Plasma protein marker for identifying or assisting in identifying parathyroid cancer and parathyroid adenoma and application thereof

Abstract

The invention discloses a plasma protein marker for identifying or assisting in identifying parathyroid cancer and parathyroid adenoma and application of the plasma protein marker. The method comprises the following steps: firstly, collecting plasma specimens of a parathyroid carcinoma patient and a parathyroid adenoma patient before an operation, and screening through an unmarked quantitative proteomics technology to obtain 24 differential expression proteins; on the basis of the other part of plasma specimens of the parathyroid carcinoma patient and the parathyroid adenoma patient collected before the operation, the 24 differential expression proteins are verified through a parallel reaction monitoring technology; finally, three plasma protein markers (myoglobin, coronin-1A and a polyimmunoglobulin receptor) which can be used for identifying the parathyroid carcinoma and the parathyroid adenoma are obtained. Experiments prove that the plasma protein marker or the combination thereof can be used for auxiliary differential diagnosis of parathyroid carcinoma and parathyroid adenoma, and has important significance for improving the accuracy of preoperative diagnosis of parathyroid carcinoma.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a plasma protein marker for identifying or assisting in identifying parathyroid carcinoma and parathyroid adenoma and application thereof. BACKGROUND

[0002] Primary hyperparathyroidism is the third most common endocrine disease after thyroid and diabetes. Parathyroid tumor is the main cause of primary hyperparathyroidism. The parathyroid hormone abnormally secreted by parathyroid tumor can cause hypercalcemia, and even high calcium crisis, which endangers the life of patients. According to the classification of WHO in 2022, parathyroid tumor can be divided into parathyroid adenoma, atypical parathyroid tumor and parathyroid carcinoma.

[0003] Parathyroid carcinoma is a rare endocrine malignancy, accounting for about 1% of primary hyperparathyroidism.

[0004] Parathyroid carcinoma not only lacks obvious clinical features, but also cannot be clearly diagnosed by existing preoperative ultrasound and imaging examination. Therefore, the preoperative diagnosis of parathyroid carcinoma is still a big problem in clinical practice. At present, the preoperative diagnosis of parathyroid carcinoma mainly relies on the preoperative clinical manifestations of patients and the intraoperative tumor manifestations for speculation; if the patient has a significant increase in parathyroid hormone or high calcium crisis, or if the tumor invades the surrounding tissue during the exploration, parathyroid carcinoma is considered. However, some large parathyroid adenomas or atypical parathyroid tumors may have the above-mentioned manifestations. Therefore, such manifestations are not specific for diagnosis, and misdiagnosis is easy to occur. In addition, the diagnostic accuracy of intraoperative rapid frozen pathological examination of parathyroid carcinoma is only about 15%.

[0005] Since the existing method speculates the benign and malignant of parathyroid tumor according to the preoperative clinical manifestations and intraoperative exploration, it lacks objective indicators, and the specificity and sensitivity are not good, which is easy to cause misdiagnosis or missed diagnosis. The preoperative diagnosis of parathyroid carcinoma directly affects the surgical approach. Misdiagnosis leads to the expansion of the surgical range, bringing more surgical complications, while missed diagnosis affects the prognosis of patients, bringing secondary surgical trauma, tumor spread or metastasis. Therefore, it is urgent to find a biomarker that can assist in the diagnosis of parathyroid tumor to improve the preoperative diagnostic accuracy of parathyroid carcinoma, so as to select the best surgical approach for patients and improve the quality of life and survival time of patients. SUMMARY

[0006] The technical problem to be solved by the present application is how to distinguish or identify parathyroid carcinoma and parathyroid adenoma.

[0007] In order to solve the above technical problem, the present application first provides a new use of a substance for detecting the content of myoglobin and / or coronin-1A and / or polymeric immunoglobulin receptor.

[0008] The present application provides the use of a substance for detecting the content of myoglobin and / or coronin-1A and / or polymeric immunoglobulin receptor in A1) or A2) as follows: A1) preparing a product for identifying or assisting in identifying parathyroid carcinoma and parathyroid adenoma; A2) preparing a product for screening or assisting in screening a patient with parathyroid carcinoma or a patient with parathyroid adenoma.

[0009] To solve the above technical problems, the present application further provides a kit; The kit functions as any one of B1) or B2) as follows: B1) identifying or assisting in identifying parathyroid carcinoma and parathyroid adenoma; B2) screening or assisting in screening a patient with parathyroid carcinoma or a patient with parathyroid adenoma.

[0010] The kit provided by the present application comprises a substance for detecting the content of myoglobin and / or coronin-1A and / or polymeric immunoglobulin receptor.

[0011] In any of the above-mentioned applications or kits, the substance for detecting the content of myoglobin and / or coronin-1A and / or polymeric immunoglobulin receptor can be a substance for detecting the content of myoglobin and / or coronin-1A and / or polymeric immunoglobulin receptor in blood plasma.

[0012] Further, the substance for detecting the content of myoglobin and / or coronin-1A and / or polymeric immunoglobulin receptor in blood plasma comprises reagents and / or instruments for detecting the content of myoglobin and / or coronin-1A and / or polymeric immunoglobulin receptor in blood plasma.

[0013] Still further, the reagents and / or instruments for detecting the content of myoglobin and / or coronin-1A and / or polymeric immunoglobulin receptor in blood plasma are reagents and / or instruments for detecting the content of myoglobin and / or coronin-1A and / or polymeric immunoglobulin receptor in blood plasma by using label-free quantitative proteomics technology or reagents and / or instruments for detecting the content of myoglobin and / or coronin-1A and / or polymeric immunoglobulin receptor in blood plasma by using parallel reaction monitoring technology.

[0014] In a specific embodiment of the present application, the kit further comprises a data processing device A; the data processing device A is composed of a data input module A, a data recording module A, a data comparison module A and a conclusion output module A; The data input module A is configured to input the content of myoglobin in the blood plasma sample of the subject to be tested; The data recording module A is configured to store the myoglobin content in the plasma sample of the subject and a judgment threshold value; The data comparison module A is configured to receive the myoglobin content in the plasma sample of the subject sent by the data input module A, and compare the judgment threshold value with the myoglobin content in the plasma sample of the subject from the data recording module A; The conclusion output module A is configured to receive the comparison result sent by the data comparison module A, and determine whether the subject is a parathyroid carcinoma patient or a parathyroid adenoma patient according to a predetermined determination condition.

[0015] In a specific embodiment of the present application, the kit further comprises a data processing device B; the data processing device B is composed of a data input module B, a data recording module B, a data comparison module B and a conclusion output module B; The data input module B is configured to input the coronin-1A content in the plasma sample of the subject; The data recording module B is configured to store the coronin-1A content in the plasma sample of the subject and a judgment threshold value; The data comparison module B is configured to receive the coronin-1A content in the plasma sample of the subject sent by the data input module B, and compare the judgment threshold value with the coronin-1A content in the plasma sample of the subject from the data recording module B; The conclusion output module B is configured to receive the comparison result sent by the data comparison module B, and determine whether the subject is a parathyroid carcinoma patient or a parathyroid adenoma patient according to a predetermined determination condition.

[0016] In a specific embodiment of the present application, the kit further comprises a data processing device C; the data processing device C is composed of a data input module C, a data recording module C, a data comparison module C and a conclusion output module C; The data input module C is configured to input the polymeric immunoglobulin receptor content in the plasma sample of the subject; The data recording module C is configured to store the polymeric immunoglobulin receptor content in the plasma sample of the subject and a judgment threshold value; The data comparison module C is configured to receive the polymeric immunoglobulin receptor content in the plasma sample of the subject sent by the data input module C, and compare the judgment threshold value with the polymeric immunoglobulin receptor content in the plasma sample of the subject from the data recording module C; The conclusion output module C is configured to receive the comparison result sent by the data comparison module C, and determine whether the subject is a parathyroid carcinoma patient or a parathyroid adenoma patient according to a predetermined determination condition.

[0017] In one specific embodiment of the present application, the kit further comprises a data processing device D; the data processing device D is composed of a data input module D, a data recording module D, a data comparison module D and a conclusion output module D; The data input module D is configured to input the myoglobin content, coronin-1A content and polymeric immunoglobulin receptor content in the plasma sample of the subject to be tested; The data recording module D is configured to store the myoglobin content, coronin-1A content, polymeric immunoglobulin receptor content in the plasma sample of the subject to be tested and the judgment threshold value; The data comparison module D is configured to receive the myoglobin content, coronin-1A content and polymeric immunoglobulin receptor content in the plasma sample of the subject to be tested sent by the data input module D, and call the judgment threshold value from the data recording module D and compare it with the myoglobin content, coronin-1A content and polymeric immunoglobulin receptor content in the plasma sample of the subject to be tested; The conclusion output module D is configured to receive the comparison result sent by the data comparison module D, and determine whether the subject to be tested is a parathyroid carcinoma patient or a parathyroid adenoma patient according to the predetermined judgment condition.

[0018] Any of the above-mentioned myoglobin content, coronin-1A content or polymeric immunoglobulin receptor content is the relative quantitative value of myoglobin, coronin-1A or polymeric immunoglobulin receptor in the plasma sample. The relative quantitative value can be detected by using the label-free quantitative proteomics technology or the parallel reaction monitoring technology according to the conventional method in the technical field.

[0019] Any of the above-mentioned judgment threshold value can be obtained by referring to the method known in the technical field, and specifically can be the cut-off value of the myoglobin content or coronin-1A content or polymeric immunoglobulin receptor content obtained from the plasma of parathyroid carcinoma patients and parathyroid adenoma patients as the judgment threshold value.

[0020] In one specific embodiment of the present application, the judgment threshold value corresponding to the myoglobin content can be 38.94. When the myoglobin content in the plasma sample of the subject to be tested is detected by using the PRM method with myoglobin as the marker for differential diagnosis, the identification can be performed according to the following standard: if the relative quantitative value of myoglobin in the plasma of the subject to be tested is greater than 38.94, the subject to be tested is or is suspected to be a parathyroid carcinoma patient; if the relative quantitative value of myoglobin in the plasma of the subject to be tested is less than or equal to 38.94, the subject to be tested is or is suspected to be a parathyroid adenoma patient.

[0021] In another specific embodiment of the present application, the judgment threshold value corresponding to the content of coronin-1A can be 432.85. When the content of myoglobin in the plasma sample of the testee is detected by PRM method using coronin-1A as a marker for differential diagnosis, the identification can be performed according to the following standard: if the relative quantitative value of coronin-1A in the plasma of the testee is greater than 432.85, the testee is or is suspected to be a patient with parathyroid carcinoma; if the relative quantitative value of coronin-1A in the plasma of the testee is less than or equal to 432.85, the testee is or is suspected to be a patient with parathyroid adenoma.

[0022] In another specific embodiment of the present application, the judgment threshold value corresponding to the content of coronin-1A can be 432.85. When the content of myoglobin in the plasma sample of the testee is detected by PRM method using coronin-1A as a marker for differential diagnosis, the identification can be performed according to the following standard: if the relative quantitative value of coronin-1A in the plasma of the testee is greater than 432.85, the testee is or is suspected to be a patient with parathyroid carcinoma; if the relative quantitative value of coronin-1A in the plasma of the testee is less than or equal to 432.85, the testee is or is suspected to be a patient with parathyroid adenoma.

[0023] In a preferred embodiment of the present application, the three differentially expressed proteins myoglobin, coronin-1A and polymeric immunoglobulin receptor discovered by the present application are used as a combined marker for differential diagnosis. When the contents of myoglobin, coronin-1A and polymeric immunoglobulin receptor in the plasma sample of the testee are detected by PRM method using myoglobin, coronin-1A and polymeric immunoglobulin receptor as a combined marker for differential diagnosis, if the relative quantitative value of myoglobin in the plasma of the testee is greater than 38.94, and the relative quantitative value of coronin-1A is greater than 432.85, and the relative quantitative value of polymeric immunoglobulin receptor is greater than 59.34, the testee is or is suspected to be a patient with parathyroid carcinoma; otherwise, the testee is or is suspected to be a patient with parathyroid adenoma.

[0024] In any of the above-mentioned applications or products, the differential diagnosis is preoperative differential diagnosis.

[0025] In any of the above-mentioned applications or products, the parathyroid carcinoma is sporadic parathyroid carcinoma.

[0026] In any of the above-mentioned applications or products, the parathyroid adenoma is sporadic parathyroid adenoma.

[0027] The amino acid sequence of myoglobin in any of the above-mentioned applications or products is shown in SEQ ID NO: 1.

[0028] The amino acid sequence of coronin-1A in any of the above-mentioned applications or products is shown in SEQ ID NO: 2.

[0029] The amino acid sequence of any of the above-described multimeric immunoglobulin receptors is shown in SEQ ID NO: 3.

[0030] The present application collects plasma samples of parathyroid carcinoma patients and parathyroid adenoma patients before surgery, first obtains 24 differentially expressed proteins through label-free quantitative proteomics technology screening, then verifies the 24 differentially expressed proteins through parallel reaction monitoring technology based on another part of the plasma samples of parathyroid carcinoma patients and parathyroid adenoma patients collected before surgery, and finally obtains three plasma protein markers (myoglobin, coronin-1A, and multimeric immunoglobulin receptor) that can be used to identify parathyroid carcinoma and parathyroid adenoma. Experiments prove that the plasma protein markers or combinations thereof discovered by the present application can be used for the auxiliary differential diagnosis of parathyroid carcinoma and parathyroid adenoma, which not only improves the preoperative diagnostic accuracy of parathyroid carcinoma, but also helps to select the best surgical method for the patient and take more precise treatment measures to improve the patient's quality of life and survival time. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 Volcano plot of differentially expressed proteins 。

[0032] Figure 2 ROC curve analysis of the diagnostic performance of myoglobin content in the plasma of parathyroid adenoma patients and parathyroid carcinoma patients obtained through label-free quantitative proteomics technology.

[0033] Figure 3 ROC curve analysis of the diagnostic performance of coronin-1A content in the plasma of parathyroid adenoma patients and parathyroid carcinoma patients obtained through label-free quantitative proteomics technology.

[0034] Figure 4 ROC curve analysis of the diagnostic performance of multimeric immunoglobulin receptor content in the plasma of parathyroid adenoma patients and parathyroid carcinoma patients obtained through label-free quantitative proteomics technology.

[0035] Figure 5 ROC curve analysis of the diagnostic performance of myoglobin, coronin-1A, and multimeric immunoglobulin receptor content in the plasma of parathyroid adenoma patients and parathyroid carcinoma patients obtained through label-free quantitative proteomics technology.

[0036] Figure 6 ROC curve analysis of the diagnostic performance of myoglobin content in the plasma of parathyroid adenoma patients and parathyroid carcinoma patients obtained through parallel reaction monitoring technology.

[0037] Figure 7ROC curve analysis of the diagnostic efficiency of the coronin-1A content in the plasma of parathyroid adenoma patients and parathyroid cancer patients obtained by parallel reaction monitoring technology.

[0038] Figure 8 ROC curve analysis of the diagnostic efficiency of the polymeric immunoglobulin receptor content in the plasma of parathyroid adenoma patients and parathyroid cancer patients obtained by parallel reaction monitoring technology.

[0039] Figure 9 ROC curve analysis of the diagnostic efficiency of the myoglobin, coronin-1A and polymeric immunoglobulin receptor contents in the plasma of parathyroid adenoma patients and parathyroid cancer patients obtained by parallel reaction monitoring technology. DETAILED DESCRIPTION

[0040] The application will be further described in conjunction with the specific embodiments. The examples given are only to illustrate the application, and are not intended to limit the scope of the application. The examples provided below can serve as a guide for further improvement by those skilled in the art, and do not in any way constitute a limitation on the application.

[0041] The experimental methods in the following examples are all routine methods, unless otherwise specified, which are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained commercially.

[0042] Example 1, obtaining of plasma protein markers for identifying or assisting in identifying parathyroid cancer and parathyroid adenoma and analysis of diagnostic efficiency I. Experimental materials and methods 1. Test subjects A total of 10 parathyroid adenoma (PA) patients and 10 parathyroid cancer (parathyroid tumor, PC) patients were included in the study. The inclusion criteria were: (1) age > 18 and < 75; (2) patients had no family history and were diagnosed with sporadic hyperparathyroidism; (3) according to the 2017 World Health Organization standard, histopathology confirmed sporadic parathyroid adenoma or sporadic parathyroid cancer (sporadic means non-hereditary, indicating that the patient has no family history of genetic disease); (4) blood plasma samples were collected before surgery. All patients were monitored in the general surgery department of Peking Union Medical College Hospital and underwent parathyroidectomy. All test subjects gave informed consent.

[0043] 2. Experimental methods Quantitative label-free proteomics research was performed on the plasma samples of the test subjects to obtain differentially expressed proteins in the plasma of parathyroid adenoma patients and parathyroid cancer patients. The specific steps are as follows: 1) Protein preparation: 10 μL of plasma sample was mixed with an appropriate amount of RIPA lysis buffer, low-temperature grinding for 5 min, ultrasonic treatment in ice bath for 5 min, and the sample was completely lysed. Then the sample was incubated at 4°C for 10 min, and centrifuged at 12000 g for 15 min at 4°C. The supernatant was collected, and the protein concentration was quantified using the BCA protein assay kit. 40 μg of total protein was extracted from each sample, 5 times the volume of pre-cooled acetone (-20°C) was added, and the protein precipitate was dissolved at 20°C overnight. Centrifugation at 12000 rpm for 10 min at 4°C, remove the supernatant. Add 40 μL of protein resuspension solution, water bath ultrasonic for 3 min to dissolve the protein precipitate. Add DTT to 5 mM, 55°C oscillation incubation for 20 min to reduce disulfide bond. Cool the sample to room temperature, add IAA to 15 mM to react for 30 min in the dark to alkylate the reduced disulfide bond. Dissolve Trypsin to 0.5 μg / μL with Resuspension buffer, incubate at room temperature for 5 min. Then, the mixture of trypsin and sample was mixed at a ratio of 1:50, and incubated at 37°C with 1000 rpm shock overnight.

[0044] 2) Nano liquid chromatography-mass spectrometry (NanoLC-MS / MS) analysis: The sample was dissolved in 20 μL of 0.1% formic acid aqueous solution, and then analyzed by LC-MS / MS. The whole system is a timsTOF Pro2 mass spectrometer (Bruker Daltonics) of a tandem nanoElute system (Bruker Daltonics). 250 ng of sample was loaded (analysis column: 25 cm x 75 μm i.d., IonOpticks), and the sample was separated with a 60 min gradient, and the column temperature was 60°C. 1 μL (about 200 ng) of sample was loaded, and the column flow rate was controlled at 200 nL / min. A is 0.1% formic acid aqueous solution, B is 0.1% formic acid acetonitrile solution, the gradient starts from 3% B, increases to 28% within 50 min, increases to 45% within 5 min, increases to 80% within 2 min, and maintains for 3 min. The mass spectrometer was operated in positive ion mode, the full scan range was from 350-1700 m / z, and the ion accumulation and release time was set to 100 milliseconds. DDA data acquisition was performed using PASEF mode, each mass spectrometry scan acquisition cycle process took 1.17 seconds, including 1 full scan and 10 parallel accumulation sequence fragmentation (PASEF) MS / MS process. In the PASEF process, the collision energy increases linearly with the ion mobility, the mobility (1 / k0) is 0.6 Vs / cm 2 when the collision energy is 20 eV, the mobility is 1.6 Vs / cm 2The collision energy was 59 eV. Other mass spectrometry parameters were set as follows: ions with an intensity exceeding the threshold of 5000 were used as candidate precursor ions, and the mobility was selected in the range of 0.75–1.35 Vs / cm. 2 The capillary voltage was 1500V, the auxiliary gas flow rate was 3 liters / minute, the temperature was 200℃, and the column temperature was kept constant at 50℃.

[0045] 3) Database Search: Raw mass spectrometry files were searched using SpectroMine software (version 3.2.220222.52329; Biognosys AG, Switzerland). The database used was the SwissProt database downloaded from uniprot (Taxonomy: Homo sapiens, containing 20,405 manually annotated and validated sequences, downloaded on 2023-01-10). Enzyme digestion was set to whole trypsin digestion, with a maximum missed digestion count of 2. Cysteine-induced urea methylation was set to fixed modification; methionine oxidation and protein N-terminal acetylation were set to variable modification. The precursor ion mass error range was set to 20 ppm, the fragment ion mass error range was set to 0.05 Da, and the identified peptide length was 6-45 amino acids. The false positive rate (FDR) for matched spectra, peptides, and proteins was controlled to be less than or equal to 1%.

[0046] 4) Bioinformatics Analysis: To explore the similarities and differences between PC and PA, principal component analysis was performed in R (version 3.6.3). Additionally, t-tests or analysis of variance (ANOVA) were performed to examine differentially expressed proteins. GO annotation enrichment analysis, KEGG annotation enrichment analysis, and protein-protein interaction network analysis were conducted to identify related signaling and metabolic pathways.

[0047] II. Experimental Results Quantitative label-free proteomics identification of differentially expressed proteins in 10 cases of parathyroid adenoma and 10 cases of parathyroid carcinoma revealed 138 differentially expressed proteins. Compared with the parathyroid adenoma group, the parathyroid carcinoma group showed 76 upregulated proteins and 62 downregulated proteins. Figure 1 ).

[0048] After quantitative label-free proteomics analysis, 138 differentially expressed proteins were further screened through database searches. The specific screening criteria were as follows: (1) the number of unique peptides was greater than 1; (2) p < 0.03; (3) fold change ≤ 0.33 or fold change ≥ 3. Finally, 24 differentially expressed proteins were screened, including myoglobin, coronin-1A, and polyimmunoglobulin receptor.

[0049] The results of detecting the contents of myoglobin, coronin-1A and polymeric immunoglobulin receptor in the plasma of each subject are shown in Table 1. It was found by quantitative label-free proteomic analysis that the contents of myoglobin, coronin-1A and polymeric immunoglobulin receptor had significant differences between the parathyroid carcinoma group and the parathyroid adenoma group.

[0050] Table 1

[0051] Note: MEAN PA represents the average of the relative quantification value of the protein in the plasma of the parathyroid adenoma patient; MEAN PC represents the average of the relative quantification value of the protein in the plasma of the parathyroid carcinoma patient; P < 0.05 indicates a statistically significant difference.

[0052] The receiver operating characteristic (ROC) curve was used to evaluate the diagnostic efficiency of the contents of myoglobin, coronin-1A and polymeric immunoglobulin receptor in the plasma of the parathyroid adenoma patients and the parathyroid carcinoma patients, and the area under the curve (AUC) was calculated.

[0053] The results are shown in Figures 2-5 It was found that the AUC for identifying parathyroid adenoma and parathyroid carcinoma based on the content of myoglobin was 0.760 (sensitivity = 60%, specificity = 90%), the AUC for identifying parathyroid adenoma and parathyroid carcinoma based on the content of coronin-1A was 0.790 (sensitivity = 70%, specificity = 80%), the AUC for identifying parathyroid adenoma and parathyroid carcinoma based on the content of polymeric immunoglobulin receptor was 0.800 (sensitivity = 70%, specificity = 100%), and the accuracy of identifying parathyroid adenoma and parathyroid carcinoma based on the combined contents of myoglobin, coronin-1A and polymeric immunoglobulin receptor was higher (AUC = 0.910, sensitivity = 70%, specificity = 100%). Therefore, whether it is a single myoglobin, coronin-1A or polymeric immunoglobulin receptor or a combination of the three, it has diagnostic significance.

[0054] Example 2, verification and diagnostic efficiency analysis of plasma protein markers for identifying or assisting in identifying parathyroid carcinoma and parathyroid adenoma I. Experimental materials and methods 1. Subjects A total of 46 patients with parathyroid adenoma (PA) and 24 patients with parathyroid carcinoma (parathyroid tumor, PC) were enrolled in this study. The inclusion criteria were: (1) age > 18 and < 75; (2) patients without family history, diagnosed as sporadic hyperparathyroidism; (3) histopathology confirmed as sporadic parathyroid adenoma or sporadic parathyroid carcinoma according to the 2017 World Health Organization standard (sporadic means non-hereditary, indicating that the patient has no family history of genetic diseases); (4) blood plasma samples were collected before surgery. All patients were monitored in the Department of General Surgery of Peking Union Medical College Hospital and underwent parathyroidectomy. All subjects gave informed consent.

[0055] 2. Experimental method The 24 differentially expressed proteins screened in Example 1 were verified by parallel reaction monitoring (PRM) technology according to the method in the literature "Rauniyar N. Parallel reaction monitoring: A targeted experiment performed using high resolution and high mass accuracy mass spectrometry. INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES. 2015; 16(12): 28566-28581." The relative quantification value of each sample was calculated according to the peak area (mAU) ， The protein content that was again determined to have a significant difference between the plasma of patients with parathyroid adenoma and parathyroid carcinoma was subjected to ROC curve analysis.

[0056] 1) PRM analysis: All samples were processed according to the method of 1) in step 1 of Example 1, to obtain protein samples. 2 μg of protein sample (polypeptide) was taken for PRM analysis. The whole system of PRM analysis was nanoUPLC (nanoElute2) coupled with timsTOF Pro2 instrument (Bruker) with nano-electrospray ion source. A reversed-phase column (PeP Sep C18, 1.9 μm, 75 μm x 25 cm, Bruker, Germany) was used. The mobile phase was H2O with 0.1% FA (phase A) and ACN with 0.1% FA (phase B). The sample separation was performed at a flow rate of 300 nL / min with a gradient of 60 min. Gradient B: 2%, 0 min, 222%, 45 min, 2237%, 5 min, 3780%, 5 min. The mass spectrometer was used in DDA-PaSEF mode for DDA data acquisition, with a scan range of 100-1700 m / z for MS1. During PASEF MS / MS scanning, the impact energy increased linearly with ion mobility, from 20 eV (1 / K0=0.6 Vs / cm 2 ) to 59 eV (1 / K0=1.6 Vs / cm 2 ).

[0057] 2) Analysis of the differential protein content in parathyroid carcinoma and parathyroid adenoma using ROC curve analysis to explore its diagnostic value.

[0058] II. Experimental results 1. Analysis of differential protein content detection results Through PRM analysis, it was found that the contents of myoglobin (p=0.048), coronin-1A (p=0.024) and polymeric immunoglobulin receptor (p=0.021) also showed significant differences between the parathyroid carcinoma group and the parathyroid adenoma group (Table 2).

[0059] Table 2

[0060] Note: MEAN PA represents the mean relative quantification value of the protein in the plasma of parathyroid adenoma patients; MEAN PC represents the mean relative quantification value of the protein in the plasma of parathyroid carcinoma patients, P<0.05 indicates a statistically significant difference.

[0061] 2. ROC curve analysis The results of ROC curve analysis are as follows Figures 6-9The results showed that myoglobin (AUC = 0.670, sensitivity = 58.3%, specificity = 78.3%), coronin-1A (AUC = 0.641, sensitivity = 29.2%, specificity = 100%) and polymeric immunoglobulin receptor (AUC = 0.681, sensitivity = 66.7%, specificity = 76.1%) were of diagnostic significance. More importantly, the combination of myoglobin, coronin-1A and polymeric immunoglobulin receptor had higher accuracy (AUC = 0.808, sensitivity = 79.2%, specificity = 71.7%) in differential diagnosis.

[0062] The present application has been described in detail. For those skilled in the art, without departing from the purpose and scope of the present application, and without unnecessary experiments, the present application can be implemented in a wider range under the same parameters, concentrations and conditions. Although the present application gives a special example, it should be understood that the present application can be further improved. In summary, according to the principle of the present application, this application intends to include any change, use or improvement of the present application, including the change made by the conventional technology known in the art, which is out of the range disclosed in this application. Some basic features can be applied according to the scope of the following attached claims.

Claims

1. Application of substances used to detect myoglobin and / or coronin-1A and / or polyimmunoglobulin receptor levels in the following A1) or A2): A1) Prepare products for the identification or auxiliary identification of parathyroid carcinoma and parathyroid adenoma; A2) Prepare products for screening or assisting in the screening of patients with parathyroid cancer or parathyroid adenoma.

2. The application according to claim 1, characterized in that: The substance used to detect the content of myoglobin and / or coronin-1A and / or polyimmunoglobulin receptor is a substance used to detect the content of myoglobin and / or coronin-1A and / or polyimmunoglobulin receptor in plasma.

3. The application according to claim 2, characterized in that: The substances used to detect the levels of myoglobin and / or coronin-1A and / or polyimmunoglobulin receptors in plasma include reagents and / or instruments for detecting the levels of myoglobin and / or coronin-1A and / or polyimmunoglobulin receptors in plasma.

4. The application according to any one of claims 1-3, characterized in that: The parathyroid carcinoma mentioned is sporadic parathyroid carcinoma.

5. The application according to any one of claims 1-3, characterized in that: The parathyroid adenoma was a sporadic parathyroid adenoma.

6. A kit comprising substances for detecting myoglobin and / or coronin-1A and / or polyimmunoglobulin receptor levels; The kit has the function of either B1) or B2) below: B1) To differentiate or assist in the differentiation between parathyroid carcinoma and parathyroid adenoma; B2) Screening or auxiliary screening for patients with parathyroid cancer or parathyroid adenoma.

7. The reagent kit according to claim 6, characterized in that: The substance used to detect the content of myoglobin and / or coronin-1A and / or polyimmunoglobulin receptor is a substance used to detect the content of myoglobin and / or coronin-1A and / or polyimmunoglobulin receptor in plasma.

8. The reagent kit according to claim 7, characterized in that: The substances used to detect the levels of myoglobin and / or coronin-1A and / or polyimmunoglobulin receptors in plasma include reagents and / or instruments for detecting the levels of myoglobin and / or coronin-1A and / or polyimmunoglobulin receptors in plasma.

9. The reagent kit according to any one of claims 6-8, characterized in that: The parathyroid carcinoma mentioned is sporadic parathyroid carcinoma.

10. The kit according to any one of claims 6-8, characterized in that: The parathyroid adenoma was a sporadic parathyroid adenoma.

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