shown in Table 3.

[0093] Table 3

[0094]

[0095] (3) Data processing

[0096] Samples were separated by online reversed-phase chromatography using an Ultimate 3000 RSLC nano-liquid chromatograph, and then injected into a Q Exactive mass spectrometer (Thermo Fisher Scientific) via a nano-electrospray ionization source. Mass spectrometry data were identified using the Human Protein Database and processed using Proteome Discoverer software (Thermo Fisher Scientific).

[0097] Example 2: Methodological Validation of the Detection Method of the Present Invention

[0098] 1. Precision

[0099] Kidney tissue samples were collected from healthy mice and processed according to the test solution preparation method in Example 1. The samples were then analyzed according to the method described in Example 1, and the results were matched with a database to obtain the peptide and protein counts. Each sample was measured 20 times. Inter-batch precision was determined by measuring twice per batch, with measurements performed continuously for 10 days, for a total of 20 measurements. The CV of peptide and protein counts was evaluated, and the results are shown in Table 4.

[0100] Table 4

[0101]

[0102] As shown in Table 4, the method has good precision, with repeatability precision <6.0% and batch-to-batch precision <6.0%.

[0103] 2. Identification of 14 target antigens for membranous nephropathy

[0104] Renal biopsy tissues with a confirmed pathological diagnosis of membranous nephropathy (containing 14 nephrotic antigens, with sample 8 including two antigens, sourced from the applicant's laboratory) were selected and excised at 0.3 mm diameters under a laser microscope. 2 The glomeruli were processed according to the test solution preparation method of Example 1, and tested on the instrument according to the method of Example 1. The results are shown in Table 5.

[0105] Table 5

[0106]

[0107] Note: The data in the figure represents the number of spectra read.

[0108] As shown in Table 5, the detection method of the present invention can successfully detect 14 reported target antigens of membranous nephropathy.

[0109] Example 3: Comparison of the effects of different protein extraction solutions on sample extraction efficiency

[0110] This example compares the effects of three protein extraction solutions (protein extraction solution ①, protein extraction solution ②, and protein extraction solution ③) on the extraction efficiency of renal biopsy tissues (from the applicant's laboratory) from three patients with a confirmed pathological diagnosis of membranous nephropathy. Except for step 3 of the test sample preparation method, which uses different protein extraction solutions, all other steps are the same as in Example 1.

[0111] Protein extraction solution ①: Weigh out sodium deoxycholate at a concentration of 12.0 mmol / L, tris(hydroxymethyl)aminomethane at a concentration of 20.0 mmol / L, ethylenediaminetetraacetic acid at a concentration of 2.0 mmol / L, and pH 8.0.

[0112] Protein extraction solution ②: ammonium bicarbonate concentration 50.0 mmol / L, tris(hydroxymethyl)aminomethane concentration 20.0 mmol / L, ethylenediaminetetraacetic acid concentration 2.0 mmol / L, pH 8.0.

[0113] Protein extraction solution ③: SDS (sodium dodecyl sulfate) concentration 12.0 mmol / L, tris(hydroxymethyl)aminomethane concentration 20.0 mmol / L, ethylenediaminetetraacetic acid concentration 2.0 mmol / L, pH 8.0.

[0114] The number of peptides and proteins obtained by different protein extraction solutions are shown in Table 6.

[0115] Table 6

[0116]

[0117] As shown in Table 6, the number of peptides and proteins obtained using protein extraction solution ① is significantly better than that obtained using protein extraction solutions ② and ③.

[0118] Example 4: Comparison of the effects of different sodium deoxycholate concentrations in protein extraction solution on sample extraction efficiency

[0119] This example compares the effects of four different concentrations of sodium deoxycholate in protein extraction solutions on extraction efficiency. Except for step 3 of the sample preparation method, which uses different protein extraction solutions, all other steps are the same as in Example 1.

[0120] Protein extraction solution ①: sodium deoxycholate concentration of 12.0 mmol / L, tris(hydroxymethyl)aminomethane concentration of 20.0 mmol / L, ethylenediaminetetraacetic acid concentration of 2.0 mmol / L, pH 8.0.

[0121] Protein extract ②: sodium deoxycholate concentration 10.8 mmol / L, tris(hydroxymethyl)aminomethane concentration 20.0 mmol / L, ethylenediaminetetraacetic acid concentration 2.0 mmol / L, pH 8.0.

[0122] Protein extract ③: sodium deoxycholate concentration of 9.6 mmol / L, tris(hydroxymethyl)aminomethane concentration of 20.0 mmol / L, ethylenediaminetetraacetic acid concentration of 2.0 mmol / L, pH 8.0.

[0123] Protein extraction solution ④: sodium deoxycholate concentration of 8.4 mmol / L, tris(hydroxymethyl)aminomethane concentration of 20.0 mmol / L, ethylenediaminetetraacetic acid concentration of 2.0 mmol / L, pH 8.0.

[0124] When the sodium deoxycholate concentration drops to 9.6 mmol / L or below (i.e., protein extracts ③ and ④), the solution becomes colloidal when the precipitant is added to remove the protein extract, making it impossible to obtain sufficient supernatant for subsequent experiments, resulting in protein loss. Only when the sodium deoxycholate concentration is 10.8 mmol / L or above (i.e., protein extracts ① and ②) can sufficient supernatant be obtained for subsequent experiments, avoiding protein loss.

[0125] The prepared test solution was further tested, and the results are shown in Table 7.

[0126] Table 7

[0127]

[0128] As shown in Table 7, when the sodium deoxycholate concentration was ≥10.8 mmol / L, the number of proteins and peptides obtained was significantly higher, indicating that the protein extraction efficiency was very high.

[0129] Example 5: Comparison of the effects of different types of enzymes on sample digestion efficiency

[0130] The method of this invention detects peptides after enzymatic hydrolysis. If the length of the enzymatically hydrolyzed peptide is too short or too long, it cannot be detected by chromatography-tandem mass spectrometry, and no clear sequence information can be obtained. This example compares the effects of trypsin and a mixed enzyme (LysC+Trypsin) on enzymatic hydrolysis efficiency. Except for step 5 of the sample preparation method, which uses a different enzyme, all other steps are the same as in Example 1.

[0131] The results of the number of peptides detected, the lysine (K) cleavage failure rate, and the arginine (R) cleavage failure rate in the test solution prepared using different enzymes are shown in Table 8.

[0132] Table 8

[0133]

[0134] As shown in Table 8, the test solution prepared using the mixed enzyme LysC+Trypsin showed better detection of peptides and lysine (K) cleavage failure rate than the test solution prepared using trypsin, while there was no significant difference in arginine (R) cleavage failure rate between the two.

[0135] Example 6: Comparison of the effects of different enzyme dosages on sample enzymatic digestion efficiency

[0136] This example compares the effect of different enzyme (LysC+Trypsin) dosages on enzymatic hydrolysis efficiency. Except for step 5 of the sample preparation method, which uses different enzyme dosages (0.5 μg, 0.05 μg, 0.025 μg), all other steps are the same as in Example 1.

[0137] Table 9 shows the proportion of autoclastized peptides of the mixed enzyme LysC+Trypsin in the test solution prepared with different amounts of enzyme.

[0138] Table 9

[0139]

[0140] As shown in Table 9, compared with an enzyme dosage of 0.5 μg, the proportion of Trypsin autoclasts decreased to below 1% when the enzyme dosage was 0.05 μg and 0.025 μg, meeting the requirements. Compared with 0.05 μg, the number of peptides and proteins both increased significantly when the enzyme dosage was 0.025 μg.

[0141] Furthermore, taking the membranous nephropathy target antigen PLA2R as an example, the effect of different enzyme dosages on the analyte was analyzed. The results are shown in Table 10 and... Figures 1-3 As shown.

[0142] Table 10

[0143]

[0144] As shown in Table 10, when the enzyme dosage is 0.025 μg, the number of peptides and protein coverage are significantly better than when the enzyme dosage is 0.05 μg and 0.5 μg.

[0145] When the enzyme dosage was 0.5 μg, the chromatogram of the test solution showed a high trypsin autolysis peak (maximum peak at 15.35 min) within the effective elution gradient time (40 min). Figure 1This is because, under high concentrations and prolonged incubation, enzymes undergo a certain degree of self-degradation or autocleavage, producing a considerable number of trypsin autocleavage peptides. These peptides compete with sample peptides for ionization during mass spectrometry analysis, diluting the sample peptide signal and causing many low-abundance peptides to go undetected. Therefore, excessive enzyme may increase the mass spectrometry background signal, masking low-abundance target peptides. However, when the enzyme dosage was 0.05 μg and 0.025 μg, no obvious trypsin autocleavage peak was found in the chromatogram of the test solution. Figure 2 , Figure 3 ).

[0146] Based on the results of this embodiment, when the enzyme dosage is 0.025 μg, while ensuring good enzymatic hydrolysis efficiency, interference with the target protein can be reduced, and the coverage of the target protein and data reliability can be improved.

[0147] Example 7: Comparison of the effects of different enzyme reaction times on sample enzymatic digestion efficiency

[0148] Enzyme reaction time directly affects the efficiency and thoroughness of enzymatic digestion. Too short a reaction time may lead to incomplete digestion, resulting in longer peptides or uncuttered protein fragments, reducing the sensitivity and peptide coverage of mass spectrometry detection. Too long a reaction time may lead to over-digestion, potentially causing non-specific cleavage and producing unexpected peptides, interfering with data analysis. This example compares the effects of different enzyme reaction times on digestion efficiency to find a balance between specificity and efficiency, improving the yield and detectability of target peptides.

[0149] Except for step 5 of the test sample preparation method, which involves adding 0.025 μg of mixed enzyme and incubating at 37°C for different times, all other steps are the same as in Example 1. The number of peptides detected, the lysine (K) cleavage failure rate, and the arginine (R) cleavage failure rate of the test sample solutions prepared with different enzyme reaction times are shown in Table 11.

[0150] Table 11

[0151]

[0152] As can be seen from the results in Table 11, the number of K and R missing cuts was high when the enzyme reaction was 2 h. There was no significant difference in the number of K and R missing cuts when the enzyme reaction was 3 h, 4 h and 8 h. Therefore, the optimal reaction time for the protease is 3 h to 4 h.

[0153] Example 8: Comparison of the effects of different precipitants on vacuum concentration and drying time

[0154] In the protein extraction solution of this invention, sodium deoxycholate is used as a protein lysis reagent; however, it interferes with mass spectrometry ionization and needs to be removed during the preparation of the test solution. This embodiment compares the removal effects of sodium deoxycholate using 4% formic acid solution and 4% trifluoroacetic acid solution as precipitants.

[0155] Except for step 8 of the sample preparation method, which involves adding different precipitants, all other steps are the same as in Example 1.

[0156] The results showed that both 4% formic acid solution and 4% trifluoroacetic acid solution could precipitate sodium deoxycholate. However, when using 4% formic acid solution as the precipitant, it took 95 minutes to vacuum concentrate and dry 50 μL of the sample, while when using 4% trifluoroacetic acid solution as the precipitant, it only took 60 minutes.

[0157] Therefore, a 4% trifluoroacetic acid solution was chosen as the precipitant for the preparation of the test solution in this invention.

[0158] Example 9: Comparison of the effects of different tissue dosages on test results

[0159] This embodiment compares the effect of different tissue amounts on the detection results. Except for step 2 of the sample preparation method, different amounts of tissue (160,000 μm) were used. 2 220000 μm 2 250000 μm 2 280000 μm 2 Except for the steps described in Example 1, all other steps were the same. The number of proteins and peptides detected in the test solutions prepared using different amounts of tissue are shown in Table 12.

[0160] Table 12

[0161]

[0162] As shown in Table 12, the tissue dosage of 250,000 μm... 2 280000 μm 2 In comparison, when the tissue dosage was 220,000 μm 2 and 160,000 μm 2 At that time, the number of detected peptides and proteins will decrease significantly.

[0163] Example 10: Comparison of the effects of reductive alkylation on detection results

[0164] This example uses PLA2R, THS7A, and FAT1 membranous nephropathy as examples to compare the impact of whether or not a reductive alkylation step is performed on the detection results. Except for the absence of steps 6 and 7 in the sample preparation method, and the omission of reductive alkylation (i.e., no incubation with dithiothreitol solution and iodoacetamide solution), all other steps are the same as in Example 1. The results are shown in Table 13.

[0165] Table 13

[0166]

[0167] As shown in Table 13, compared with the samples with reductive alkylation, the number of PLA2R, THS7A, and FAT1 protein spectra and the protein coverage were significantly reduced in the samples without reductive alkylation.

[0168] Example 11: Comparison of the effects of the order of enzymatic hydrolysis-reductive alkylation steps on detection results

[0169] This embodiment uses PLA2R, THS7A, and FAT1 membranous nephropathy as examples to compare the effect of the order of enzymatic digestion-reductive alkylation steps on the detection results. Except for the exchange of the order of steps 5 and 6-7 in the sample preparation method, all other steps are the same as in Example 1. The results are shown in Table 14.

[0170] Table 14

[0171]

[0172] As shown in Table 14, the number of PLA2R, THS7A, and FAT1 protein spectra and the protein coverage were significantly lower in the samples that were first reduced and alkylated and then enzymatically digested.

[0173] Example 12 Comparison of the effects of different gradient elution programs on detection results

[0174] This embodiment compares the effects of different nano-pump liquid phase gradients on the detection results. Except for the different elution gradients (A / B / C, as shown in Tables 15-17) used in the chromatographic conditions, all other steps are the same as in Example 1.

[0175] Table 15

[0176]

[0177] Table 16

[0178]

[0179] Table 17

[0180]

[0181] The number of proteins and peptides detected using different elution programs is shown in Table 18, and the chromatograms are shown below. Figures 4-6 As shown.

[0182] Table 18

[0183]

[0184] As shown in Table 18, compared with elution programs B and C, the number of peptides and proteins detected by elution program A was significantly higher.

[0185] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0186] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for detecting target antigens of membranous nephropathy, characterized in that, Includes the following steps: (1) Add protein extraction solution to the glomerular tissue to be tested and incubate at 90℃~110℃ for 40 min~50 min; the protein extraction solution includes 10.8 mmol / L~12.0 mmol / L sodium deoxycholate, 18.0 mmol / L~22.0 mmol / L tris(hydroxymethyl)aminomethane and 1.8 mmol / L~2.2 mmol / L ethylenediaminetetraacetic acid; (2) Add a mixture of LysC enzyme and Trypsin enzyme and incubate at 36℃~38℃ for 3 h~8 h; (3) Add dithiothreitol solution and incubate at 36℃~38℃ for 20 to 40 minutes; (4) Add iodoacetamide solution and incubate at room temperature in the dark for 20 to 40 minutes; (5) Add precipitant, sonicate, centrifuge, and load the supernatant onto C. 18 Small column, elute the small column, and collect the eluent; (6) The eluent is frozen and dried, and then reconstituted with the sample solution to obtain the test solution; (7) The test solution was detected by liquid chromatography-tandem mass spectrometry.

2. The method for detecting target antigens of membranous nephropathy according to claim 1, characterized in that, The protein extraction solution in step (1) includes 11.0 mmol / L~12.0 mmol / L sodium deoxycholate, 19.0 mmol / L~21.0 mmol / L tris(hydroxymethyl)aminomethane and 1.9 mmol / L~2.1 mmol / L ethylenediaminetetraacetic acid; Preferably, the protein extract comprises 11.5 mmol / L to 12.0 mmol / L sodium deoxycholate, 20.0 mmol / L to 21.0 mmol / L tris(hydroxymethyl)aminomethane and 2.0 mmol / L to 2.1 mmol / L ethylenediaminetetraacetic acid.

3. The method for detecting target antigens of membranous nephropathy according to claim 1, characterized in that, In step (1), a laser microdissection device is used to obtain the glomerular tissue to be tested; And / or, the volume of the glomerular tissue in step (1) is (250000 μm) 2 ~300000 μm 2 (7 μm~8 μm); Preferably, the volume of the glomerular tissue is 280,000 μm. 2 ~300000 μm 2 (7 μm~8 μm).

4. The method for detecting target antigens of membranous nephropathy according to claim 1, characterized in that, The ratio of glomerular tissue to protein extract in step (1) is (250000 μm) 2 ~300000 μm 2 )*(7 μm~8 μm): 50 μL; And / or, the incubation temperature in step (1) is 95℃~105℃, and the incubation time is 44 min~46 min.

5. The method for detecting target antigens of membranous nephropathy according to claim 1, characterized in that, In step (2), the ratio of LysC enzyme to Trypsin enzyme in the mixed enzyme is 0.8~1.2:

1.

6. The method for detecting target antigens of membranous nephropathy according to claim 1, characterized in that, The ratio of the mixed enzyme in step (2) to the glomerular tissue in step (1) is 0.02 μg~0.04 μg: (250000 μm 2 ~300000 μm 2 (7 μm~8 μm), preferably 0.025 μg~0.030 μg: (250000 μm) 2 ~300000 μm 2 (7 μm~8 μm); and / or the incubation time described in step (2) is 3 h~4 h.

7. The method for detecting target antigens of membranous nephropathy according to claim 1, characterized in that, The concentration of the dithiothreitol solution mentioned in step (3) is 4 mmol / L ~ 6 mmol / L; And / or, the concentration of the iodoacetamide solution in step (4) is 14 mmol / L ~ 16 mmol / L.

8. The method for detecting target antigens of membranous nephropathy according to claim 1, characterized in that, The precipitant mentioned in step (5) is a 3.5%~4.5% aqueous solution of trifluoroacetic acid or a 3.5%~4.5% aqueous solution of formic acid, preferably a 3.8%~4.2% aqueous solution of trifluoroacetic acid; And / or, the ratio of the precipitant in step (5) to the protein extract in step (1) is 4 μL~6 μL:50 μL, preferably 4.5 μL~5.5 μL:50 μL; And / or, the solvent used for elution in step (5) is a mixture of acetonitrile, water and trifluoroacetic acid in a volume ratio of 55~65:35~45:0.08~0.12; And / or, the freezing temperature in step (6) is -82℃ to -78℃, and the freezing time is 15 min to 25 min; And / or, the loading solution in step (6) is a 0.08%~0.12% aqueous solution of trifluoroacetic acid.

9. The method for detecting target antigens of membranous nephropathy according to any one of claims 1 to 8, characterized in that, The chromatographic conditions for liquid chromatography described in step (7) include: Mobile phase A: 0.08%~0.12% formic acid aqueous solution; Mobile phase B: 75%~85% acetonitrile solution containing 0.08%~0.12% formic acid; Injection volume: 14 μL~16 μL; The liquid phase gradient program for the sample loading pump was as follows: 0 → 5 min, flow rate 5.000 μL / min; 5 min → 6 min, flow rate 5.000 μL / min → 1.000 μL / min; 6 min → 45 min, flow rate 1.000 μL / min; 45 min → 46 min, flow rate 1.000 μL / min → 5.000 μL / min; 46 min → 50 min, flow rate 5.000 μL / min; 0 → 50 min, 100% mobile phase A; The liquid phase gradient program for the nano-pump is as follows: 0 → 5 min, 90% mobile phase A; 5 min → 35 min, 90% mobile phase A → 55% mobile phase A; 35 min → 40 min, 55% mobile phase A → 10% mobile phase A; 40 min → 45 min, 10% mobile phase A; 45 min → 46 min, 10% mobile phase A → 90% mobile phase A; 46 min → 50 min, 90% mobile phase A; 0 → 50 min, flow rate 0.3 μL / min; And / or, the parameters of the mass spectrometer described in step (7) include: Resolution: 70000 (MS1), 17500 (MS2); MS1 scan range: 300 m / z - 2000 m / z; Collision energy: 30% HCD; Isolation window: 2.0 m / z (DDA); Maximum injection time: 50 ms (MS1) / 200 ms (MS2).

10. The method for detecting target antigens of membranous nephropathy according to any one of claims 1 to 8, characterized in that, The target antigens for membranous nephropathy include PLA2R, THSD7A, NELL1, SEMA3B, VASN, HTRA1, NTNG1, EXT1, EXT2, NCAM1, CNTN1, FAT1, NDNF, and / or PCSK6.

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