CN115754293A

Movatterモバイル変換

Info

Publication number: CN115754293A
Application number: CN202211385304.5A
Authority: CN
Inventors: 周斌; 张�林; 王艳云; 李芝隆; 宋雅平; 粟闵
Original assignee: West China Second University Hospital of Sichuan University
Current assignee: West China Second University Hospital of Sichuan University
Priority date: 2022-11-07
Filing date: 2022-11-07
Publication date: 2023-03-07

Abstract

本发明提供了VWF糖基化蛋白的检测试剂在制备筛查患肺癌风险的试剂盒中的用途，属于体外诊断试剂领域。本发明的试剂盒通过检测血浆中VWF糖基化蛋白的水平，可以筛查待检人群患肺癌的风险，可用于临床肺癌的辅助诊断，为患者采取相关的治疗措施或者决策提供有效的依据，临床应用前景良好。

The invention provides the use of a detection reagent of VWF glycosylated protein in the preparation of a kit for screening the risk of lung cancer, and belongs to the field of in vitro diagnostic reagents. By detecting the level of VWF glycosylated protein in the plasma, the kit of the present invention can screen the risk of lung cancer in the population to be tested, can be used for auxiliary diagnosis of clinical lung cancer, and provides an effective basis for patients to take relevant treatment measures or decision-making, The prospect of clinical application is good.

Description

Translated fromChinese

VWF糖基化蛋白的检测试剂在制备筛查患肺癌风险的试剂盒中的用途The detection reagent of VWF glycosylated protein is preparing a kit for screening the risk of lung cancerUses in

技术领域technical field

本发明属于体外诊断试剂领域，具体涉及VWF糖基化蛋白的检测试剂在制备筛查患肺癌风险的试剂盒中的用途。The invention belongs to the field of in vitro diagnostic reagents, and in particular relates to the use of a detection reagent for VWF glycosylated protein in the preparation of a kit for screening the risk of lung cancer.

背景技术Background technique

肺癌是世界上最常见的恶性肿瘤之一，其发病率和死亡率呈逐年上升趋势，目前发病率居世界首位，严重威胁着人类健康和生命。Lung cancer is one of the most common malignant tumors in the world. Its morbidity and mortality are increasing year by year. Currently, the incidence rate ranks first in the world, which seriously threatens human health and life.

肺癌是一种善于隐匿的疾病，经常在疾病发展到晚期才表现出临床症状，70～80％的肺癌患者在诊断出患有肺癌症状时已是中、晚期，癌细胞已经扩散，错过了最佳治愈时机，五年生存率低。对于早期的肺癌患者，经过及时治疗可大大提高患者的5年及以上生存率和生存质量。因此肺癌的早期诊断和进行有效的筛查至关重要。Lung cancer is a disease that is good at concealment. It often shows clinical symptoms when the disease develops to an advanced stage. 70-80% of lung cancer patients are already in the middle or late stage when they are diagnosed with lung cancer symptoms. The best cure time, five-year survival rate is low. For patients with early lung cancer, timely treatment can greatly improve the 5-year survival rate and quality of life of the patients. Therefore, early diagnosis and effective screening of lung cancer are very important.

肺癌的筛查，是指对那些没有肺癌相关症状的人群进行常规体检，在出现症状前及时发现肺癌。如果可以找到血浆或其外泌体里面的肺癌分子标志物，用于提示临床医生早期对患者采取相关的治疗措施或者决策具有重要的意义。Screening for lung cancer refers to routine physical examinations for those without lung cancer-related symptoms to detect lung cancer in time before symptoms appear. If the molecular markers of lung cancer in plasma or exosomes can be found, it is of great significance to prompt clinicians to take relevant treatment measures or decision-making for patients in the early stage.

VWF蛋白(UniProtKB编号为:P04275)是一种大分子量的多亚基糖蛋白，由多个220kD的亚基组成。一方面，VWF可以介导血小板粘附于受损的血管壁，形成血栓得以止血；另一方面，正常情况下，VWF与人凝血因子VIII基因(hFVIII)在血浆中紧密结合形成稳定的复合物，VWF复合物的形成在体内阻碍了可能由细胞表面受体介导的hFVIII分解，对维持hFVIII的半衰期至关重要。糖基化(Glycosylation)是一种重要且广泛存在的蛋白质翻译后修饰方式。目前尚未见VWF糖基化蛋白与肺癌相关的报道。VWF protein (UniProtKB number: P04275) is a large molecular weight multi-subunit glycoprotein consisting of multiple 220kD subunits. On the one hand, VWF can mediate the adhesion of platelets to the damaged blood vessel wall, forming a thrombus to stop bleeding; on the other hand, under normal circumstances, VWF and human coagulation factor VIII gene (hFVIII) are tightly combined in plasma to form a stable complex , the formation of the VWF complex hinders the breakdown of hFVIII in vivo, possibly mediated by cell surface receptors, and is critical for maintaining the half-life of hFVIII. Glycosylation is an important and widespread protein post-translational modification. There is no report on the relationship between VWF glycosylated protein and lung cancer.

发明内容Contents of the invention

本发明的第一个目的在于提供VWF糖基化蛋白的检测试剂在制备筛查患肺癌风险的试剂盒中的用途。The first object of the present invention is to provide the use of the detection reagent of VWF glycosylated protein in the preparation of a kit for screening the risk of lung cancer.

本发明的第二个目的在于提供检测VWF糖基化蛋白的仪器在制备筛查患肺癌风险的设备中的用途。The second object of the present invention is to provide the use of the instrument for detecting VWF glycosylated protein in the preparation of equipment for screening the risk of lung cancer.

本发明的第三个目的在于提供一种新的筛查患肺癌风险的试剂盒或设备。The third object of the present invention is to provide a new kit or device for screening the risk of lung cancer.

本发明提供了检测VWF糖基化蛋白的试剂在制备筛查患肺癌风险的试剂盒中的用途。The invention provides the use of a reagent for detecting VWF glycosylated protein in the preparation of a kit for screening the risk of lung cancer.

进一步地，所述VWF糖基化蛋白为VWF蛋白的第2357位点上发生N-糖基化修饰后的产物。Further, the VWF glycosylated protein is a product after N-glycosylation modification at the 2357th position of the VWF protein.

进一步地，所述检测VWF糖基化蛋白的试剂为检测人血浆中VWF糖基化蛋白的试剂。Further, the reagent for detecting VWF glycosylated protein is a reagent for detecting VWF glycosylated protein in human plasma.

进一步地，所述检测VWF糖基化蛋白的试剂为酶联免疫吸附试验用试剂或westernblot试剂。Further, the reagent for detecting VWF glycosylated protein is an enzyme-linked immunosorbent assay reagent or a western blot reagent.

本发明还提供了检测VWF糖基化蛋白的仪器在制备筛查患肺癌风险的设备中的用途。The present invention also provides the use of the instrument for detecting VWF glycosylated protein in the preparation of equipment for screening the risk of lung cancer.

进一步地，所述检测VWF糖基化蛋白的仪器为检测人血浆中VWF糖基化蛋白的仪器。Further, the instrument for detecting VWF glycosylated protein is an instrument for detecting VWF glycosylated protein in human plasma.

进一步地，所述检测VWF糖基化蛋白的仪器为液相色谱-质谱联用仪。Further, the instrument for detecting VWF glycosylated protein is liquid chromatography-mass spectrometry.

本发明还提供了一种筛查患肺癌风险的试剂盒或设备，它包括用于检测VWF糖基化蛋白的试剂或仪器。The present invention also provides a kit or device for screening the risk of lung cancer, which includes a reagent or instrument for detecting VWF glycosylated protein.

本发明通过质谱蛋白组学方法鉴定发现VWF蛋白的第2357位点上能够发生N-糖基化修饰。The present invention finds that N-glycosylation modification can occur at the 2357th position of the VWF protein through mass spectrometry proteomics method identification.

蛋白质糖基化按照氨基酸和糖的连接方式可以分为四类：O位糖基化、N位糖基化、C位甘露糖化以及GPI(glycophosphatidlyinositol)锚定连接。其中，N位糖基化(N-linkedglycosylation，简称N-糖基化)是通过糖链的还原端N-乙酰胺基葡萄糖(Glc-Nac)和肽链中某些Asn侧链酰胺基上的N原子相连，能接有糖链的Asn必须处于Asn-X-Ser/Thr(X！＝P)残基构成的基序中。糖为N-乙酰葡糖胺。Protein glycosylation can be divided into four types according to the connection mode of amino acid and sugar: O-position glycosylation, N-position glycosylation, C-position mannosylation and GPI (glycophosphatidlyinositol) anchor connection. Among them, N-linkedglycosylation (N-linkedglycosylation, referred to as N-glycosylation) is through the reducing end N-acetamidoglucose (Glc-Nac) of the sugar chain and some Asn side chain amide groups in the peptide chain. The N atoms are connected, and the Asn that can be connected with sugar chains must be in the motif composed of Asn-X-Ser/Thr (X!=P) residues. The sugar is N-acetylglucosamine.

本发明的关键在于，确定了人体血浆中VWF糖基化蛋白水平与患肺癌的风险显著相关，因此可以通过检测人体血浆中VWF糖基化蛋白水平来判断患肺癌的风险，至于具体检测人体血浆中VWF糖基化蛋白水平的手段，可以采用现有技术公开的各种手段，本发明实施例具体采用液相色谱-质谱联用分析法进行检测，单不仅仅限于该手段，任何能够检测人体血浆中VWF糖基化蛋白水平的方法均可用于肺癌筛查。The key point of the present invention is to determine that the level of VWF glycosylated protein in human plasma is significantly related to the risk of lung cancer, so the risk of lung cancer can be judged by detecting the level of VWF glycosylated protein in human plasma. As for the specific detection of human plasma The means for the level of VWF glycosylated protein in the medium can adopt various means disclosed in the prior art. The embodiment of the present invention specifically adopts the liquid chromatography-mass spectrometry analysis method for detection, and it is not limited to this means, any method that can detect human body The method of VWF glycosylated protein level in plasma can be used for lung cancer screening.

本发明提供了一种新的肺癌筛查标记物和一种新的肺癌筛查试剂盒，可以筛查待检人群患肺癌的风险，可用于临床肺癌的辅助诊断，为患者采取相关的治疗措施或者决策提供有效的依据，临床应用前景良好。且能以血浆作为检测样品，对患者伤害很低。The present invention provides a new lung cancer screening marker and a new lung cancer screening kit, which can screen the risk of lung cancer in people to be checked, can be used for auxiliary diagnosis of clinical lung cancer, and take relevant treatment measures for patients Or provide an effective basis for decision-making, and the clinical application prospect is good. Moreover, blood plasma can be used as a test sample, and the harm to patients is very low.

显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。Apparently, according to the above content of the present invention, according to common technical knowledge and conventional means in this field, without departing from the above basic technical idea of the present invention, other various forms of modification, replacement or change can also be made.

以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。The above-mentioned content of the present invention will be further described in detail below through specific implementation in the form of examples. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention.

附图说明Description of drawings

图1：肺癌患者(LC)与健康人群(NC)血浆中VWF糖基化蛋白水平对比图。Figure 1: Comparison of plasma levels of VWF glycosylated proteins in lung cancer patients (LC) and healthy people (NC).

图2：肺癌患者(LC)与健康人群(NC)的ROC分析。Figure 2: ROC analysis of lung cancer patients (LC) and healthy people (NC).

具体实施方式Detailed ways

本发明所用原料与设备均为已知产品，通过购买市售产品所得。The raw materials and equipment used in the present invention are known products obtained by purchasing commercially available products.

实施例1：VWF糖基化蛋白的检测试剂在制备筛查患肺癌风险的试剂盒中的用途Example 1: The use of the detection reagent of VWF glycosylated protein in the preparation of a kit for screening the risk of lung cancer

1.检测原理1. Detection principle

糖基化是一种重要且广泛存在的蛋白质翻译后修饰方式。血浆N-糖基化检测原理为：提取血浆全蛋白，经胰酶酶解后形成的肽段使用亲水作用色谱富集N糖基化肽段，再用N-糖酰胺酶F(PNGase F)在H₂O¹⁸中切除糖链，使得糖基化修饰过的位点引入O¹⁸，分子量的增加使得在液质联用系统中能够轻松辦别出该位点，从而确定N-糖基化发生的位点。Glycosylation is an important and ubiquitous protein post-translational modification. The detection principle of plasma N-glycosylation is as follows: extract plasma whole protein, use hydrophilic interaction chromatography to enrich N-glycosylated peptides, and then use N-glycoamidase F (PNGase F ) cut the sugar chain in H₂ O¹⁸ , so that the glycosylation-modified site is introduced into O¹⁸ , and the increase in molecular weight makes it easy to identify the site in the liquid chromatography-mass spectrometry system, thereby determining the N-glycosyl group the site where chemicalization occurs.

2.检测方法2. Detection method

(1)蛋白提取(1) Protein extraction

表1.样品来源人群信息Table 1. Sample source population information

人群crowd人数number of people肺癌患者lung cancer patients2020健康人群healthy people2020

样品来源于肺癌患者和健康人群血浆，受试人群的年龄范围为29～82岁，男性占32.5％，女性占67.5％。The samples were derived from the blood plasma of lung cancer patients and healthy people. The age range of the subjects was 29-82 years old, 32.5% were male and 67.5% were female.

样品从-80℃取出，4℃，12000g离心10min，去除红细胞碎片，上清液转移至新的离心管，利用BCA试剂盒进行蛋白浓度测定。The sample was taken out from -80°C, centrifuged at 12000g for 10min at 4°C to remove red blood cell fragments, and the supernatant was transferred to a new centrifuge tube, and the protein concentration was determined using the BCA kit.

(2)胰酶酶解(2) Trypsinization

各样品蛋白取等量进行酶解，用裂解液将体积调整至一致，加入适量标准蛋白，再加入二硫苏糖醇(DTT)使其终浓度为5mM，56℃还原30min。之后加入碘乙酰胺(IAA)使其终浓度为11mM，室温避光孵育15min。将烷基化好的样本转移至超滤管，室温12000g离心20min，用8M尿素置换3次，再用置换buffer置换尿素3次，以1:50的比例(蛋白酶：蛋白，m/m)加入胰蛋白酶，酶解过夜。室温12000g离心10min回收肽段，再用超纯水回收肽段一次，合并两次肽段溶液。An equal amount of protein from each sample was used for enzymatic hydrolysis, and the volume was adjusted to be consistent with the lysate, an appropriate amount of standard protein was added, and then dithiothreitol (DTT) was added to make the final concentration 5 mM, and reduced at 56°C for 30 min. Afterwards, iodoacetamide (IAA) was added to make the final concentration 11 mM, and incubated at room temperature in the dark for 15 min. Transfer the alkylated sample to an ultrafiltration tube, centrifuge at 12000g at room temperature for 20min, replace with 8M urea for 3 times, then replace urea with replacement buffer for 3 times, add in a ratio of 1:50 (protease: protein, m/m) Trypsin, digested overnight. Centrifuge at 12000g at room temperature for 10 minutes to recover the peptides, then use ultrapure water to recover the peptides once, and combine the peptide solutions twice.

(3)TMT标记(3) TMT mark

胰酶酶解的肽段用Strata X C18(Phenomenex)除盐后真空冷冻干燥。以0.5MTEAB溶解肽段，根据TMT试剂盒操作说明标记肽段。简单的操作如下：标记试剂解冻后用乙腈溶解，与肽段混合后室温孵育2h，标记后的肽段混合后除盐，真空冷冻干燥。Trypsinized peptides were desalted with Strata X C18 (Phenomenex) and then vacuum freeze-dried. Dissolve the peptide with 0.5 MTEAB, and label the peptide according to the operating instructions of the TMT kit. The simple operation is as follows: after thawing the labeling reagent, dissolve it with acetonitrile, mix it with the peptide and incubate at room temperature for 2 hours, desalt the labeled peptide after mixing, and vacuum freeze-dry.

(4)HPLC分级(4) HPLC classification

肽段用高pH反相HPLC分级，色谱柱为Agilent 300Extend C18(5μm粒径，4.6mm内径，250mm长)。操作如下：肽段分级梯度为8％-32％乙腈、pH 9，60min时间分离60个组分，随后肽段合并为4个组分，合并后的组分经真空冷冻干燥后进行后续操作。Peptides were fractionated by high-pH reverse-phase HPLC, and the chromatographic column was Agilent 300Extend C18 (5 μm particle size, 4.6 mm inner diameter, 250 mm long). The operation is as follows: the stepwise peptide gradient is 8%-32% acetonitrile,pH 9, 60 fractions are separated in 60 minutes, and then the peptides are merged into 4 fractions, and the combined fractions are subjected to vacuum freeze-drying before subsequent operations.

(5)修饰富集(5) Modification and enrichment

将肽段溶解在200μL富集缓冲溶液中(80％乙腈/5％三氟乙酸)，转移上清液至亲水(HILIC)微柱中，1000g离心约15min完成富集。接着使用富集缓冲液洗涤亲水微柱3次。然后使用分别使用0.1％三氟乙酸，50mM碳酸氢铵溶液和50％乙腈洗脱糖肽，收集合并洗脱液并真空冷冻抽干。抽干后复溶在50μL重氧水溶解的50mM碳酸氢铵缓冲液中，并加入2μLPNGase F糖苷酶(植物样本复溶在50μL重氧水溶解的50mM柠檬酸钠缓冲液中，并加入2μLPNGase A和PNGase F糖苷酶)，37℃酶切过夜。最后，按照C18ZipTips说明书除盐，真空冷冻抽干后供液质联用分析。The peptides were dissolved in 200 μL enrichment buffer solution (80% acetonitrile/5% trifluoroacetic acid), the supernatant was transferred to a hydrophilic (HILIC) micro-column, and the enrichment was completed by centrifugation at 1000 g for about 15 min. Then the hydrophilic micro-column was washed 3 times with enrichment buffer. The glycopeptides were then eluted using 0.1% trifluoroacetic acid, 50 mM ammonium bicarbonate solution and 50% acetonitrile, respectively, and the combined eluates were collected and vacuum freeze-dried. After pumping dry, redissolve in 50mM ammonium bicarbonate buffer dissolved in 50μL heavy oxygen water, and add 2μL PNGase F glycosidase (plant samples are redissolved in 50mM sodium citrate buffer dissolved in 50μL heavy oxygen water, and add 2μL PNGase A and PNGase F glycosidase), digest overnight at 37°C. Finally, according to the instructions of C18ZipTips, desalting was carried out, and after vacuum freeze-drying, it was used for liquid mass spectrometry analysis.

(6)液相色谱-质谱联用分析(6) Liquid chromatography-mass spectrometry analysis

肽段用液相色谱流动相A相溶解后使用EASY-nLC 1200超高效液相系统进行分离。流动相A为含0.1％甲酸和2％乙腈的水溶液；流动相B为含0.1％甲酸和90％乙腈的水溶液。液相梯度设置：0-38min，4％～20％B；38-54min，20％～32％B；54-57min，32％～80％B；57-60min，80％B，流速维持在500nL/min。肽段经由超高效液相系统分离后被注入NSI离子源中进行电离然后进Q Exactive^TM HF-X质谱进行分析。离子源电压设置为2.1kV，肽段母离子及其二级碎片都使用高分辨的Orbitrap进行检测和分析。一级质谱扫描范围设置为350-1400m/z，扫描分辨率设置为120000；二级质谱扫描范围则固定起点为100m/z，二级扫描分辨率设置为30000。数据采集模式使用数据依赖型扫描(DDA)程序，即在一级扫描后选择信号强度最高的前20肽段母离子依次进入HCD碰撞池使用28％的碎裂能量进行碎裂，同样依次进行二级质谱分析。为了提高质谱的有效利用率，自动增益控制(AGC)设置为1E5，信号阈值设置为1E5 ions/s，最大注入时间设置为100ms，串联质谱扫描的动态排除时间设置为15s秒避免母离子的重复扫描。Peptides were dissolved in phase A of liquid chromatography mobile phase and then separated using EASY-nLC 1200 ultra-high performance liquid phase system. Mobile phase A is an aqueous solution containing 0.1% formic acid and 2% acetonitrile; mobile phase B is an aqueous solution containing 0.1% formic acid and 90% acetonitrile. Liquid phase gradient setting: 0-38min, 4%～20%B; 38-54min, 20%～32%B; 54-57min, 32%～80%B; 57-60min, 80%B, the flow rate is maintained at 500nL /min. Peptides were separated by an ultra-high performance liquid phase system and then injected into the NSI ion source for ionization and then analyzed by Q Exactive^TM HF-X mass spectrometry. The ion source voltage was set at 2.1kV, and the peptide precursor ions and their secondary fragments were detected and analyzed using a high-resolution Orbitrap. The scanning range of the primary mass spectrometer is set to 350-1400m/z, and the scanning resolution is set to 120000; the scanning range of the secondary mass spectrometry is fixed at 100m/z, and the scanning resolution of the secondary mass spectrometry is set to 30000. The data acquisition mode uses the data-dependent scanning (DDA) program, that is, after the first-level scanning, the precursor ions of the top 20 peptides with the highest signal intensity are selected to enter the HCD collision cell in sequence and use 28% fragmentation energy for fragmentation, and the second stage is also carried out in sequence. level mass spectrometry. In order to improve the effective utilization of the mass spectrometer, the automatic gain control (AGC) was set to 1E5, the signal threshold was set to 1E5 ions/s, the maximum injection time was set to 100ms, and the dynamic exclusion time of the tandem mass spectrometry scan was set to 15s to avoid the repetition of precursor ions scanning.

3.检测结果3. Test results

本发明通过前述质谱蛋白组学方法鉴定发现VWF蛋白的第2357位点上能够发生N-糖基化修饰，且该N-糖基化修饰水平在肺癌患者和健康人群之间有显著性差异。The present invention finds that N-glycosylation modification can occur at the 2357th position of VWF protein through the aforementioned mass spectrometry proteomics method, and the N-glycosylation modification level is significantly different between lung cancer patients and healthy people.

表2.不同人群血浆VWF糖基化蛋白水平的检测结果Table 2. Detection results of plasma VWF glycosylated protein levels in different populations

根据表2和图1可以看出，与健康人群相比，肺癌患者血浆中的VWF糖基化蛋白水平显著降低，差异具有统计学意义(P<0.01)。According to Table 2 and Figure 1, it can be seen that compared with healthy people, the level of VWF glycosylated protein in the plasma of lung cancer patients was significantly reduced, and the difference was statistically significant (P<0.01).

根据ROC曲线(图2)可以看出，ROC曲线下面积为76.8，说明通过检测血浆中VWF糖基化蛋白水平来筛查或诊断肺癌患者的方法具有优良的准确度、敏感度和特异性。According to the ROC curve (Figure 2), it can be seen that the area under the ROC curve is 76.8, indicating that the method of screening or diagnosing lung cancer patients by detecting the level of VWF glycosylated protein in plasma has excellent accuracy, sensitivity and specificity.

综上，本发明首次发现，与健康人群相比，肺癌患者血浆中的VWF糖基化蛋白水平显著降低，差异具有统计学意义(P<0.01)。说明通过检测血浆中VWF糖基化蛋白水平，可以筛查待检人群患肺癌的风险：若VWF糖基化蛋白水平低(相对于健康人群而言)，则患肺癌的风险高，若VWF糖基化蛋白水平高(相对于健康人群而言)，则患肺癌的风险低。可用于临床肺癌的辅助诊断，为患者采取相关的治疗措施或者决策提供有效的依据，临床应用前景良好。In summary, the present invention found for the first time that compared with healthy people, the level of VWF glycosylated protein in the plasma of lung cancer patients was significantly reduced, and the difference was statistically significant (P<0.01). It shows that by detecting the level of VWF glycosylated protein in plasma, the risk of lung cancer in the population to be tested can be screened: if the level of VWF glycosylated protein is low (relative to healthy people), the risk of lung cancer is high, and if the level of VWF glycosylated Higher levels of ylated protein (relative to healthy people) were associated with a lower risk of lung cancer. It can be used for auxiliary diagnosis of clinical lung cancer, and provide effective basis for patients to take relevant treatment measures or decision-making, and has a good clinical application prospect.

Claims

Translated fromChinese

1.检测VWF糖基化蛋白的试剂在制备筛查患肺癌风险的试剂盒中的用途。1. Use of a reagent for detecting VWF glycosylated protein in the preparation of a kit for screening the risk of lung cancer.

2.如权利要求1所述的用途，其特征在于，所述VWF糖基化蛋白为VWF蛋白的第2357位点上发生N-糖基化修饰后的产物。2. The use according to claim 1, characterized in that the VWF glycosylated protein is a product of N-glycosylation modification at the 2357th position of the VWF protein.

3.如权利要求1所述的用途，其特征在于，所述检测VWF糖基化蛋白的试剂为检测人血浆中VWF糖基化蛋白的试剂。3. The use according to claim 1, wherein the reagent for detecting VWF glycosylated protein is a reagent for detecting VWF glycosylated protein in human plasma.

4.如权利要求1-3任一项所述的用途，其特征在于，所述检测VWF糖基化蛋白的试剂为酶联免疫吸附试验用试剂或western blot试剂。4. The use according to any one of claims 1-3, characterized in that the reagent for detecting VWF glycosylated protein is an enzyme-linked immunosorbent assay reagent or a western blot reagent.

5.检测VWF糖基化蛋白的仪器在制备筛查患肺癌风险的设备中的用途。5. Use of the instrument for detecting VWF glycosylated protein in the preparation of equipment for screening the risk of lung cancer.

6.如权利要求5所述的用途，其特征在于，所述VWF糖基化蛋白为VWF蛋白的第2357位点上发生N-糖基化修饰后的产物。6 . The use according to claim 5 , wherein the VWF glycosylated protein is a product of N-glycosylation modification at position 2357 of the VWF protein.

7.如权利要求5所述的用途，其特征在于，所述检测VWF糖基化蛋白的仪器为检测人血浆中VWF糖基化蛋白的仪器。7. The use according to claim 5, wherein the instrument for detecting VWF glycosylated protein is an instrument for detecting VWF glycosylated protein in human plasma.

8.如权利要求5-7任一项所述的用途，其特征在于，所述检测VWF糖基化蛋白的仪器为液相色谱-质谱联用仪。8. The use according to any one of claims 5-7, characterized in that the instrument for detecting VWF glycosylated protein is a liquid chromatography-mass spectrometry instrument.

9.一种筛查患肺癌风险的试剂盒或设备，其特征在于，它包括用于检测VWF糖基化蛋白的试剂或仪器。9. A kit or device for screening the risk of lung cancer, characterized in that it includes a reagent or instrument for detecting VWF glycosylated protein.

10.如权利要求9所述的试剂盒，其特征在于，所述VWF糖基化蛋白为VWF蛋白的第2357位点上发生N-糖基化修饰后的产物。10 . The kit according to claim 9 , wherein the VWF glycosylated protein is a product of N-glycosylation modification at position 2357 of the VWF protein. 11 .