Urine marker for ulcerative colitis as well as analysis method and application thereof

Abstract

The ulcerative colitis urine marker can be effectively used for diagnosis and treatment monitoring of ulcerative colitis, and is non-invasive, high in sensitivity and accurate and reliable in result. The ulcerative colitis urine marker at least comprises one of an immune globulin kappa connecting region 1, an immune globulin kappa variable region 6-21 and the like; the urine marker can be used for disease monitoring and at least comprises one of protein 4 rich in glutamic acid, presumed teratoma-derived growth factors 3 and the like.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, and more particularly to a set of urinary biomarkers for ulcerative colitis, analytical methods for these urinary biomarkers for ulcerative colitis, and the application of these urinary biomarkers for ulcerative colitis in the preparation of diagnostic kits for differential diagnosis of ulcerative colitis. Background Technology

[0002] Ulcerative colitis is a common intestinal disease in clinical practice. Current diagnostic methods mainly rely on colonoscopy. However, colonoscopy is a poor patient experience, and pathological examination is invasive, making it unsuitable for disease screening and monitoring. In recent years, urinary proteomics technology has been increasingly applied to the early screening and diagnosis of the disease. Related studies have shown that various disease-related proteins exist in urine and can serve as potential biomarkers.

[0003] Currently, urinary proteomics technology is developing rapidly and has become an important non-invasive detection method. Through techniques such as mass spectrometry, comprehensive qualitative and quantitative analysis of proteins in urine can be performed. Researchers have discovered that the expression levels of certain proteins in urine differ significantly across different disease states. These protein changes can reflect the pathological state of the body, providing new insights for early disease diagnosis. Although some studies have explored the application of urinary proteomics in other diseases, research on ulcerative colitis remains relatively scarce. Currently, there are no urinary protein markers that can be used to diagnose ulcerative colitis. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, the technical problem to be solved by the present invention is to provide a set of urinary biomarkers for ulcerative colitis, which can be effectively used for the diagnosis and monitoring of ulcerative colitis, and are non-invasive, highly sensitive, and provide accurate and reliable results.

[0005] The technical solution of the present invention is: this urinary marker for ulcerative colitis includes at least one of the following substances: immunoglobulin κ linker 1, immunoglobulin κ variable region 6-21, kinin light chain 2, keratin, and type II cytoskeleton 1b.

[0006] It also provides urine markers for monitoring ulcerative colitis, including at least one of the following: glutamate-rich protein 4, and putative teratoma-derived growth factor 3.

[0007] The urinary biomarkers and analytical methods for ulcerative colitis of the present invention can be effectively used for the diagnosis or monitoring of ulcerative colitis. They are non-invasive, highly sensitive, and provide accurate and reliable results, providing a basis for clinical decision-making. They also provide a foundation for subsequent basic and clinical research, and have significant application and research value.

[0008] It also provides analytical methods for biomarkers, which can be used to obtain the biomarker levels in biological samples of the test subject through one or more of the following methods: mass spectrometry, chemical analysis, and immunoassay.

[0009] It also provides applications for urinary markers of ulcerative colitis in the preparation of diagnostic kits for ulcerative colitis.

[0010] It also provides applications for urine markers in monitoring ulcerative colitis, used to prepare kits for monitoring ulcerative colitis. Attached Figure Description

[0011] Figure 1 This is a basic flowchart illustrating the research on differential diagnostic markers for ulcerative colitis according to the present invention.

[0012] Figure 2 The diagram shows the differentially expressed protein HCA plot and volcano plot between the ulcerative colitis patient group and the healthy control group in Example 1 of this invention. Figure 2 A is the HCA cluster analysis diagram, where light blue represents the healthy control group and pink represents the ulcerative colitis group. The results show that the two groups of patients can be significantly distinguished. Figure 2 B is a volcano plot, which shows the differential protein distribution between the two groups of patients. Detailed Implementation

[0013] This urinary biomarker for ulcerative colitis includes at least one of the following substances: immunoglobulin κ linker 1, immunoglobulin κ variable region 6-21, kinin light chain 2, keratin, and type II cytoskeleton 1b.

[0014] Furthermore, urinary markers for ulcerative colitis include at least one of the following substances: natural killer cell antigen CD94, natural cytotoxicity trigger receptor 1, glutathione peroxidase 1, and serum amyloid A-2.

[0015] It also provides urine markers for monitoring ulcerative colitis, including at least one of the following: glutamate-rich protein 4, and putative teratoma-derived growth factor 3.

[0016] Furthermore, urine markers for monitoring ulcerative colitis include at least one of the following substances: C-type lectin domain family 7 member A, transforming growth factor-β inducible protein ig-h3, or pendrin protein.

[0017] The urinary biomarkers and analytical methods for ulcerative colitis of the present invention can be effectively used for the diagnosis or monitoring of ulcerative colitis. They are non-invasive, highly sensitive, and provide accurate and reliable results, providing a basis for clinical decision-making. They also provide a foundation for subsequent basic and clinical research, and have significant application and research value.

[0018] It also provides analytical methods for biomarkers, which can be used to obtain the biomarker levels in biological samples of the test subject through one or more of the following methods: mass spectrometry, chemical analysis, and immunoassay.

[0019] Preferably, the chemical analysis method includes electrochemical analysis, radiochemical analysis, and enzymatic methods; the immunoassay method includes radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), time-resolved fluorescence immunoassay, gold nanoparticle immunoassay, and immunosensor analysis; the mass spectrometry method is triple quadrupole mass spectrometry, ion trap mass spectrometry, orbital trap mass spectrometry, and matrix-assisted laser desorption / ionization time-of-flight mass spectrometry. The method for detecting protein biomarkers using mass spectrometry involves first performing gradient elution on a chromatographic column, followed by data acquisition under electrospray ionization (ESI). In this mass spectrometry method, all biomarkers are analyzed in a single injection.

[0020] Preferably, the biological sample is urine.

[0021] Preferably, the urine is precipitated with acetone organic solvent, then dissolved in a lysis solution containing 8 mol / L urea, and the protein concentration is determined using a Bicinchoninic acid (BCA) kit or Bradford protein quantification method for mass spectrometry pretreatment.

[0022] It also provides applications for urinary markers of ulcerative colitis in the preparation of diagnostic kits for ulcerative colitis.

[0023] It also provides applications for urine markers in monitoring ulcerative colitis, used to prepare kits for monitoring ulcerative colitis.

[0024] The embodiments of the present invention will be described in detail below.

[0025] Example 1

[0026] Urine proteomics technology was used to screen biomarkers for the differential diagnosis of ulcerative colitis.

[0027] 1. Sample source:

[0028] With the approval of the Ethics Committee of the China-Japan Friendship Hospital, samples were collected from 17 healthy individuals and 32 individuals with ulcerative colitis. All participants were from the China-Japan Friendship Hospital, and all patients were definitively diagnosed through colonoscopy and histopathological examination. Specimens were collected after clinical testing. All samples were stored at -80℃ until use.

[0029] 2. Instrument: Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Scientific).

[0030] 3. Main reagents:

[0031] Trypsin (Promega); C18 solid-phase extraction column (3CC, 60mg, Waters); C18 reversed-phase chromatography column (4.6mm × 250mm, C18, 3μm, Waters); LC / MS grade acetonitrile was purchased from Merck, HPLC grade methanol from Merck, and formic acid from CNW. All other reagents were commercially available analytical grade. Deionized water was prepared using the Milli-Q ultrapure water system from Millipore.

[0032] 4. Research Methods:

[0033] 4.1 Sample preparation:

[0034] Urine samples were extracted using acetone precipitation and then digested with enzymes on membrane (FASP). The protein samples to be digested were diluted with 25 mM NH4HCO3 (sample:25 mM NH4HCO3 = 1:3, volume ratio), vortexed for 30 s–1 min. 1 M DTT was added to the sample tube to achieve a final concentration of 20 mM (DTT is a reducing agent that breaks disulfide bonds; if the total volume after dilution is 100 μL, 2 μL of 1 M DTT should be added, i.e., 2 μL of 1 M DTT in 100 μL of solution results in a final concentration of 20 mM). The samples were fixed on a water float and placed in a water bath at 95°C for 5 min. Remove the sample from the water bath and allow it to reach room temperature. Then, add 1M IAA to the tube to achieve a final concentration of 50mM in the solution (alkylation; if the total volume after dilution is 100ul, add 5ul of 1M IAA, i.e., 5ul of 1M IAA in 100ul solution results in a final concentration of 50mM). Incubate the sample in the dark for 45 minutes, then centrifuge at 12000rpm for 5-10 minutes (make sure to balance the liquid). Take out a new 30K filter membrane and add 100ul of UA to the filter tube inside the 30K membrane (the filter tube consists of two parts: an outer white collection tube and an inner tube with a membrane at the bottom, in which all solutions will be added). Cover the tube and centrifuge at 14000g for 1 minute. This step can be repeated 2-3 times. After centrifugation, the reductively alkylated sample appears as two layers in the EP tube: the lower layer is the precipitate produced by reductive alkylation, and the upper layer is the reductively alkylated protein sample. The upper layer is slowly aspirated into a 30K filter tube treated with UA (UA treatment can be performed 45 minutes after alkylation). The 10K filter membrane containing the protein sample is placed in a centrifuge and centrifuged at 14000g for 10-30 minutes (until all liquid on the inner membrane is removed). After thorough filtration, 200µl of UA is added to the inner tube, pipetted, vortexed for 1 minute, and centrifuged at 14000g for 10-30 minutes (until all liquid on the inner membrane is removed). This step can be repeated twice (this step is to remove DTT and IAA, as DTT and IAA are small molecules that affect peptide detection). Add 200 μL of 25 mM NH4HCO3 to the inner tube of the filter tube, pipette, vortex for 1 min, centrifuge at 14000g for 15-30 min (until all liquid on the inner tube membrane is completely removed), discard the waste liquid in the collection tube, repeat this step twice (change the buffer, because we will be using Trypsin enzyme later, and Trypsin enzyme has a better enzymatic digestion effect in 25 mM NH4HCO3, so the buffer needs to be changed).Add 200 μL of 25 mM NH4HCO3 to the inner sleeve of the filter tube, add an appropriate amount of Trypsin enzyme (at a protein:Trypsin enzyme mass ratio of 1:50), and vortex for 1 min. Fix the sample with added trypsin on a water float. Fill a 1 L beaker with water, place the float on the water surface, and microwave on high for 1 min. Finally, place the sample in a water bath at 37°C for 16-24 h. The next day, remove the sample, centrifuge at 14000 g for 1-10 min, add 50-100 μL of 500 mM NaCl, repeatedly pipette, centrifuge at 14000 g for 1-10 min, and transfer the liquid from the collection tube into a new EP tube. (A solid-phase extraction step can be added here; see the solid-phase extraction steps below for details.) Ideally, the volume of each EP tube should be approximately 500 μL to facilitate the subsequent vacuum drying. Place the sample from the previous step into a vacuum dryer at -1 level, and after drying, dissolve it with 1 / 1000 FA (the dissolution concentration is generally 5ug / ul).

[0035] 4.2 Chromatographic / Mass Spectrometry Conditions:

[0036] An Orbitrap Exploris 480 (Thermo Scientific) and an EASY nLC 1000 were used in combination for data analysis in DIA-MS mode. Digested peptides were separated on an RP C18 self-filled capillary LC column (75 μm × 100 mm; particle size 3 μm). The elution gradient was 5–30% buffer B2 (0.1% formic acid, 99.9% ACN; flow rate, 0.3 μL / min), with peptide elution for 25 min.

[0037] For DIA analysis, MS acquisition was performed using a variable isolation window with 60 windows. The number of precursor ions was equal in each isolation window based on the precursor m / z distribution of the pooled samples. The full scan range was set to 350 to 1200 m / z with a resolution of 120,000, followed by a DIA scan at a resolution of 30,000 (High-energy C-trap dissociation [HCD] collision energy: 30%; AGC target: 200%; Maximum injection time: 50 ms).

[0038] 4.3 Data Processing:

[0039] Raw DIA data were analyzed using Spectronaut Pulsar 17.1 (Biognosys) at default settings. In short, the retention time prediction type was set to Dynamic iRT. Interference correction at the MS2 level was enabled. Peptide intensities were calculated by summing the peak areas of the respective fragment ions in MS2, and protein intensities were calculated by summing the intensities of the respective peptides. Cross-run normalization was enabled to correct for systematic variance in LC-MS / MS performance, and a local normalization strategy was used. Normalization was based on the assumption that, on average, a similar number of peptides are upregulated and downregulated, and that most peptides in the sample are not regulated at different runs or retention times. Protein inference was performed using the ID selector algorithm implemented in Spectronaut. All results were filtered with a Q-value cutoff of 0.01 (corresponding to 1% FDR).

[0040] 5. Data processing and statistical analysis:

[0041] Missing proteomics data in the samples were independently imputed using the sequential k-nearest neighbor method. A t-test was used for statistical analysis of all quantitative data. Statistical significance was defined as a fold change >2-fold, with a corrected P < 0.01. Candidate biomarkers for the differential diagnosis of ulcerative colitis were screened.

[0042] 6. Results:

[0043] A total of 4672 proteins were identified, and 2653 of them were quantitatively analyzed. Significant changes were found in 451 proteins, with a fold change >2-fold (corrected P < 0.01). Using MetaboAnalyst 6.0, the area under the ROC curve was calculated, and 220 protein molecules with an AUC value ≥ 0.8 were selected. These can be considered as candidate biomarkers for the diagnosis of ulcerative colitis. The results are shown in Table 1.

[0044] Table 1

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052] Example 2

[0053] 1. Validation of candidate markers for the diagnosis of ulcerative colitis using proteomics techniques.

[0054] 1. Sample collection:

[0055] Eleven individuals with normal colonoscopy results and ten patients diagnosed with ulcerative colitis were included in this study. All participants were from the China-Japan Friendship Hospital, and all patients were definitively diagnosed through colonoscopy and histopathological examination. Specimens were collected after clinical testing. All samples were stored at -80℃ until use.

[0056] 2. Instrument: Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Scientific).

[0057] 3. Main reagents:

[0058] Trypsin (Promega); C18 solid-phase extraction column (3CC, 60mg, Waters); C18 reversed-phase chromatography column (4.6mm × 250mm, C18, 3μm, Waters); LC / MS grade acetonitrile was purchased from Merck, HPLC grade methanol from Merck, and formic acid from CNW. All other reagents were commercially available analytical grade. Deionized water was prepared using the Milli-Q ultrapure water system from Millipore.

[0059] 4. Research Methods:

[0060] 4.1 Sample preparation:

[0061] Urine samples were extracted using acetone precipitation and then digested with enzymes on membrane (FASP). The protein samples to be digested were diluted with 25 mM NH4HCO3 (sample:25 mM NH4HCO3 = 1:3, volume ratio), vortexed for 30 s–1 min. 1 M DTT was added to the sample tube to achieve a final concentration of 20 mM (DTT is a reducing agent that breaks disulfide bonds; if the total volume after dilution is 100 μL, 2 μL of 1 M DTT should be added, i.e., 2 μL of 1 M DTT in 100 μL of solution results in a final concentration of 20 mM). The samples were fixed on a water float and placed in a water bath at 95°C for 5 min. Remove the sample from the water bath and allow it to reach room temperature. Then, add 1M IAA to the tube to achieve a final concentration of 50mM in the solution (alkylation; if the total volume after dilution is 100ul, add 5ul of 1M IAA, i.e., 5ul of 1M IAA in 100ul solution results in a final concentration of 50mM). Incubate the sample in the dark for 45 minutes, then centrifuge at 12000rpm for 5-10 minutes (make sure to balance the liquid). Take out a new 30K filter membrane and add 100ul of UA to the filter tube inside the 30K membrane (the filter tube consists of two parts: an outer white collection tube and an inner tube with a membrane at the bottom, in which all solutions will be added). Cover the tube and centrifuge at 14000g for 1 minute. This step can be repeated 2-3 times. After centrifugation, the reductively alkylated sample appears as two layers in the EP tube: the lower layer is the precipitate produced by reductive alkylation, and the upper layer is the reductively alkylated protein sample. The upper layer is slowly aspirated into a 30K filter tube treated with UA (UA treatment can be performed 45 minutes after alkylation). The 10K filter membrane containing the protein sample is placed in a centrifuge and centrifuged at 14000g for 10-30 minutes (until all liquid on the inner membrane is removed). After thorough filtration, 200µl of UA is added to the inner tube, pipetted, vortexed for 1 minute, and centrifuged at 14000g for 10-30 minutes (until all liquid on the inner membrane is removed). This step can be repeated twice (this step is to remove DTT and IAA, as DTT and IAA are small molecules that affect peptide detection). Add 200 μL of 25 mM NH4HCO3 to the inner tube of the filter tube, pipette, vortex for 1 min, centrifuge at 14000g for 15-30 min (until all liquid on the inner tube membrane is completely removed), discard the waste liquid in the collection tube, repeat this step twice (change the buffer, because we will be using Trypsin enzyme later, and Trypsin enzyme has a better enzymatic digestion effect in 25 mM NH4HCO3, so the buffer needs to be changed).Add 200 μL of 25 mM NH4HCO3 to the inner sleeve of the filter tube, add an appropriate amount of Trypsin enzyme (at a protein:Trypsin enzyme mass ratio of 1:50), and vortex for 1 min. Fix the sample with added trypsin on a water float. Fill a 1 L beaker with water, place the float on the water surface, and microwave on high for 1 min. Finally, place the sample in a water bath at 37°C for 16-24 h. The next day, remove the sample, centrifuge at 14000 g for 1-10 min, add 50-100 μL of 500 mM NaCl, repeatedly pipette, centrifuge at 14000 g for 1-10 min, and transfer the liquid from the collection tube into a new EP tube. (A solid-phase extraction step can be added here; see the solid-phase extraction steps below for details.) Ideally, the volume of each EP tube should be approximately 500 μL to facilitate the subsequent vacuum drying. Place the sample from the previous step into a vacuum dryer at -1 level, and after drying, dissolve it with 1 / 1000 FA (the dissolution concentration is generally 5ug / ul).

[0062] 4.2 Chromatographic / Mass Spectrometry Conditions:

[0063] An Orbitrap Exploris 480 (Thermo Scientific) and an EASY nLC 1000 were used in combination for data analysis in DIA-MS mode. Digested peptides were separated on an RP C18 self-filled capillary LC column (75 μm × 100 mm; particle size 3 μm). The elution gradient was 5–30% buffer B2 (0.1% formic acid, 99.9% ACN; flow rate, 0.3 μL / min), with peptide elution for 25 min.

[0064] For DIA analysis, MS acquisition was performed using a variable isolation window with 60 windows. The number of precursor ions was equal in each isolation window based on the precursor m / z distribution of the pooled samples. The full scan range was set to 350 to 1200 m / z with a resolution of 120,000, followed by a DIA scan at a resolution of 30,000 (High-energy C-trap dissociation [HCD] collision energy: 30%; AGC target: 200%; Maximum injection time: 50 ms).

[0065] 4.3 Data Processing:

[0066] Raw DIA data were analyzed using Spectronaut Pulsar 17.1 (Biognosys) at default settings. In short, the retention time prediction type was set to Dynamic iRT. Interference correction at the MS2 level was enabled. Peptide intensities were calculated by summing the peak areas of the respective fragment ions in MS2, and protein intensities were calculated by summing the intensities of the respective peptides. Cross-run normalization was enabled to correct for systematic variance in LC-MS / MS performance, and a local normalization strategy was used. Normalization was based on the assumption that, on average, a similar number of peptides are upregulated and downregulated, and that most peptides in the sample are not regulated at different runs or retention times. Protein inference was performed using the ID selector algorithm implemented in Spectronaut. All results were filtered with a Q-value cutoff of 0.01 (corresponding to 1% FDR).

[0067] 4.4 Data Processing and Statistical Analysis:

[0068] Missing proteomics data in the samples were independently imputed using the sequential k-nearest neighbor method. A t-test was used for statistical analysis of all quantitative data. The area under the ROC curve was calculated using MetaboAnalyst 6.0, and protein molecules with an AUC value ≥ 0.8 were selected as high-efficiency markers.

[0069] 5. Analysis of verification results:

[0070] Candidate protein marker molecules were validated using urinary proteomics technology. The area under the ROC curve was calculated, and markers with an AUC value greater than or equal to 0.8 in both the experimental and validation groups were considered highly efficient. The validation results are shown in Table 2.

[0071] Table 2

[0072] protein name Database entry name AUC value of the experimental group AUC value of validation group Natural killer cell antigen CD94 KLRD1_HUMAN 0.97 0.86 kinin light chain 2 KLC2_HUMAN 0.95 0.85 Immunoglobulin κ linker 1 KJ01_HUMAN 0.94 0.92 Immunoglobulin κ variable region 6-21 KV621_HUMAN 0.92 0.84 Natural cytotoxicity triggering receptor 1 NCTR1_HUMAN 0.89 0.85 Glutathione peroxidase 1 GPX1_HUMAN 0.85 0.85 Keratin, type II cytoskeleton 1b K2C1B_HUMAN 0.91 0.93 Serum amyloid A-2 SAA2_HUMAN 0.85 0.97

[0073] Example 3

[0074] The use of proteomics techniques to validate candidate diagnostic markers for ulcerative colitis is applicable to disease monitoring.

[0075] 1. Sample collection:

[0076] Ten patients diagnosed with ulcerative colitis were included in this study. Urine samples were collected before treatment and after significant improvement in their condition following treatment. All participants were from the China-Japan Friendship Hospital. Specimens were collected after clinical testing. All samples were stored at -80℃ until use.

[0077] 2. Instrument: Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Scientific).

[0078] 3. Main reagents:

[0079] Trypsin (Promega); C18 solid-phase extraction column (3CC, 60mg, Waters); C18 reversed-phase chromatography column (4.6mm × 250mm, C18, 3μm, Waters); LC / MS grade acetonitrile was purchased from Merck, HPLC grade methanol from Merck, and formic acid from CNW. All other reagents were commercially available analytical grade. Deionized water was prepared using the Milli-Q ultrapure water system from Millipore.

[0080] 4. Research Methods:

[0081] 4.1 Sample preparation:

[0082] Urine samples were extracted using acetone precipitation and then digested with enzymes on membrane (FASP). The protein samples to be digested were diluted with 25 mM NH4HCO3 (sample:25 mM NH4HCO3 = 1:3, volume ratio), vortexed for 30 s–1 min. 1 M DTT was added to the sample tube to achieve a final concentration of 20 mM (DTT is a reducing agent that breaks disulfide bonds; if the total volume after dilution is 100 μL, 2 μL of 1 M DTT should be added, i.e., 2 μL of 1 M DTT in 100 μL of solution results in a final concentration of 20 mM). The samples were fixed on a water float and placed in a water bath at 95°C for 5 min. Remove the sample from the water bath and allow it to reach room temperature. Then, add 1M IAA to the tube to achieve a final concentration of 50mM in the solution (alkylation; if the total volume after dilution is 100ul, add 5ul of 1M IAA, i.e., 5ul of 1M IAA in 100ul solution results in a final concentration of 50mM). Incubate the sample in the dark for 45 minutes, then centrifuge at 12000rpm for 5-10 minutes (make sure to balance the liquid). Take out a new 30K filter membrane and add 100ul of UA to the filter tube inside the 30K membrane (the filter tube consists of two parts: an outer white collection tube and an inner tube with a membrane at the bottom, in which all solutions will be added). Cover the tube and centrifuge at 14000g for 1 minute. This step can be repeated 2-3 times. After centrifugation, the reductively alkylated sample appears as two layers in the EP tube: the lower layer is the precipitate produced by reductive alkylation, and the upper layer is the reductively alkylated protein sample. The upper layer is slowly aspirated into a 30K filter tube treated with UA (UA treatment can be performed 45 minutes after alkylation). The 10K filter membrane containing the protein sample is placed in a centrifuge and centrifuged at 14000g for 10-30 minutes (until all liquid on the inner membrane is removed). After thorough filtration, 200µl of UA is added to the inner tube, pipetted, vortexed for 1 minute, and centrifuged at 14000g for 10-30 minutes (until all liquid on the inner membrane is removed). This step can be repeated twice (this step is to remove DTT and IAA, as DTT and IAA are small molecules that affect peptide detection). Add 200 μL of 25 mM NH4HCO3 to the inner tube of the filter tube, pipette, vortex for 1 min, centrifuge at 14000g for 15-30 min (until all liquid on the inner tube membrane is completely removed), discard the waste liquid in the collection tube, repeat this step twice (change the buffer, because we will be using Trypsin enzyme later, and Trypsin enzyme has a better enzymatic digestion effect in 25 mM NH4HCO3, so the buffer needs to be changed).Add 200 μL of 25 mM NH4HCO3 to the inner sleeve of the filter tube, add an appropriate amount of Trypsin enzyme (at a protein:Trypsin enzyme mass ratio of 1:50), and vortex for 1 min. Fix the sample with added trypsin on a water float. Fill a 1 L beaker with water, place the float on the water surface, and microwave on high for 1 min. Finally, place the sample in a water bath at 37°C for 16-24 h. The next day, remove the sample, centrifuge at 14000 g for 1-10 min, add 50-100 μL of 500 mM NaCl, repeatedly pipette, centrifuge at 14000 g for 1-10 min, and transfer the liquid from the collection tube into a new EP tube. (A solid-phase extraction step can be added here; see the solid-phase extraction steps below for details.) Ideally, the volume of each EP tube should be approximately 500 μL to facilitate the subsequent vacuum drying. Place the sample from the previous step into a vacuum dryer at -1 level, and after drying, dissolve it with 1 / 1000 FA (the dissolution concentration is generally 5ug / ul).

[0083] 4.2 Chromatographic / Mass Spectrometry Conditions:

[0084] An Orbitrap Exploris 480 (Thermo Scientific) and an EASY nLC 1000 were used in combination for data analysis in DIA-MS mode. Digested peptides were separated on an RP C18 self-filled capillary LC column (75 μm × 100 mm; particle size 3 μm). The elution gradient was 5–30% buffer B2 (0.1% formic acid, 99.9% ACN; flow rate, 0.3 μL / min), with peptide elution for 25 min.

[0085] For DIA analysis, MS acquisition was performed using a variable isolation window with 60 windows. The number of precursor ions was equal in each isolation window based on the precursor m / z distribution of the pooled samples. The full scan range was set to 350 to 1200 m / z with a resolution of 120,000, followed by a DIA scan at a resolution of 30,000 (High-energy C-trap dissociation [HCD] collision energy: 30%; AGC target: 200%; Maximum injection time: 50 ms).

[0086] 4.3 Data Processing:

[0087] Raw DIA data were analyzed using Spectronaut Pulsar 17.1 (Biognosys) at default settings. In short, the retention time prediction type was set to Dynamic iRT. Interference correction at the MS2 level was enabled. Peptide intensities were calculated by summing the peak areas of the respective fragment ions in MS2, and protein intensities were calculated by summing the intensities of the respective peptides. Cross-run normalization was enabled to correct for systematic variance in LC-MS / MS performance, and a local normalization strategy was used. Normalization was based on the assumption that, on average, a similar number of peptides are upregulated and downregulated, and that most peptides in the sample are not regulated at different runs or retention times. Protein inference was performed using the ID selector algorithm implemented in Spectronaut. All results were filtered with a Q-value cutoff of 0.01 (corresponding to 1% FDR).

[0088] 4.4 Data Processing and Statistical Analysis:

[0089] Missing proteomics data in the samples were independently filled using the sequential k-nearest neighbor method. A t-test was used for statistical analysis of all quantitative data. The area under the ROC curve was calculated using MetaboAnalyst 6.0, and protein molecules with an AUC value ≥ 0.8 were selected as suitable biomarkers for disease monitoring.

[0090] 5. Analysis of verification results:

[0091] Candidate protein marker molecules were validated using urinary proteomics technology. The area under the ROC curve was calculated, and markers with an AUC value greater than or equal to 0.8 in both the experimental and validation groups were deemed suitable for disease monitoring. The validation results are shown in Table 3.

[0092] Table 3

[0093] protein name Database entry name AUC value of the experimental group AUC value of validation group Protein 4 rich in glutamate ERIC4_HUMAN 0.89 0.96 Presumed teratoma-derived growth factor 3 TDGF3_HUMAN 0.91 0.82 C-type lectin domain family 7 member A CLC7A_HUMAN 0.94 0.90 Transforming growth factor-β inducible protein ig-h3 BGH3_HUMAN 0.84 0.85 Pendrin protein S26A4_HUMAN 0.97 0.97

[0094] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. Urinary markers for ulcerative colitis, characterized in that: It includes at least one of the following substances: immunoglobulin κ linker 1, immunoglobulin κ variable region 6-21, kinin light chain 2, keratin, and type II cytoskeleton 1b.

2. The urinary marker for ulcerative colitis according to claim 1, characterized in that: It includes at least one of the following substances: natural killer cell antigen CD94, natural cytotoxicity trigger receptor 1, glutathione peroxidase 1, and serum amyloid A-2.

3. Urinary markers for monitoring ulcerative colitis, characterized in that: It includes at least one of the following substances:

4. Glutamic acid-rich protein; 3. Presumed teratoma-derived growth factor.

4. The urinary marker for monitoring ulcerative colitis according to claim 3, characterized in that: It includes at least one of the following substances: C-type lectin domain family 7 member A, transforming growth factor-β inducible protein ig-h3, and Pendrin protein.

5. The method for analyzing markers according to any one of claims 1-4, characterized in that: The levels of markers in biological samples of the test subject can be obtained through one or more of the following methods: mass spectrometry, chemical analysis, and immunoassay.

6. The method for analyzing markers according to claim 5, characterized in that: The chemical analysis methods include electrochemical analysis, radiochemical analysis, and enzymatic methods; The immunoassays include radioimmunoassay, enzyme-linked immunosorbent assay, time-resolved fluorescence immunoassay, gold nanoparticle immunoassay, and immunosensor analysis. The mass spectrometry method is triple quadrupole mass spectrometry, ion trap mass spectrometry, orbital trap mass spectrometry, and matrix-assisted laser desorption / ionization time-of-flight mass spectrometry. The method for detecting protein biomarkers by mass spectrometry is to first perform gradient elution using a chromatographic column, and then collect data under electrospray ionization (ESI). In the mass spectrometry method, all biomarkers are detected by analyzing a single injection.

7. The method for analyzing markers according to claim 6, characterized in that: The biological sample was urine.

8. The method for analyzing markers according to claim 7, characterized in that: The urine was precipitated with acetone organic solvent, then dissolved in a lysis solution containing 8 mol / L urea, and the protein concentration was determined using the dioctanine acid method BCA kit or the Bradford protein quantification method for mass spectrometry pretreatment.

9. The application of the urinary marker for ulcerative colitis according to claim 1 or 2, characterized in that: A kit for the preparation of diagnostic reagents for ulcerative colitis.

10. The application of the urine marker for monitoring ulcerative colitis according to claim 3 or 4, characterized in that: A kit for preparing a monitoring kit for ulcerative colitis.

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