Benign and malignant urine marker of thyroid nodule as well as analysis method and application thereof

Abstract

The invention discloses a benign and malignant thyroid nodule urine marker, an analysis method and application, which can be effectively used for differential diagnosis of benign and malignant thyroid nodules and are noninvasive, high in sensitivity and accurate and reliable in result. The benign and malignant urine marker for thyroid nodules at least comprises one of ubiquitin-like modification activating enzyme 6, sulfotransferase 2B1 and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical detection, and particularly to a urine biomarker for differentiating benign and malignant thyroid nodules, an analysis method for this urine biomarker for differentiating benign and malignant thyroid nodules, and the application of this urine biomarker for differentiating benign and malignant thyroid nodules in the preparation of a kit for differentiating benign and malignant thyroid nodules. Background Art

[0002] Thyroid nodules are common endocrine diseases clinically, usually divided into two categories: benign and malignant. Existing diagnostic methods mainly rely on means such as ultrasound examination and fine needle aspiration biopsy. However, these methods have certain limitations. For example, the subjectivity of ultrasound examination and the invasiveness of fine needle aspiration biopsy may lead to misdiagnosis or missed diagnosis. In recent years, urine proteomics technology has gradually been applied to the early screening and diagnosis of diseases. Relevant research shows that there are various disease-related proteins in urine, which can be used as potential biomarkers.

[0003] Currently, the development of urine proteomics technology is rapid and has become an important non-invasive detection means. Through technologies such as mass spectrometry analysis, comprehensive qualitative and quantitative analysis of proteins in urine can be carried out. Researchers have found that the expression levels of certain specific proteins in urine are significantly different under different disease states. The changes in these proteins can reflect the pathological state of the body and provide new ideas for the early diagnosis of diseases. Although some studies have explored the application of urine proteomics in other diseases, the research on benign and malignant thyroid nodules is still relatively scarce. Currently, there is no urine protein biomarker available for differentiating benign and malignant thyroid nodules. Summary of the Invention

[0004] To overcome the defects of the prior art, the technical problem to be solved by the present invention is to provide a urine biomarker for differentiating benign and malignant thyroid nodules, which can be effectively used for differentiating benign and malignant thyroid nodules, is non-invasive, has high sensitivity, and the results are accurate and reliable.

[0005] The technical solution of the present invention is as follows: This kind of urine biomarker for benign and malignant thyroid nodules includes at least one of the following substances: ubiquitin-like modifier activating enzyme 6; sulfotransferase 2B1; exportin 1; kallikrein 8; carboxypeptidase D; protein containing SAM and SH3 domains 1; transforming acidic coiled-coil protein 2; apolipoprotein M; adaptor-related protein complex 2 alpha 1 subunit; nucleophosmin; amine oxidase; mitochondrial cytochrome b-c1 complex subunit 2; cytoplasmic threonyl-tRNA synthetase 1; cystathionine gamma-lyase; succinyl-CoA ligase; large ribosomal subunit protein uL5; large ribosomal subunit protein eL38; small ribosomal subunit protein eS21; metallothionein 1H; lamin B2; annexin A8-like protein 1; syntaxin-binding protein 4; ferritin family homolog 3; deaminated glutathione amidase; uncharacterized protein C4orf19; dual specificity tyrosine phosphorylation-regulated kinase 2; transmembrane BAX inhibitor 1; neurogenic locus notch homolog protein 4; semaphorin-4B; CYFIP-related Rac1-interacting protein B; type XVII collagen alpha-1 chain; E3 ubiquitin-protein ligase CHIP; SH3 and multiple ankyrin repeat domains protein 2.

[0006] The urine biomarker for benign and malignant thyroid nodules and the analysis method of the present invention can be effectively used for the differential diagnosis of benign and malignant thyroid nodules, are non-invasive, have high sensitivity, and the results are accurate and reliable, providing a basis for clinical decision-making, and at the same time providing a certain foundation for subsequent basic research and clinical research, and having great application and research value.

[0007] An analysis method for the urine biomarker for benign and malignant thyroid nodules is also provided, and the marker level in the biological sample of the detection object is obtained by one or more of the following methods: mass spectrometry, chemical analysis, immunoassay.

[0008] An application of the urine biomarker for benign and malignant thyroid nodules is also provided, which is used for preparing a kit for differential diagnosis of benign and malignant thyroid nodules. Description of the Drawings

[0009] Figure 1 It shows the basic flow chart of the research on the differential diagnosis marker for benign and malignant thyroid nodules of the present invention.

[0010] Figure 2Shown are the OPLS-DA and Permutation model score plots, the HCA plot of differential proteins, and the volcano plot for the benign thyroid nodule group and the malignant thyroid nodule group in an embodiment of the present invention. Among them, Figure A is the OPLS-DA model score plot, where the green circles represent the benign group and the blue circles represent the malignant group. Figure B is the Permutation model score plot, where the green circles represent the benign group and the blue circles represent the malignant group. It can be clearly seen from the above figures that the model can clearly distinguish between benign nodules and malignant nodules. Figure C is the HCA clustering analysis plot, where light blue represents the benign thyroid nodule group and pink represents the malignant thyroid nodule group, and the results show that the two groups of patients can be significantly distinguished. Figure D is the volcano plot, from which the distribution of differential proteins between the two groups of patients can be seen. Detailed implementation mode

[0011] This urinary biomarker for benign and malignant thyroid nodules includes at least one of the following substances:

[0012] Ubiquitin-like modifier activating enzyme 6; Sulfotransferase 2B1; Exportin 1; Kallikrein 8; Carboxypeptidase D; Protein containing SAM and SH3 domains 1; Transforming acidic coiled-coil protein 2; Apolipoprotein M; Adaptor-related protein complex 2 alpha-1 subunit; Nucleophosmin; Amine oxidase; Mitochondrial cytochrome b-c1 complex subunit 2; Cytoplasmic threonyl-tRNA synthetase 1; Cystathionine gamma-lyase; Succinyl-CoA ligase; Large ribosomal subunit protein uL5; Large ribosomal subunit protein eL38; Small ribosomal subunit protein eS21; Metallothionein 1H; Nuclear lamina B2; Laminin A8-like protein 1; Syntaxin-binding protein 4; Ferritin family homolog 3; Deaminated glutathione amidase; Uncharacterized protein C4orf19; Dual-specificity tyrosine phosphorylation-regulated kinase 2; Transmembrane BAX inhibitor 1; Neuralized E3 ubiquitin-protein ligase 4; Semaphorin-4B; CYFIP-related Rac1-interacting protein B; Type XVII collagen alpha-1 chain; E3 ubiquitin-protein ligase CHIP; SH3 and multiple ankyrin repeat domains protein 2.

[0013] The urinary biomarker for benign and malignant thyroid nodules and the analysis method of the present invention can be effectively used for the differential diagnosis of benign and malignant thyroid nodules, are non-invasive, have high sensitivity, and the results are accurate and reliable, providing a basis for clinical decision-making, and at the same time providing a certain basis for subsequent basic research and clinical research, and have great application and research value.

[0014] Preferably, the biomarker further includes a combination of various proteins screened by urine proteomics in claim 1.

[0015] A method for analyzing benign and malignant urine markers of thyroid nodules is also provided, and the marker levels in the biological sample of the test subject are obtained by one or more of the following methods: mass spectrometry, chemical analysis, and immunoassay.

[0016] Preferably, the chemical analysis method includes electrochemistry analysis method, radiochemistry analysis method, and enzymatic method; the immunoassay method includes radioimmunoassay, enzyme-linked immunosorbent assay, time-resolved fluorescence immunoassay, nanogold-labeled immunoassay, and immunosensor assay; the mass spectrometry method is triple quadrupole mass spectrometry, ion trap mass spectrometry, orbitrap mass spectrometry, matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The method for detecting protein markers by mass spectrometry is to first perform gradient elution using a chromatographic column, and then collect data under electrospray ionization source ESI ionization. In the mass spectrometry method, all markers are detected by the method of one-time injection analysis.

[0017] Preferably, the biological sample is urine.

[0018] Preferably, the urine is precipitated with acetone organic solvent and then dissolved in a lysis solution containing 8 mol / L urea. The protein concentration is measured using a bicinchoninic acid (BCA) kit or Bradford protein quantification method for pretreatment before mass spectrometry.

[0019] The application of benign and malignant urine markers of thyroid nodules is also provided, which is used for preparing a kit for differential diagnosis of benign and malignant thyroid nodules.

[0020] The following is a detailed description of the embodiments of the present invention.

[0021] Example 1

[0022] Urine proteomics technology is used to screen markers for differential diagnosis of benign and malignant thyroid nodules.

[0023] 1. Sample source:

[0024] After being approved by the Ethics Committee of China-Japan Friendship Hospital, 31 samples of thyroid benign nodules and 30 samples of thyroid malignant nodules were collected. All participants were from China-Japan Friendship Hospital, and all patients were clearly diagnosed by histopathological examination. The specimens were retained after clinical tests. All samples were stored at -80 °C for later use.

[0025] 2. Instrument: Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Scientific)

[0026] 3. Main reagents:

[0027] Trypsin (Promega); C18 solid-phase extraction cartridge (3CC, 60 mg, Waters); C18 reversed-phase chromatography column (4.6 mm × 250 mm, C18, 3 μm, Waters): LC / MS grade acetonitrile was purchased from Merck, HPLC grade methanol was purchased from Merck, and formic acid was purchased from CNW. Other reagents were all commercially available analytical grade. Deionized water was prepared by the Milli-Q ultrapure water system from Millipore.

[0028] 4. Research methods:

[0029] 4.1 Sample preparation:

[0030] The urine sample was extracted by acetone precipitation and then subjected to proteolysis by in-solution digestion method (FASP). The protein sample to be digested was diluted with 25 mM NH4HCO3 (sample: 25 mM NH4HCO3 = 1:3 by volume), vortexed for 30 s - 1 min. 1 M DTT was added to the sample tube to a final concentration of 20 mM in the solution (DTT is a reducing agent that breaks disulfide bonds; if the total volume after dilution is 100 ul, 2 ul of 1 M DTT needs to be added, i.e., the final concentration of 2 ul of 1 M DTT in 100 ul of solution is 20 mM). The sample was fixed on a water float and placed in a water bath at 95 °C for 5 min. The sample in the water bath was taken out and allowed to cool to room temperature. Then 1 M IAA was added to the tube to a final concentration of 50 mM in the solution (alkylation; if the total volume after dilution is 100 ul, 5 ul of 1 M IAA needs to be added, i.e., the final concentration of 5 ul of 1 M IAA in 100 ul of solution is 50 mM). After the sample was protected from light for 45 min, it was centrifuged at 12000 rpm for 5 - 10 min (pay attention to balancing). Take out a new 30K filtration membrane, add 100 ul of UA to the filtration tube inside the 30K membrane (the filtration tube is divided into two parts: one part is the outer collection tube which is white, and the other part is the inner sleeve with a membrane at the bottom which is blue. All solutions in this experiment will be added to the inner sleeve), cover the lid, and centrifuge at 14000 g for 1 min. This step can be repeated 2 - 3 times. After centrifugation of the reduced and alkylated sample, it can be seen that the EP tube is divided into two layers: the lower layer is the precipitate produced after reduction and alkylation, and the upper layer is the protein sample after reduction and alkylation; use a pipette to slowly aspirate the upper layer into the 30K filtration tube treated with UA (the UA filtration tube can be treated during the 45 min of alkylation). Place the 10K filtration membrane with the protein sample added in the centrifuge and centrifuge at 14000 g for 10 - 30 min (until all the liquid on the inner sleeve membrane is completely centrifuged out). After the sample has been fully filtered through the filtration tube, add 200 ul of UA to the inner sleeve, pipette up and down, vortex for 1 min, and centrifuge at 14000 g for 10 - 30 min (until all the liquid on the inner sleeve membrane is completely centrifuged out). This step can be repeated 2 times (this step is to remove DTT and IAA because DTT and IAA are small molecules that affect polypeptide detection). Add 200 ul of 25 mM NH4HCO3 to the inner sleeve of the filtration tube, pipette up and down, vortex for 1 min, and centrifuge at 14000 g for 15 - 30 min (until all the liquid on the inner sleeve membrane is completely centrifuged out). Discard the waste liquid in the collection tube. This step is repeated 2 times (change the buffer. Since we will use Trypsin enzyme later and Trypsin enzyme has good digestion effect in 25 mM NH4HCO3, so we need to change the buffer).Add 200 μL of 25 mM NH4HCO3 to the inner sleeve of the filtration tube, add an appropriate amount of Trypsin enzyme (added according to the mass ratio of protein:Trypsin enzyme = 1:50), and vortex for 1 min. Fix the sample with trypsin on a water float, take a 1 L beaker filled with clear water, place the water float on the water surface, and put it into the microwave oven for 1 min at high power. Finally, put the sample in the beaker into a water bath at 37 °C for 16 - 24 h. Take out the sample the next day, centrifuge at 14000 g for 1 - 10 min, add 50 - 100 μL of 500 mM NaCl and pipette repeatedly, centrifuge at 14000 g for 1 - 10 min, and aspirate the liquid in the collection tube into a new EP tube. (Here, a step of solid-phase extraction can be added, and the detailed steps are referred to the following solid-phase extraction steps). It is best that the volume in each EP tube is about 500 μL, which is beneficial for the next step of vacuum drying. Put the above sample into a vacuum dryer on the -1 layer, and dissolve it with one-thousandth FA after drying (the dissolution concentration is generally 5 μg / μL).

[0031] 4.2 Chromatography / Mass Spectrometry Conditions:

[0032] Orbitrap Exploris 480 (Thermo Scientific) is combined with EASY nLC 1000 for data analysis in data-independent acquisition mass spectrometry (DIA-MS) mode. The digested peptides are separated on an RP C18 self-packed capillary LC column (75 μm × 100 mm; particle size 3 μm). The elution gradient is 5 - 30% buffer B2 (0.1% formic acid, 99.9% ACN; flow rate, 0.3 μL / min), and the peptides are eluted for 25 minutes.

[0033] For DIA analysis, variable isolation windows with 60 windows are used for MS acquisition. According to the precursor m / z distribution of the pooled samples, the number of precursor ions is equal in each separation window. The full scan range is set to 350 to 1200 m / z with a resolution of 120000, followed by a DIA scan with a resolution of 30000 (higher-energy C-trap dissociation [HCD] collision energy: 30%; AGC target: 200%; maximum injection time: 50 ms).

[0034] 4.3 Data Processing:

[0035] The original DIA data was analyzed by Spectronaut Pulsar 17.1 (Biognosys) under default settings. Briefly, 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 of 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, similar numbers of peptides were upregulated and downregulated, and that most peptides in the samples were not regulated across 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).

[0036] 5. Data processing and statistical analysis:

[0037] Missing values in the proteomics data of the samples were independently imputed using the sequential k-nearest neighbor method. The t-test was used for the statistical analysis of all quantitative data. Statistical significance was defined as fold change > 1.5-fold and P < 0.05. Candidate markers for the differential diagnosis of benign and malignant thyroid nodules were screened out.

[0038] 6. Results:

[0039] A total of 3,842 proteins were identified, and quantitative analysis was performed on 2,479 proteins. A total of 196 proteins showed significant changes, with fold change > 1.5-fold (P < 0.05); the area under the ROC curve was calculated using the MetaboAnalyst 6.0 website. These could be candidate markers for the differential diagnosis of benign and malignant thyroid nodules. The results are shown in Table 1.

[0040] Table 1

[0041]

[0042]

[0043]

[0044]

[0045]

[0046]

[0047] Example 2

[0048] The candidate markers for the differential diagnosis of benign and malignant thyroid nodules in Example 1 were verified using proteomics techniques.

[0049] 1. Sample collection:

[0050] Collect 10 cases of benign thyroid nodules and 11 cases of malignant thyroid nodules.

[0051] All participants are from China-Japan Friendship Hospital, and all patients are clearly diagnosed by histopathological examination. The specimens are retained after clinical tests. All samples are stored at -80 °C for later use.

[0052] 2. Instrument: Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Scientific)

[0053] 3. Main reagents:

[0054] Trypsin (Promega); C18 solid-phase extraction cartridge (3CC, 60mg, Waters); C18 reversed-phase chromatography column (4.6mm×250mm, C18, 3μm, Waters): LC / MS grade acetonitrile is purchased from Merck, HPLC grade methanol is purchased from Merck, and formic acid is purchased from CNW. Other reagents are all commercially available analytical pure. Deionized water is prepared by the Milli-Q ultrapure water system of Millipore.

[0055] 4. Research methods:

[0056] 4.1 Sample preparation:

[0057] The urine sample was extracted by acetone precipitation and then proteolyzed by in-solution trypsin digestion method (FASP). The protein sample to be digested was diluted with 25 mM NH4HCO3 (sample: 25 mM NH4HCO3 = 1:3, this ratio is the volume ratio), vortexed for 30 s - 1 min. 1 M DTT was added to the sample tube to make its final concentration in the solution 20 mM (DTT is a reducing agent that breaks disulfide bonds; if the total volume after dilution is 100 ul, 2 ul of 1 M DTT needs to be added, that is, the final concentration of 2 ul of 1 M DTT in 100 ul of solution is 20 mM). The sample was fixed on a water float and placed in a water bath at 95 °C for 5 min. The sample in the water bath was taken out and allowed to reach room temperature. Then 1 M IAA was added to the tube to make its final concentration in the solution 50 mM (alkylation; if the total volume after dilution is 100 ul, 5 ul of 1 M IAA needs to be added, that is, the final concentration of 5 ul of 1 M IAA in 100 ul of solution is 50 mM). After the sample was protected from light for 45 min, it was centrifuged at 12000 rpm for 5 - 10 min (pay attention to balancing). Take out a new 30K filtration membrane, add 100 ul of UA to the filtration tube in the 30K membrane (the filtration tube is divided into two parts: one part is the outer collection tube which is white, and the other part is the inner sleeve with a membrane at the bottom which is blue. All solutions in this experiment will be added to the inner sleeve), cover the lid and centrifuge at 14000 g for 1 min. This step can be repeated 2 - 3 times. After centrifugation of the reduced and alkylated sample, it can be seen that the EP tube is divided into two layers: the lower layer is the precipitate produced after reduction and alkylation, and the upper layer is the reduced and alkylated protein sample; use a pipette to slowly aspirate the upper layer into the 30K filtration tube treated with UA (the UA filtration tube can be treated during the 45 min of alkylation). Place the 10K filtration membrane with the protein sample added in the centrifuge and centrifuge at 14000 g for 10 - 30 min (until all the liquid on the membrane of the inner sleeve is centrifuged clean). After the sample has been fully filtered through the filtration tube, add 200 ul of UA to the inner sleeve, pipette and vortex for 1 min, then centrifuge at 14000 g for 10 - 30 min (until all the liquid on the membrane of the inner sleeve is centrifuged clean). This step can be repeated 2 times (this step is to remove DTT and IAA because DTT and IAA are small molecules that affect polypeptide detection). Add 200 ul of 25 mM NH4HCO3 to the inner sleeve of the filtration tube, pipette and vortex for 1 min, then centrifuge at 14000 g for 15 - 30 min (until all the liquid on the membrane of the inner sleeve is centrifuged clean), discard the waste liquid in the collection tube, and repeat this step 2 times (change the buffer solution. Since we will use Trypsin enzyme later and Trypsin enzyme has good digestion effect in 25 mM NH4HCO3, so we need to change the buffer solution).Add 200 μl of 25 mM NH4HCO3 to the inner sleeve of the filtration tube, add an appropriate amount of Trypsin enzyme (added according to the mass ratio of protein:Trypsin enzyme = 1:50), and vortex for 1 min. Fix the sample added with trypsin on the water float, take a 1-L beaker filled with clear water, place the water float on the water surface, and put it into the microwave oven for 1 min at high power. Finally, put the sample in the beaker into a water bath at 37 °C for 16 - 24 h. Take out the sample the next day, centrifuge at 14000 g for 1 - 10 min, add 50 - 100 μL of 500 mM NaCl and pipette repeatedly, centrifuge at 14000 g for 1 - 10 min, and aspirate the liquid in the collection tube into a new EP tube. (A solid-phase extraction step can be added here, and the detailed steps are referred to the following solid-phase extraction steps.) It is best that the volume in each EP tube is about 500 μl, which is beneficial for the next vacuum drying. Put the above sample into the vacuum dryer on the -1 layer, and dissolve it with one-thousandth FA after drying (the dissolution concentration is generally 5 μg / μl).

[0058] 4.2 Chromatography / Mass Spectrometry Conditions:

[0059] Orbitrap Exploris 480 (Thermo Scientific) is combined with EASY nLC 1000 for data analysis in data-independent acquisition mass spectrometry (DIA-MS) mode. The digested peptides are separated on an RP C18 self-packed capillary LC column (75 μm × 100 mm; particle size 3 μm). The elution gradient is 5 - 30% buffer B2 (0.1% formic acid, 99.9% ACN; flow rate, 0.3 μL / min), and the peptides are eluted for 25 minutes.

[0060] For DIA analysis, MS acquisition is performed using variable isolation windows with 60 windows. According to the precursor m / z distribution of the pooled samples, the number of precursor ions is equal in each separation window. The full scan range is set to 350 to 1200 m / z with a resolution of 120000, followed by a DIA scan with a resolution of 30000 (higher-energy C-trap dissociation [HCD] collision energy: 30%; AGC target: 200%; maximum injection time: 50 ms).

[0061] 4.3 Data Processing:

[0062] The original DIA data was analyzed by Spectronaut Pulsar 17.1 (Biognosys) under default settings. Briefly, the retention time prediction type was set to dynamic iRT. Interference correction at the MS2 level was enabled. Peptide intensity was calculated by summing the peak areas of the respective fragment ions of MS2, and protein intensity was 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 were upregulated and downregulated, and that most peptides in the sample were not regulated across 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).

[0063] 4.4 Data processing and statistical analysis:

[0064] Missing values in the proteomics data of the samples were independently imputed using the sequential k-nearest neighbor method. The t-test was used for the statistical analysis of all quantitative data. Statistical significance was defined as fold change > 1.5-fold and P < 0.05. The area under the ROC curve was calculated using the MetaboAnalyst 6.0 website.

[0065] 5 Validation result analysis:

[0066] 5.1 The candidate protein biomarker molecules were validated using urine proteomics technology, and the validation results are shown in Table 2.

[0067] Table 2

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074] 5.2 Analyzing the validation results, to improve the reliability of the experiment, the data of the experimental group and the validation group were analyzed to obtain stable differential proteins that are commonly used as biomarkers for benign and malignant thyroid nodules. The screening criteria were that the AUC value of the ROC curve of the experimental group was greater than 0.800, and the AUC value of the ROC curve of the validation group was greater than 0.850. See Table 3.

[0075] Table 3

[0076]

[0077]

[0078] As described above, it is only a preferred embodiment of the present invention and does not impose any form of limitation on the present invention. Any simple modification, equivalent change, and modification made to the above embodiments based on the technical essence of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. Benign and malignant urine markers for thyroid nodules, characterized in that: At least one of the following substances: ubiquitin-like modifier activating enzyme 6; sulfotransferase 2B1; exportin 1; kallikrein 8; carboxypeptidase D; SAM and SH3 domain-containing protein 1; transforming acidic coiled-coil protein 2; apolipoprotein M; adaptor-related protein complex 2 alpha-1 subunit; nucleophosmin; amine oxidase; mitochondrial cytochrome b-c1 complex subunit 2; cytoplasmic threonyl-tRNA synthetase 1; cystathionine gamma-lyase; succinyl-CoA ligase; large ribosomal subunit protein uL5; large ribosomal subunit protein eL38; small ribosomal subunit protein eS21; metallothionein 1H; lamin B2; emilin A8-like protein 1; synaptotagmin-binding protein 4; ferritin family homolog 3; deaminated glutathione amidase; uncharacterized protein C4orf19; dual specificity tyrosine phosphorylation-regulated kinase 2; transmembrane BAX inhibitor 1; neurogenic locus notch homolog protein 4; semaphorin-4B; CYFIP-related Rac1-interacting protein B; type XVII collagen alpha-1 chain; E3 ubiquitin-protein ligase CHIP; SH3 and multiple ankyrin repeat domains protein 2.

2. The urine marker for benign and malignant thyroid nodules according to claim 1, wherein: The biomarker also includes a combination of various proteins obtained by urine proteomics screening according to claim 1.

3. The method for analyzing the benign and malignant urine markers of thyroid nodules according to claim 2, wherein: The level of the biomarker in the biological sample of the test subject is obtained by one or more of the following methods: mass spectrometry, chemical analysis, immunoassay.

4. The analysis method of the benign and malignant urine markers of thyroid nodules according to claim 3, wherein: The chemical analysis method includes electrochemistry analysis, radiochemistry analysis, enzymatic method; the immunoassay method includes radioimmunoassay, enzyme-linked immunosorbent assay, time-resolved fluorescence immunoassay, nano-gold-labeled immunoassay, and immunosensor assay; the mass spectrometry method is triple quadrupole mass spectrometry, ion trap mass spectrometry, orbitrap mass spectrometry, 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 source ESI ionization. In the mass spectrometry method, all biomarkers are detected by the method of one-time injection analysis.

5. The analysis method of the benign and malignant urine markers of thyroid nodules according to claim 4, characterized in that: The biological sample is urine.

6. The analysis method of the benign and malignant urine markers of thyroid nodules according to claim 5, characterized in that: The urine is precipitated with acetone organic solvent and then dissolved in a lysis solution containing 8 mol / L urea. The protein concentration is measured using a bicinchoninic acid method kit or Bradford protein quantification method for pretreatment before mass spectrometry.

7. Use of the urine marker for benign and malignant thyroid nodules according to claim 1, characterized in that: For the preparation of a kit for differential diagnosis of benign and malignant thyroid nodules.

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