WO2023098804A1 - 尿液蛋白标志物在诊断遗传性血管水肿中的用途 - Google Patents

尿液蛋白标志物在诊断遗传性血管水肿中的用途 Download PDF

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WO2023098804A1
WO2023098804A1 PCT/CN2022/135877 CN2022135877W WO2023098804A1 WO 2023098804 A1 WO2023098804 A1 WO 2023098804A1 CN 2022135877 W CN2022135877 W CN 2022135877W WO 2023098804 A1 WO2023098804 A1 WO 2023098804A1
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protein
cathepsin
inhibitor
complement component
growth factor
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PCT/CN2022/135877
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English (en)
French (fr)
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支玉香
吴建强
刘鹏
汤晓悦
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中国医学科学院北京协和医院
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/86—Signal analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry

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  • the present disclosure relates to clinical medicine, in particular to urine protein markers related to human hereditary angioedema. More specifically, the present disclosure relates to collecting urine samples from patients with hereditary angioedema and mass spectrometry analysis proteomics technology to obtain urine protein markers for diagnosing hereditary angioedema, and provides methods and applications thereof.
  • Urine is an ideal source of clinical samples for disease markers, which can be obtained noninvasively in large quantities. Moreover, the urine lacks the regulation of the homeostasis mechanism, which can enrich various changes excreted by the human body. These changes are potential marker sources (see Wu J&Gao Y.Physiological conditions can be reflected in human urine proteome and metabolome.Expert Review of Proteomics, 2015; 12(6):623-636).
  • Urinary proteomics aims to establish a non-invasive "liquid biopsy" diagnostic method, which partially replaces tissue biopsy by detecting specific disease markers in urine. It has become an important research direction and has a huge potential. Prospects for clinical translation.
  • DIA Data independent acquisition
  • DIA is the latest data-independent proteome quantification technology, which divides the entire full scan range of mass spectrometry into several windows, so as to obtain all fragment information of all ions in the sample without difference or omission.
  • DIA technology can obtain information of all peptides in clinical samples without discrimination without loss of low-abundance proteins, it is especially suitable for robust and accurate quantification of proteins under different laboratory conditions (see Navarro P et al. A multicenter study benchmarks software tools for label-free proteome quantification.
  • PRM Parallel reaction monitoring
  • Orbitrap high-resolution mass spectrometry
  • HAE Hereditary angioedema
  • HAE Hereditary angioedema
  • Skin and abdominal attacks are the most common features of HAE, which can easily be misdiagnosed as edema or acute abdomen leading to ineffective treatment or unnecessary surgery.
  • more than 50% of HAE patients will develop laryngeal edema, and they will die of suffocation if they are not rescued in time. Therefore, the discovery of early diagnostic markers is crucial for the clinical management of HAE.
  • the present invention provides the use of an identification reagent for any one of the following proteins or a combination of proteins in the preparation of a product for diagnosing hereditary angioedema:
  • Plasma protease C1 inhibitor ketohexokinase, phenazine biosynthetic domain protein, contractile protein alpha chain, prefibrillin 1, cathepsin H precursor, IST1 homologue, malate dehydrogenase, Cochlin, containing Disintegrin and metalloprotease domain protein 10, basement membrane-specific heparan sulfate proteoglycan core protein, kininogen 1 protein, V-set domain-containing inhibitor of T-cell activation 1, epidermal growth factor precursor, junction Adhesion molecule A, N-acetylated ⁇ -linked acid dipeptidase 2, transmembrane glycoprotein NMB, procollagen c endopeptidase enhancer 2, urokinase plasminogen activator surface receptor, thrombospondin proteolysis Integrin Metallopeptidase 1, Cathepsin Z, Cathepsin L1, Endothelial Cell Selective Adhesion Molecule, Kallikrein 1,
  • the present invention uses some proteins as examples in specific examples to demonstrate the diagnostic effect of the diagnostic markers provided by the present invention.
  • malate dehydrogenase, kininogen 1, kallikrein 1, thrombospondin disintegrin metallopeptidase 1 were detected in cohort 2 of the present invention in HAE patients and subjects, and the experimental results It is proved that the fold difference in the expression of the above proteins between HAE patients and subjects is enough to distinguish the two groups of people and achieve the purpose of diagnosis; , epidermal growth factor precursor, kininogen 1, and aspartic proteinase A to calculate the AUC value, and the AUC values of the above proteins in the diagnosis of HAE alone are above 0.7, reaching the standard for accurate diagnosis; the above exemplary verification provides The above-mentioned profibrin 1, thrombospondin disintegrin metallopeptidase 1, malate dehydrogenase, kallikrein 1, plasma protease C1 inhibitor, epidermal growth factor precursor, kininogen 1, aspart
  • the present invention provides the combination of at least 3 proteins among the above 35 proteins, and the application of the identification reagent for protein combination in the preparation of products for diagnosing angioedema.
  • plasma protease C1 inhibitor, kallikrein 1 and thrombospondin disintegrin metallopeptidase 1 were randomly selected from 35 proteins for combination to verify the diagnostic effect of the protein combination on HAE, The AUC value was 0.925, proving that it has an excellent diagnostic effect and can accurately distinguish healthy people from patients.
  • ROC curve receiver operator characteristic curve
  • AUC value Absolute under Curve
  • the identification reagents include reagents for quantitative and/or qualitative detection of proteins; more specifically, reagents for detecting protein expression.
  • the reagents for detecting protein expression include, but are not limited to, reagents used in any one or more of the following methods: mass spectrometry, immunohistochemistry (Immunohistochemistry) staining, hematoxylin-eosin staining (hematoxylin -eosin staining, referred to as HE staining), safranin O-fast green staining, Western Blot (Western Blot), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), sandwich assay, immunoprecipitation analysis, Complement fixation assay, flow cytometry and protein microarray.
  • mass spectrometry immunohistochemistry staining
  • hematoxylin-eosin staining hematoxylin -eosin stain staining, referred to as HE staining
  • HE staining hematoxylin -eosin stain staining
  • the reagents include antibodies, nucleic acid probes or other reagents that have a certain affinity (have specificity) for a particular marker sufficient to distinguish the particular marker from other markers that may be present in the sample of interest.
  • the measurement of protein expression can be performed by any means known in the art.
  • identification reagent and “detection reagent” have the same meaning and can be used interchangeably. As described in the present invention, “identification” and “detection” have the same meaning and can be used interchangeably. Both “identifying” and “detecting” can be quantitative and/or qualitative.
  • said identification is an identification (detection) performed on a sample from a subject.
  • the sample may include, but not limited to, blood, urine, mucus, amniotic fluid, plasma, ascites, cerebrospinal fluid, sputum, bone marrow, synovial fluid, aqueous humor, breast milk, sweat, fecal matter, tears, peritoneal fluid, Lymph, vaginal fluid, blastocoel fluid, cord blood, and/or saliva.
  • said sample is urine.
  • said urine is morning urine.
  • the definition of the term "morning urine” is common knowledge in the field, and all urine samples meeting the urine routine examination fall within the scope of the samples described in the present invention.
  • the term "subject” refers to any animal (e.g., mammal), including, but not limited to, humans, non-human primates (e.g., apes, monkeys, chimpanzees, gorillas), rodents, etc. .
  • the subject is a human.
  • the products include diagnostic devices, diagnostic kits, diagnostic chips and the like.
  • kits or chip for diagnosing hereditary hemoedema comprising identification reagents for any one or more of the following proteins:
  • Plasma protease C1 inhibitor ketohexokinase, phenazine biosynthetic domain protein, contractile protein alpha chain, prefibrillin 1, cathepsin H precursor, IST1 homologue, malate dehydrogenase, Cochlin, containing Disintegrin and metalloprotease domain protein 10, basement membrane-specific heparan sulfate proteoglycan core protein, kininogen 1, V-set domain-containing inhibitor of T-cell activation 1, epidermal growth factor precursor, junctional adhesion Adnexin A, N-acetylated ⁇ -linked acid dipeptidase 2, transmembrane glycoprotein NMB, procollagen c endopeptidase enhancer 2, urokinase plasminogen activator surface receptor, thrombospondin proteolytic integration Cathepsin Metallopeptidase 1, Cathepsin Z, Cathepsin L1, Endothelial Cell Selective Adhesion Molecule,
  • the kit or chip contains identification reagents for any one or more of the following proteins: plasma protease C1 inhibitor, prefibrin 1, epidermal growth factor precursor, malate dehydrogenase, kininogen 1, Kallikrein 1, thrombospondin proteolytic integrin metallopeptidase 1, aspartic proteinase A.
  • the kit or chip contains identification reagents for the following protein combinations, the protein combination comprising: plasma protease C1 inhibitor, kallikrein 1 and thrombospondin disintegrin metallopeptidase 1.
  • the protein combination consists of plasma protease C1 inhibitor, kallikrein 1 and thrombospondin disintegrin metallopeptidase 1.
  • chip may refer to a solid substrate having specific reagents (eg, antibodies, nucleic acid probes) attached to its surface.
  • specific reagents eg, antibodies, nucleic acid probes
  • the chip can use any solid-phase carrier, and specific examples of the material of the solid-phase carrier can include: polystyrene, polyvinyl chloride, agarose, liposome, membrane carrier, polymer magnetic particles wait.
  • a computer readable medium may be included in the kit.
  • kits described herein comprise packaging materials.
  • packaging material may refer to the physical structure that houses the components of the kit.
  • the packaging material maintains the sterility of the kit components and can be made from materials commonly used for such purposes (eg, paper, glass, plastic, metallic materials, etc.).
  • the kit may also contain buffers, preservatives or protein/nucleic acid stabilizers.
  • the present invention provides a set of systems for diagnosing hereditary angioedema, the system includes a calculation module for calculating whether a subject suffers from hereditary angioedema according to the detection results of any one or more of the following proteins:
  • Plasma protease C1 inhibitor ketohexokinase, phenazine biosynthetic domain protein, contractile protein alpha chain, prefibrillin 1, cathepsin H precursor, IST1 homologue, malate dehydrogenase, Cochlin, containing Disintegrin and metalloprotease domain protein 10, basement membrane-specific heparan sulfate proteoglycan core protein, kininogen 1 protein, V-set domain-containing inhibitor of T-cell activation 1, epidermal growth factor precursor, junction Adhesion molecule A, N-acetylated ⁇ -linked acid dipeptidase 2, transmembrane glycoprotein NMB, procollagen c endopeptidase enhancer 2, urokinase plasminogen activator surface receptor, thrombospondin proteolysis Integrin Metallopeptidase 1, Cathepsin Z, Cathepsin L1, Endothelial Cell Selective Adhesion Molecule, Kallikrein 1,
  • the protein is selected from any one or more of the following:
  • the protein comprises a combination of plasma proteinase C1 inhibitor, kallikrein 1 and thrombospondin disintegrin metallopeptidase 1.
  • the system includes the following devices in sequence:
  • a data collection module used to collect the test results of the subjects' samples, specifically, to detect the expression levels of the above-mentioned protein markers
  • a diagnosis result output module configured to output the diagnosis result of the calculation module.
  • any of the above modules can include the hospital (doctor) port and the subject (patient) port, which is convenient for both parties who diagnose the disease to upload the test results, view the diagnosis results and communicate.
  • the data collection module can digitally record the results of the analysis.
  • the computing module is capable of performing any desired mathematical processing on the data generated by the analysis.
  • a method of diagnosing whether a subject has hereditary angioedema comprising the steps of:
  • Plasma protease C1 inhibitor ketohexokinase, phenazine biosynthetic domain protein, contractile protein alpha chain, prefibrillin 1, cathepsin H precursor, IST1 homologue, malate dehydrogenase, Cochlin, containing Disintegrin and metalloprotease domain protein 10, basement membrane-specific heparan sulfate proteoglycan core protein, kininogen 1, V-set domain-containing inhibitor of T-cell activation 1, epidermal growth factor precursor, junctional adhesion Adnexin A, N-acetylated ⁇ -linked acid dipeptidase 2, transmembrane glycoprotein NMB, procollagen c endopeptidase enhancer 2, urokinase plasminogen activator surface receptor, thrombospondin proteolytic integration Cathepsin Metallopeptidase 1, Cathepsin Z, Cathepsin L1, Endothelial Cell Selective Adhesion Molecule,
  • step 3 Comparing the detection result obtained in step 2) with the standard value (threshold value), and giving a diagnosis result according to the comparison result, that is, judging whether the subject is an HAE patient.
  • said standard value may be said protein expression level in a sample obtained from a control.
  • the values obtained by different detection methods or detection instruments for the detection of the same sample may be different, but for those skilled in the art, the method for obtaining the standard value is routine.
  • control in the present invention refers to individuals who do not suffer from hereditary angioedema.
  • the control is not necessarily a clinically healthy individual, but is preferably a healthy individual.
  • a decrease in the expression level of a protein selected from the group consisting of a plasma protease C1 inhibitor, as compared to a standard value indicates that the subject suffers from hereditary angioedema or is at increased risk of suffering from hereditary angioedema , ketohexokinase, phenazine biosynthetic domain protein, malate dehydrogenase, kininogen 1, immunoglobulin heavy chain constant region ⁇ 4, V-set domain-containing T-cell activation inhibitor 1, epidermis A growth factor precursor, kallikrein 1, aspartic proteinase A, calbindin, or a combination thereof. On the contrary, the risk is reduced.
  • a control is an individual who does not suffer from hereditary angioedema.
  • a control need not be a clinically healthy individual, but is preferably a healthy individual.
  • identification reagent used in this application refers to a reagent capable of quantitatively and/or qualitatively detecting a target protein. Specifically, but without limitation, the identification reagent of the present invention can specifically identify the presence or absence of the target protein in the test sample; or can detect the level of the target protein in the test sample.
  • the present invention provides a method for treating hereditary angioedema, said method comprising screening patients with hereditary angioedema according to the above diagnosis method, and giving corresponding treatment.
  • the present invention provides a computer-readable storage medium storing computer-readable instructions, the computer-readable instructions, when executed by one or more processors, perform the aforementioned diagnosis of whether a subject suffers from genetic A method for angioedema or the aforementioned method for treating hereditary angioedema.
  • the stored computer readable instructions of the present invention may be stored permanently or temporarily in one or more memories of the computer.
  • the processor executes instructions stored in a computer-readable storage medium to process data.
  • processors and/or memory of the computers need not be physically located in the same geographic location in order to implement the methods of the present invention. That is, the various processors and memory used by the computer may be geographically disparate and connected so as to communicate in any suitable manner. Additionally, it should be understood that each processor and/or memory may be comprised of different physical components of the device.
  • Two or more different device components may be connected in any suitable manner, eg a network.
  • a memory may comprise two or more portions of memory in two or more physical locations.
  • Various techniques may be used to provide communication between various computers, processors and/or memories, as well as to allow the processors and/or memories of the present invention to communicate with any other entity; for example, to obtain further instructions or access and Use remote storage, eg.
  • the technology used to provide such communications may include a network, the Internet, an intranet, an extranet, a LAN, Ethernet or any client server system that provides the communications.
  • Such communication techniques may use any suitable protocol, such as TCP/IP, UDP or OSI.
  • various user interfaces may be utilized to allow a human user to interact with a computer or machine used to at least partially implement an implementation.
  • the user interface may be in the form of dialog screens.
  • User interfaces can also include mice, touch screens, keyboards, voice readers, voice recognizers, dialog screens, menu boxes, lists, check boxes, toggle switches, buttons or allow the user to receive information about the computer as it processes a set of instructions.
  • a user interface is any device that provides communication between a user and a computer.
  • the information a user provides to a computer through a user interface can be in the form of commands, data selections, or some other form of input.
  • the identified reagents are broadly understood and cannot be construed only as physically present chemical or biological reagents.
  • the term also includes mass spectrometric identification parameters. As an example, the entire full scan range of the mass spectrometer is divided into several windows, and all ions in each window are selected, fragmented, and detected in a high-speed and cyclic manner, so as to obtain all the ions in the sample without omission and difference. fragment information. This collection method is like carpet bombing, hitting all targets without omission; the mass-to-charge ratio information of the fragments is compared in the database to identify and quantify the protein.
  • protease digestion step may also be included.
  • proteases are used to fragment proteins in the urine sample.
  • each protein marker as described in the present invention is shown in Table 1 of the present invention, and its English abbreviation is also well known in the art. According to the above information, those skilled in the art can accurately know the information of each protein marker , and measure its expression level according to conventional methods.
  • Figure 1 shows the ROC curves for the diagnosis of HAE when malate dehydrogenase, kallikrein 1, plasma protease C1 inhibitor, epidermal growth factor precursor, kininogen 1, and aspartic protease A were used alone.
  • Figure 2 shows the ROC curve for the diagnosis of HAE when the combination of plasma protease C1 inhibitor, kallikrein 1 and thrombospondin disintegrin metallopeptidase 1 is used.
  • Example 1 Collection and preparation of urine samples for mass spectrometry detection
  • Cohort 1 Obtained morning urine samples from 28 patients with hereditary angioedema (including 19 females and 9 males; average age 42.6 years old) from Peking Union Medical College Hospital of Chinese Academy of Medical Sciences; compared with 29 healthy controls for urine protein expression For comparison (including 20 females and 9 males; mean age 43.0 years).
  • Cohort 2 Obtain morning urine samples from 24 patients with hereditary angioedema from Peking Union Medical College Hospital of Chinese Academy of Medical Sciences (including 15 females and 9 males; average age 42.8 years old); urine protein expression of 18 healthy controls For comparison (including 15 females and 3 males; average age 41.8 years old). This study was approved by the Ethics Committee, and the patients signed an informed consent.
  • Orbitrap Q Exactive HF high-resolution mass spectrometer was purchased from Thermo Fisher; high-performance liquid chromatography EASY-nLC 1200 was purchased from Thermo Fisher.
  • Use Orbitrap Q Exactive HF high-resolution mass spectrometer to perform tandem mass spectrometry analysis on the enzyme-cleaved peptides in cohort 1, use DIA quantitative proteomics technology to collect mass spectrometry data, and then use PRM targeting protein for the screened target protein in cohort 2 Group quantification techniques were validated.
  • DIA parameter setting modify the mass spectrometer variable window, the scan time is 60min, the collision energy: 28%; the primary scan resolution is set to 60000, the secondary resolution is 30000; the maximum precursor ion injection time is 80ms; the precursor ion scan range: 300 -1300m/z; product ion scanning range: start from 350m/z; set 40 scanning windows; the size of the window is determined according to the number of primary precursor ions, and the average distribution.
  • PRM parameter setting each full scan was followed by 25 targeted scans, and the scan time was 60 min. The primary full scan resolution is 60000, the scan range is 300-1250m/z, AGC 3e6, the maximum injection time is 80ms; the collision energy is 28%, and the secondary scan resolution is 30000.
  • the obtained DIA mass spectrum data was retrieved by Proteome Discoverer software (version 2.1), and the result file was imported into Spectronaut software to establish a spectral library.
  • the original mass spectrometry data of DIA official samples were imported into Spectronaut software, and matched with the data in the spectral library, the q value was ⁇ 1.0%, and the retention time was corrected by iRT reagent.
  • Proteins with more than 2 specific polypeptides were screened and identified for subsequent proteome quantification. Proteins with a fold change in protein expression of more than 1.5 times and a p value less than 0.05 were screened out as differential proteins.
  • the mixed sample was first used to collect 6 needles of DDA data, and the Proteome Discoverer software was used to search the database, and the search results were imported into the Skyline software to establish a spectral library. Import the targeted quantitative protein sequence Fasta file into the Skyline spectral library, and export the m/z and retention time tables of the peptides corresponding to the targeted protein. Import the collected PRM raw data back to Skyline, screen the quantitative fragment ions, and export the peak area of the secondary fragment ions of each peptide for PRM quantification.
  • differential proteins with a fold change of more than 1.5 times and independent sample t-test p ⁇ 0.05 were screened out, and 35 differential proteins could be used as diagnostic markers.
  • Table 1 shows that there are 35 diagnostic markers in total, 24 proteins are increased and 11 proteins are decreased compared with the control group.
  • the specific steps are as follows: 24 HAE patients (including 15 females and 9 males; average age 42.8 years old) and 18 healthy control subjects (including 15 females and 3 males; average age 41.8 years old) were newly collected in cohort 2. years old) urine samples, using the PRM targeted proteome quantitative technology to test the expression levels of some differential proteins in the samples, including plasma protease C1 inhibitor, profibrin 1, epidermal growth factor precursor, malate Hydrogenase, kininogen 1, kallikrein 1, thrombospondin proteointegrin metallopeptidase 1.
  • the combination of plasma protease C1 inhibitor, kallikrein 1 and thrombospondin disintegrin metallopeptidase 1 has a good diagnostic effect on the diagnosis of HAE, the area under the ROC curve AUC The value is 0.925 ( FIG. 1 ), which proves that the diagnostic markers provided by the present invention can have extremely high accuracy when used in combination.

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Abstract

一种尿液蛋白标志物在诊断遗传性血管水肿中的用途。同时，涉及鉴定试剂在制备用于诊断受试者中遗传性血管水肿的装置中的用途。另一方面，还涉及用于诊断遗传性血管水肿的试剂盒或芯片，其包含所述鉴定试剂。

Description

尿液蛋白标志物在诊断遗传性血管水肿中的用途 技术领域
本公开涉及临床医学，具体而言涉及人遗传性血管水肿相关的尿液蛋白标志物。更具体地，本公开涉及收集遗传性血管水肿患者尿液样品并质谱分析蛋白组学技术获得诊断遗传性血管水肿的尿液蛋白标志物，并且提供其使用方法和用途。
背景技术
疾病标志物是与病理生理过程相关的可测量的变化，可以用于疾病诊断、病程监测、疗效评估等。尿液是一种理想的疾病标志物临床样品来源，可以无创性地大量获得。而且尿液中缺乏内稳态机制的调控，可以富集人体排出的各种变化，这些变化就是潜在的标志物来源(参见Wu J&Gao Y.Physiological conditions can be reflected in human urine proteome and metabolome.Expert Review of Proteomics,2015；12(6):623-636)。
尿液蛋白质组(urinary proteomics)旨在建立一种无创的“液体活检”诊断方法，通过检测尿液中特异的疾病标志物来部分替代组织穿刺活检,己成为重要的研究方向，具有十分巨大的临床转化应用前景。DIA(Data independent acquisition)是一种最新的数据非依赖蛋白组定量技术，它将质谱整个全扫描范围分成若干窗口，实现无差异无遗漏地获取样本中所有离子的全部碎片信息。因为DIA技术可以无歧视地获得临床样品中所有肽段的信息而不会造成低丰度蛋白的丢失，尤其适合不同实验室条件下对蛋白质进行稳健而准确的定量(参见Navarro P等人A multicenter study benchmarks software tools for label-free proteome quantification.Nature Biotechnology,2016；34(11)：1130-1136)。PRM(Parallel reaction monitoring)是一种靶向蛋白质组学技术，基于Orbitrap高分辨质谱的离子监视技术，能够对目标蛋白目标肽段进行选择性的检测从而实现靶向定量。
遗传性血管性水肿(Hereditary angioedema，HAE)是一种罕见的、潜在威胁生命的疾病，其特征是皮下和粘膜下肿胀的反复发作。HAE是一种罕见疾病， 发病率为每5万人中有1例，通常不容易被发现。皮肤和腹部发作是HAE最常见的特征，很容易被误诊为水肿或急腹症导致患者接受无效的治疗或不必要的手术。此外，超过50％的HAE患者会发生喉头水肿，来不及抢救则会窒息死亡。因此，早期诊断标志物的发现对HAE的临床管理至关重要。
发明内容
鉴于本领域的上述需求，本发明提供了以下技术方案：
用途
根据本公开的一些实施方案，本发明提供了以下任意一种蛋白或其蛋白组合的鉴定试剂在制备诊断遗传性血管水肿的产品中的用途：
血浆蛋白酶C1抑制剂、己酮糖激酶、非那嗪类生物合成结构域蛋白、收缩蛋白α链、前纤维蛋白1、组织蛋白酶H前体、IST1同族体、苹果酸脱氢酶、Cochlin、含有崩解素和金属蛋白酶结构域的蛋白10、基底膜特异性硫酸肝素蛋白聚糖核心蛋白、激肽原1蛋白、含v-集域的t细胞活化抑制剂1、表皮生长因子前体、连接粘附分子A、N-乙酰化α-联酸二肽酶2、跨膜糖蛋白NMB、前胶原c内肽酶增强剂2、尿激酶纤溶酶原激活物表面受体、血小板反应蛋白解整合素金属肽酶1、组织蛋白酶Z、组织蛋白酶L1、内皮细胞选择性粘附分子、激肽释放酶1、补体成分C9、补体成分C1、钙结合蛋白、补体成分C7、水通道蛋白2、促卵泡激素β亚基、原钙粘附蛋白α-C2、血管内皮生长因子受体2、钙粘附相关家族成员5、免疫球蛋白重链恒定区γ4和天冬氨酸蛋白酶A。
具体地，本发明在具体实施例中以部分蛋白为例，证明了本发明所提供的诊断标志物的诊断效果，一方面，针对血浆蛋白酶C1抑制剂、前纤维蛋白1、表皮生长因子前体、苹果酸脱氢酶、激肽原1、激肽释放酶1、血小板反应蛋白解整合素金属肽酶1在本发明所述队列2中检测HAE患者和受试者中的表达量，实验结果证明以上蛋白在HAE患者和受试者之间的表达量倍数差异足以区分两组人群，达到诊断的目的；另一方面，针对苹果酸脱氢酶、激肽释放酶1、血浆蛋白酶C1抑制剂、表皮生长因子前体、激肽原1、天冬氨酸蛋白酶A计算AUC值，以上蛋白在单独诊断HAE时的AUC值均达到0.7之上，达到准确诊断的标准；以上示例性的验证提供了上述前纤维蛋白1、血小板反应蛋白解整合素金属 肽酶1、苹果酸脱氢酶、激肽释放酶1、血浆蛋白酶C1抑制剂、表皮生长因子前体、激肽原1、天冬氨酸蛋白酶A中任意一种作为生物标志物的技术方案，并证明了应用价值；进一步也代表了本发明所筛选得到的35个差异蛋白均可作为诊断标志物，均具有应用到临床的价值。
更优选地，本发明提供了以上35个蛋白中至少3个蛋白进行组合后，对于蛋白组合的鉴定试剂在制备诊断血管水肿的产品中的应用。
具体如实施例记载，在35个蛋白中任意挑选了血浆蛋白酶C1抑制剂、激肽释放酶1和血小板反应蛋白解整合素金属肽酶1进行组合，验证该蛋白组合的对HAE的诊断效果，AUC值为0.925，证明了其诊断效果极佳，可以准确区分健康者和患者。
本发明所述术语“ROC曲线(receiver operator characteristic curve)”，又称为受试者工作特征曲线，是反映灵敏度和特异度连续变量的综合指标，通常被用来直观地确定某一种诊断方法的最优诊断界值。ROC曲线下面的面积为AUC值(Area under Curve)，一般来说AUC的最小值为0.5，最大值为1，数值越大代表诊断越准确。
在本申请的一个实施方案中，所述鉴定试剂包括定量和/或定性检测蛋白的试剂；更具体地，检测蛋白表达量的试剂。
优选地，所述检测蛋白表达量的试剂包括但不限于以下任意一种或多种方法中使用的试剂：质谱法、免疫组织化学(Immunohistochemistry)染色方法、苏木精-伊红染色法(hematoxylin-eosin staining，简称HE染色法)、番红O-固绿染色、蛋白质印迹(Western Blot法)、酶联免疫吸附测定(ELISA)、放射性免疫测定(RIA)、夹心测定、免疫沉淀分析法、补体结合分析法、流式细胞荧光分边技术和蛋白质芯片法。如本发明具体实施例中所使用的是质谱法。
更优选地，所述试剂包括抗体、核酸探针或其他试剂，其对特定标志物具有特定亲和力(具有特异性)，足以区分特定标志物和可能存在于感兴趣样品中的其他标志物。
优选地，所述测蛋白表达量可以通过本领域已知的任何方式进行。
如本发明所述“鉴定试剂”和“检测试剂”是同种含义，可互换使用。如本发明所述“鉴定”和“检测”是同种含义，可互换使用。所述“鉴定”和“检测”都可以定量和/或定性地。
优选地，所述鉴定(检测)是针对来自受试者的样本进行的鉴定(检测)。
优选地，所述样本可以包括但不限于血液、尿液、粘液、羊水、血浆、腹水、脑脊髓液、痰、骨髓、滑液、房水、乳汁、汗水、粪便物、眼泪、腹膜液、淋巴液、阴道分泌物、囊胚腔液、脐带血和/或唾液。
优选地，所述样本是尿液。
更具体地，所述尿液是晨尿。所述术语“晨尿”的定义是本领域的公知常识，凡满足尿常规检查的尿液样本均落入本发明所述样本的范围。
本文中使用的术语“受试者”是指任何动物(例如，哺乳动物)，包括但不限于人、非人灵长类动物(例如，猿、猴、黑猩猩、大猩猩)、啮齿类动物等。
优选地，所述受试者是人。
优选地，所述产品包括诊断装置、诊断试剂盒、诊断芯片等。
产品
根据另一些实施方案，还提供了一种用于诊断遗传性血水肿的试剂盒或芯片，所述试剂盒或芯片包含以下任意一种或多种蛋白的鉴定试剂：
血浆蛋白酶C1抑制剂、己酮糖激酶、非那嗪类生物合成结构域蛋白、收缩蛋白α链、前纤维蛋白1、组织蛋白酶H前体、IST1同族体、苹果酸脱氢酶、Cochlin、含有崩解素和金属蛋白酶结构域的蛋白10、基底膜特异性硫酸肝素蛋白聚糖核心蛋白、激肽原1、含v-集域的t细胞活化抑制剂1、表皮生长因子前体、连接粘附分子A、N-乙酰化α-联酸二肽酶2、跨膜糖蛋白NMB、前胶原c内肽酶增强剂2、尿激酶纤溶酶原激活物表面受体、血小板反应蛋白解整合素金属肽酶1、组织蛋白酶Z、组织蛋白酶L1、内皮细胞选择性粘附分子、激肽释放酶1、补体成分C9、补体成分C1、钙结合蛋白、补体成分C7、水通道蛋白2、促卵泡激素β亚基、原钙粘附蛋白α-C2、血管内皮生长因子受体2、钙粘附相关 家族成员5、免疫球蛋白重链恒定区γ4、天冬氨酸蛋白酶A或其组合。
优选地，所述试剂盒或芯片包含以下任意一种或多种蛋白的鉴定试剂：血浆蛋白酶C1抑制剂、前纤维蛋白1、表皮生长因子前体、苹果酸脱氢酶、激肽原1、激肽释放酶1、血小板反应蛋白解整合素金属肽酶1、天冬氨酸蛋白酶A。
优选地，所述试剂盒或芯片包含以下蛋白组合的鉴定试剂，所述蛋白组合包括：血浆蛋白酶C1抑制剂、激肽释放酶1和血小板反应蛋白解整合素金属肽酶1。
更优选地，所述蛋白组合由血浆蛋白酶C1抑制剂、激肽释放酶1和血小板反应蛋白解整合素金属肽酶1组成。
术语“芯片”可指表面附着有特异性试剂(例如抗体、核酸探针)的固体基底。
优选地，所述芯片可以使用任意的固相载体，所述固相载体的材质的具体例子可举出：聚苯乙烯、聚氯乙烯、琼脂糖、脂质体、膜载体、高分子磁微粒等。
优选地，所述试剂盒中可以包括计算机可读介质。
在一些实施方案中，本文所述的试剂盒包含包装材料。如本文所用的，术语“包装材料”可指容纳试剂盒的组件的物理结构。该包装材料可维持试剂盒组件的无菌性，并可由常用于这类目的的材料(例如，纸、玻璃、塑料、金属材料等)制成。试剂盒还可包含缓冲剂、防腐剂或蛋白质/核酸稳定剂。
系统
另一方面，本发明提供了一组诊断遗传性血管水肿的系统，所述系统中包括根据以下任意一种或多种蛋白的检测结果计算受试者是否患有遗传性血管水肿的计算模块：
血浆蛋白酶C1抑制剂、己酮糖激酶、非那嗪类生物合成结构域蛋白、收缩蛋白α链、前纤维蛋白1、组织蛋白酶H前体、IST1同族体、苹果酸脱氢酶、Cochlin、含有崩解素和金属蛋白酶结构域的蛋白10、基底膜特异性硫酸肝素蛋白聚糖核心蛋白、激肽原1蛋白、含v-集域的t细胞活化抑制剂1、表皮生长因子前体、连接粘附分子A、N-乙酰化α-联酸二肽酶2、跨膜糖蛋白NMB、前胶 原c内肽酶增强剂2、尿激酶纤溶酶原激活物表面受体、血小板反应蛋白解整合素金属肽酶1、组织蛋白酶Z、组织蛋白酶L1、内皮细胞选择性粘附分子、激肽释放酶1、补体成分C9、补体成分C1、钙结合蛋白、补体成分C7、水通道蛋白2、促卵泡激素β亚基、原钙粘附蛋白α-C2、血管内皮生长因子受体2、钙粘附相关家族成员5、免疫球蛋白重链恒定区γ4和天冬氨酸蛋白酶A。
优选地，所述蛋白选自以下任意一种或多种：
苹果酸脱氢酶、激肽释放酶1、血浆蛋白酶C1抑制剂、表皮生长因子前体、激肽原1、天冬氨酸蛋白酶A、血前纤维蛋白1、血小板反应蛋白解整合素金属肽酶1。
更优选地，所述蛋白包括血浆蛋白酶C1抑制剂、激肽释放酶1和血小板反应蛋白解整合素金属肽酶1的组合。
更具体地，所述系统依次包括以下装置：
1)数据收集模块，用于收集对受试者样品进行检测的结果，具体地，对以上所述蛋白标志物的表达量进行检测；
2)计算模块，用于根据所述数据收集模块中收集的检测结果计算受试者是否患有遗传性血管水肿；
3)诊断结果输出模块，用于输出计算模块的诊断结果。
以上任意一种模块的使用都可以包括医院(医生)端口和受试者(患者)端口，方便诊断疾病的双方上传检测结果，查看诊断结果和沟通。
在一些实施方式中，数据收集模块能够以数字方式记录分析的结果。
在一些实施方式中，计算模块能够对分析产生的数据进行任何所需的数学处理。
方法
根据一些实施方案，提供了一种诊断受试者是否患有遗传性血管水肿的方法，包括步骤：
1)获得受试者或对照的尿液样本，
2)确定所述受试者的尿液样本中以下任意蛋白的表达水平：
血浆蛋白酶C1抑制剂、己酮糖激酶、非那嗪类生物合成结构域蛋白、收缩蛋白α链、前纤维蛋白1、组织蛋白酶H前体、IST1同族体、苹果酸脱氢酶、Cochlin、含有崩解素和金属蛋白酶结构域的蛋白10、基底膜特异性硫酸肝素蛋白聚糖核心蛋白、激肽原1、含v-集域的t细胞活化抑制剂1、表皮生长因子前体、连接粘附分子A、N-乙酰化α-联酸二肽酶2、跨膜糖蛋白NMB、前胶原c内肽酶增强剂2、尿激酶纤溶酶原激活物表面受体、血小板反应蛋白解整合素金属肽酶1、组织蛋白酶Z、组织蛋白酶L1、内皮细胞选择性粘附分子、激肽释放酶-1、补体成分C9、补体成分C1、钙结合蛋白、补体成分C7、水通道蛋白2、促卵泡激素β亚基、原钙粘附蛋白α-C2、血管内皮生长因子受体2、钙粘附相关家族成员5、免疫球蛋白重链恒定区γ4、天冬氨酸蛋白酶A或其组合；
3)将步骤2)检测得到的结果和标准值(阈值)比较，根据比较结果给出诊断结果，也即判断受试者是否是HAE患者。
优选地，所述标准值可以是获自对照的样本中所述蛋白表达水平。不同检测方法或检测仪器针对同一样本进行检测的得到的数值可能不同，但是对于本领域技术人员，获得标准值的方法是常规的。
更具体地，本发明所述对照(健康对照组)是指未患有遗传性血管水肿的个体。对照(健康对照组)不一定是临床意义上健康的个体，优选是健康的个体。
在具体的实施方案中，和标准值相比较，选自以下的蛋白的表达水平提高，指示所述受试者患有遗传性血管水肿或者患遗传性血管水肿的风险增加：收缩蛋白α链、前纤维蛋白1、组织蛋白酶H前体、IST1同族体、Cochlin、含有崩解素和金属蛋白酶结构域的蛋白10、基底膜特异性硫酸肝素蛋白聚糖核心蛋白、连接粘附分子A、N-乙酰化α-联酸二肽酶2、跨膜糖蛋白NMB、前胶原c内肽酶增强剂2、尿激酶纤溶酶原激活物表面受体、血小板反应蛋白解整合素金属肽酶1、组织蛋白酶Z、组织蛋白酶L1、内皮细胞选择性粘附分子、补体成分C9、补体成分C1、补体成分C7、水通道蛋白2、促卵泡激素β亚基、原钙粘附蛋白α-C2、血管内皮生长因子受体2、钙粘附相关家族成员5或其组合。
在具体的实施方案中，和标准值相比较，选自以下的蛋白的表达水平降低，指示所述受试者患有遗传性血管水肿或者患遗传性血管水肿的风险增加：血浆蛋白酶C1抑制剂、己酮糖激酶、非那嗪类生物合成结构域蛋白、苹果酸脱氢酶、激肽原1、免疫球蛋白重链恒定区γ4、含v-集域的t细胞活化抑制剂1、表皮生长因子前体、激肽释放酶1、天冬氨酸蛋白酶A、钙结合蛋白或其组合。反之，则风险降低。
更具体地，本发明在涉及遗传性血管水肿诊断的实施方案中，对照是指未患有遗传性血管水肿的个体。对照不一定是临床意义上健康的个体，优选是健康的个体。
本申请中所用的术语“鉴定试剂”是指能够定量和/或定性检测目标蛋白的试剂。具体地，但非限制性地，本发明的鉴定试剂能够特异性地识别受试样本中目标蛋白的存在或不存在；或者能够检测受试样本中目标蛋白的水平。
另一方面，本发明提供了一种治疗遗传性血管水肿的方法，所述方法包括根据上述诊断方法筛选出遗传性血管水肿的患者，并给于相应的治疗。
计算机可读存储介质
另一方面，本发明提供了一种存储计算机可读指令的计算机可读存储介质，所述计算机可读指令在被一个或多个处理器执行时，执行前述诊断受试者是否患有遗传性血管水肿的方法或者前述治疗遗传性血管水肿的方法。
本发明所述存储计算机可读指令可以永久或临时地存储在计算机的一个或多个存储器中。所述处理器执行存储在计算机可读存储介质中的指令以处理数据。
可以理解的是，为了实施本发明的方法，不需要将计算机的处理器和/或存储器物理地放置在相同的地理位置。也就是，计算机所使用的各个处理器和存储器可以位于地理上不同的位置并连接，从而以任何合适的方式进行通信。此外，应当理解的是，各处理器和/或存储器可以由设备的不同物理部件组成。
两个或更多个不同的设备部件可以任何适当的方式(如网络)连接。此外，存储器可以在两个或更多个物理位置中包括存储器的两个或更多个部分。
可以使用各种技术来提供各种计算机、处理器和/或存储器之间的通信，以 及允许本发明的处理器和/或存储器与任何其他实体进行通信；例如，从而获取进一步的指令或访问和使用远程存储器，例如。用于提供这类通信的技术可能包括网络，互联网，内联网，外联网，LAN，以太网或提供通信的任何客户端服务器系统。这类通信技术可以使用任何合适的协议，例如，TCP/IP、UDP或OSI。
在一些实施方式中，可以利用各种用户界面来允许人用户与用于至少部分地实施实施方式的计算机或机器进行交互。用户界面可以是对话屏幕的形式。用户界面还可以包括鼠标，触摸屏，键盘，语音读取器，语音识别器，对话屏幕，菜单框，列表，复选框，拨动开关，按钮或允许用户随计算机处理一组指令而接收有关计算机运行的信息和/或向计算机提供信息的其他装置。因此，用户界面是提供用户与计算机之间通信的任何装置。例如，用户通过用户界面提供给计算机的信息可以是命令，数据选择或一些其他输入形式。
通用概念
在一个实施方案中，当采用质谱鉴定试剂时，鉴定试剂作广泛理解，不能仅解释为实体存在的化学、生物试剂。该术语也包含质谱鉴定参数。作为一个示例，将质谱整个全扫描范围分为若干个窗口，高速、循环地对每个窗口中的所有离子进行选择、碎裂、检测，从而无遗漏、无差异地获得样本中所有离子的全部碎片信息。这种采集方法像地毯式轰炸，无遗漏地打击全部靶标；将碎片的质荷比信息在数据库中进行比对，以鉴定蛋白身份和定量。应当理解，尽管具体示例中采用了特定的鉴定方式，但是本申请技术效果的实现并不依赖于特定的鉴定方式(例如质谱操作步骤、质谱仪型号、质谱方法中设定的参数、质谱鉴定中所识别的特定肽段序列、色谱柱型号；抗体供应商、抗体所靶向的特定表位、抗体分型、免疫学操作步骤和参数)，这是因为本申请技术方案的核心在于发现了前述蛋白在尿液中的存在量与疾病之间的关系，因此任何能够确定蛋白含量的手段都是可用的。
当采用质谱方法确定蛋白及其表达水平时，在获得尿液样本的步骤之后，还可以包括蛋白酶的消化步骤。在具体的实施方案中，用蛋白酶使尿液样本中的蛋白片段化。
如本发明所述的各个蛋白标志物的英文名称以及Uniprot ID如本发明表1所 示，其英文缩写也是本领域公知的，根据以上信息本领域技术人员可以准确得知各个蛋白标志物的信息，并按照常规方法测量其表达量。
附图说明
图1显示苹果酸脱氢酶、激肽释放酶1、血浆蛋白酶C1抑制剂、表皮生长因子前体、激肽原1、天冬氨酸蛋白酶A单独使用时，对HAE诊断的ROC曲线。
图2显示在血浆蛋白酶C1抑制剂、激肽释放酶1和血小板反应蛋白解整合素金属肽酶1组合使用时，对HAE诊断的ROC曲线。
具体实施方式
下面结合实施例对本发明做进一步的说明，以下所述，仅是对本发明的较佳实施例而已，并非对本发明做其他形式的限制，任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更为同等变化的等效实施例。凡是未脱离本发明方案内容，依据本发明的技术实质对以下实施例所做的任何简单修改或等同变化，均落在本发明的保护范围内。
实施例1、用于质谱检测的尿液样本的收集以及制备
队列1：获取来自中国医学科学院北京协和医院的28例遗传性血管水肿患者的晨尿样品(其中女性19例，男性9例；平均年龄42.6岁)；与29例健康对照组的尿液蛋白表达进行比较(其中女性20例，男性9例；平均年龄43.0岁)。队列2：获取来自中国医学科学院北京协和医院的24例遗传性血管水肿患者的晨尿样品(其中女性15例，男性9例；平均年龄42.8岁)；与18例健康对照组的尿液蛋白表达进行比较(其中女性15例，男性3例；平均年龄41.8岁)。本研究得到了伦理委员会的批准，且患者签署了知情同意书。
对照为未患有遗传性血管水肿的健康个体。
用于质谱检测的尿液样本的制备步骤如下：
1)收集遗传性血管水肿患者和对照的尿液；
2)提取患者尿液蛋白：患者尿液5000g离心30min取上清，将尿液与丙酮按体积比1:4进行混合，-20℃过夜沉淀尿液蛋白。4℃以12000g离心30min，弃上清，沉淀自然风干后加裂解液复溶；
3)蛋白组样品制备：尿液蛋白使用FASP法进行膜上酶切，参见Wisniewski JR等人Universal sample preparation method for proteome analysis.Nature methods 2009；6:359-62。
实施例2.尿液蛋白质谱分析
1.材料与试剂
1)仪器：
Orbitrap Q Exactive HF高分辨质谱仪购自Thermo Fisher公司；高效液相色谱仪EASY-nLC 1200购自Thermo Fisher公司。
2)试剂：
色谱级乙腈、甲酸和甲醇为Waters公司生产；碘乙酰铵(IAA)、碳酸氢铵、二硫苏糖醇(DTT)从Sigma公司购买；质谱级胰酶从Promega公司购买。
2.质谱鉴定
对队列1中酶切的肽段用Orbitrap Q Exactive HF高分辨质谱仪进行串联质谱分析,使用DIA定量蛋白质组学技术采集质谱数据，然后再队列2中对筛选出的目标蛋白使用PRM靶向蛋白组定量技术进行验证。DIA参数设置：修改质谱可变窗口，扫描时间为60min，碰撞能量：28％；一级扫描分辨率设置为60000，二级分辨率为30000；母离子最大注入时间80ms；母离子扫描范围：300-1300m/z；子离子扫描范围：从350m/z开始；设置40个扫描窗口；窗口大小根据一级母离子数目确定，平均分布。PRM参数设置：每个全扫描后接25个靶向扫描，扫描时间60min。一级全扫描分辨率为60000，扫描范围为300-1250m/z，AGC 3e6，最大注射时间为80ms；碰撞能量为28％，二级扫描分辨率为30000。
3.蛋白组定量分析
所得DIA质谱数据经过Proteome Discoverer软件(版本2.1)检索，结果文件导入Spectronaut软件，建立谱图库。DIA正式样品质谱原始数据导入Spectronaut软件，与谱图库中数据进行匹配，q值<1.0％，通过iRT试剂校正保留时间。筛选鉴定到2个以上特异性多肽的蛋白用于后续蛋白组定量。筛选出蛋白表达量变化倍数在1.5倍以上，p值小于0.05的蛋白作为差异蛋白。PRM靶向定量中首先用混合样采集6针DDA数据并使用Proteome Discoverer软件进行搜库，将搜库结果导入Skyline软件建立谱图库。将靶向定量的蛋白序列Fasta 文件导入Skyline谱图库，导出靶向蛋白对应肽段的m/z及保留时间表格。将采集到的PRM原始数据导回Skyline，筛选定量碎片离子，导出每一条肽段二级碎片离子峰面积用于PRM定量。
4.质谱分析结果
队列1中收集28例确诊的遗传性血管水肿患者和29例健康人尿液样品，在至少鉴定到2个特异性多肽及蛋白鉴定FDR<1％的水平，共鉴定到蛋白数1897个。
与对照的样品比对，筛选出变化倍数在1.5倍以上且独立样本t检验p<0.05的差异蛋白，有35个差异蛋白可作为诊断标志物。
表1显示，诊断标志物共35个，与对照组相比，24个蛋白升高，11个蛋白降低。
表1.遗传性血管水肿患者和对照差异尿液蛋白标志物
Figure PCTCN2022135877-appb-000001
Figure PCTCN2022135877-appb-000002
实施例3.尿液蛋白质标记物在HAE诊断中的应用
为进一步证实所筛选的差异蛋白(或称为诊断标志物、蛋白标志物)在HAE诊断中的灵敏度与特异性，针对表1中的蛋白，我们采用PRM靶向蛋白组技术在新的临床队列中对候选标志物进行了验证，并绘制了上述标志物在区分HAE患者和受试者中的ROC曲线。
具体步骤如下：在队列2中新收集24例HAE患者(其中女性15例，男性9例；平均年龄42.8岁)和18例健康对照受试者(其中女性15例，男性3例；平均年龄41.8岁)的尿液样本，采用盲法，用PRM靶向蛋白组定量技术测试样本中部分差异蛋白的表达水平，包括血浆蛋白酶C1抑制剂、前纤维蛋白1、表皮生长因子前体、苹果酸脱氢酶、激肽原1、激肽释放酶1、血小板反应蛋白解整合素金属肽酶1。结果表明与健康对照相比，患者尿液中血浆蛋白酶C1抑制剂变化倍数为0.32、前纤维蛋白1变化倍数为1.48、表皮生长因子前体变化倍数为0.50、苹果酸脱氢酶变化倍数为0.49、激肽释放酶1变化倍数为0.50、血小板反应蛋白解整合素金属肽酶1变化倍数为2.10、天冬氨酸蛋白酶A为0.46，这些蛋白(单独或者组合使用)的表达水平，均能够区分HAE患者和受试者。
同时，示例性的选择苹果酸脱氢酶、激肽释放酶1、血浆蛋白酶C1抑制剂、表皮生长因子前体、激肽原1、天冬氨酸蛋白酶A进行ROC曲线验证，得到的AUC值都在0.7以上，代表了本发明所筛选出的差异蛋白都具备诊断疾病的功能。
表2.尿液蛋白标志物对遗传性血管水肿的诊断性能
序号 蛋白名称 英文缩写 AUC值 灵敏度 特异性
1 苹果酸脱氢酶 MDH1 0.79 83.3 72.2
2 激肽释放酶1 KLK1 0.85 70.8 88.9
3 血浆蛋白酶C1抑制剂 C1NH 0.91 83.3 94.4
4 表皮生长因子前体 EGF 0.74 95.8 50.0
5 激肽原1 KNG1 0.73 66.7 77.8
6 天冬氨酸蛋白酶A NAPSA 0.73 70.8 83.3
示例性地，如图2所示，血浆蛋白酶C1抑制剂、激肽释放酶1和血小板反应蛋白解整合素金属肽酶1组合使用时对HAE的诊断具有良好的诊断效果，ROC曲线下面积AUC值为0.925(图1)，证明了本发明所提供的诊断标志物进一步组合使用时可以具备极高的准确性。

Claims (10)

  1. 鉴定以下任意一种或两种蛋白的试剂在制备诊断遗传性血管水肿的产品中的用途：
    血浆蛋白酶C1抑制剂、己酮糖激酶、非那嗪类生物合成结构域蛋白、收缩蛋白α链、前纤维蛋白1、组织蛋白酶H前体、IST1同族体、苹果酸脱氢酶、Cochlin、含有崩解素和金属蛋白酶结构域的蛋白10、基底膜特异性硫酸肝素蛋白聚糖核心蛋白、激肽原1蛋白、含v-集域的t细胞活化抑制剂1、表皮生长因子前体、连接粘附分子A、N-乙酰化α-联酸二肽酶2、跨膜糖蛋白NMB、前胶原c内肽酶增强剂2、尿激酶纤溶酶原激活物表面受体、血小板反应蛋白解整合素金属肽酶1、组织蛋白酶Z、组织蛋白酶L1、内皮细胞选择性粘附分子、激肽释放酶1、补体成分C9、补体成分C1、钙结合蛋白、补体成分C7、水通道蛋白2、促卵泡激素β亚基、原钙粘附蛋白α-C2、血管内皮生长因子受体2、钙粘附相关家族成员5、免疫球蛋白重链恒定区γ4和天冬氨酸蛋白酶A；
    优选地，所述蛋白选自：血浆蛋白酶C1抑制剂、前纤维蛋白1、表皮生长因子前体、苹果酸脱氢酶、激肽原1、激肽释放酶1、血小板反应蛋白解整合素金属肽酶1、天冬氨酸蛋白酶A。
  2. 鉴定蛋白组合的试剂在制备诊断遗传性血管水肿的产品中的用途，所述蛋白组合包括以下至少3种：
    血浆蛋白酶C1抑制剂、己酮糖激酶、非那嗪类生物合成结构域蛋白、收缩蛋白α链、前纤维蛋白1、组织蛋白酶H前体、IST1同族体、苹果酸脱氢酶、Cochlin、含有崩解素和金属蛋白酶结构域的蛋白10、基底膜特异性硫酸肝素蛋白聚糖核心蛋白、激肽原1蛋白、含v-集域的t细胞活化抑制剂1、表皮生长因子前体、连接粘附分子A、N-乙酰化α-联酸二肽酶2、跨膜糖蛋白NMB、前胶原c内肽酶增强剂2、尿激酶纤溶酶原激活物表面受体、血小板反应蛋白解整合素金属肽酶1、组织蛋白酶Z、组织蛋白酶L1、内皮细胞选择性粘附分子、激肽释放酶1、补体成分C9、补体成分C1、钙结合蛋白、补体成分C7、水通道蛋白2、促卵泡激素β亚基、原钙粘附蛋白α-C2、血管内皮生长因子受体2、钙粘附相关家族成员5、免疫球蛋白重链恒定区γ4和天冬氨酸蛋白酶A；
    优选地，所述蛋白组合是血浆蛋白酶C1抑制剂、激肽释放酶1和血小板反应蛋白解整合素金属肽酶1的组合。
  3. 如权利要求1或2所述的用途，所述鉴定试剂包括定量和/或定性检测蛋白的试剂；
    优选地，所述鉴定试剂是检测蛋白表达量的试剂；
    优选地，所述检测蛋白表达量的试剂包括以下任意一种或多种方法中使用的试剂：质谱法、免疫组织化学染色方法、苏木精-伊红染色法、番红O-固绿染色、蛋白质印迹、酶联免疫吸附测定、放射性免疫测定、夹心测定、免疫沉淀分析法、补体结合分析法、流式细胞荧光分边技术和蛋白质芯片法；
    优选地，所述鉴定试剂是使用质谱法检测蛋白表达量中使用的试剂。
  4. 如权利要求1或2所述的用途，所述鉴定试剂包括抗体、核酸探针。
  5. 如权利要求1或2所述的用途，所述鉴定是针对来自受试者的样本进行的鉴定；
    优选地，所述样本包括血液、尿液、粘液、羊水、血浆、腹水、脑脊髓液、痰、骨髓、滑液、房水、乳汁、汗水、粪便物、眼泪、腹膜液、淋巴液、阴道分泌物、囊胚腔液、脐带血和/或唾液；
    优选地，所述样本是尿液；
    更具体地，所述尿液是晨尿；
    优选地，所述受试者包括人、非人灵长类动物、啮齿类动物；
    优选地，所述受试者是人。
  6. 如权利要求1或2所述的用途，所述产品包括诊断装置、诊断试剂盒、诊断芯片。
  7. 一种用于诊断遗传性血水肿的试剂盒或芯片，所述试剂盒或芯片包含以下任意一种或多种蛋白的鉴定试剂：
    血浆蛋白酶C1抑制剂、己酮糖激酶、非那嗪类生物合成结构域蛋白、收缩 蛋白α链、前纤维蛋白1、组织蛋白酶H前体、IST1同族体、苹果酸脱氢酶、Cochlin、含有崩解素和金属蛋白酶结构域的蛋白10、基底膜特异性硫酸肝素蛋白聚糖核心蛋白、激肽原1、含v-集域的t细胞活化抑制剂1、表皮生长因子前体、连接粘附分子A、N-乙酰化α-联酸二肽酶2、跨膜糖蛋白NMB、前胶原c内肽酶增强剂2、尿激酶纤溶酶原激活物表面受体、血小板反应蛋白解整合素金属肽酶1、组织蛋白酶Z、组织蛋白酶L1、内皮细胞选择性粘附分子、激肽释放酶1、补体成分C9、补体成分C1、钙结合蛋白、补体成分C7、水通道蛋白2、促卵泡激素β亚基、原钙粘附蛋白α-C2、血管内皮生长因子受体2、钙粘附相关家族成员5、免疫球蛋白重链恒定区γ4、天冬氨酸蛋白酶A或其组合；
    优选地，所述试剂盒或芯片包含以下任意一种或多种蛋白的鉴定试剂：血浆蛋白酶C1抑制剂、前纤维蛋白1、表皮生长因子前体、苹果酸脱氢酶、激肽原1、激肽释放酶1、血小板反应蛋白解整合素金属肽酶1、天冬氨酸蛋白酶A；
    优选地，所述试剂盒或芯片包含以下蛋白组合的鉴定试剂，所述蛋白组合包括以下蛋白中的任意3种或更多种：血浆蛋白酶C1抑制剂、己酮糖激酶、非那嗪类生物合成结构域蛋白、收缩蛋白α链、前纤维蛋白1、组织蛋白酶H前体、IST1同族体、苹果酸脱氢酶、Cochlin、含有崩解素和金属蛋白酶结构域的蛋白10、基底膜特异性硫酸肝素蛋白聚糖核心蛋白、激肽原1、含v-集域的t细胞活化抑制剂1、表皮生长因子前体、连接粘附分子A、N-乙酰化α-联酸二肽酶2、跨膜糖蛋白NMB、前胶原c内肽酶增强剂2、尿激酶纤溶酶原激活物表面受体、血小板反应蛋白解整合素金属肽酶1、组织蛋白酶Z、组织蛋白酶L1、内皮细胞选择性粘附分子、激肽释放酶1、补体成分C9、补体成分C1、钙结合蛋白、补体成分C7、水通道蛋白2、促卵泡激素β亚基、原钙粘附蛋白α-C2、血管内皮生长因子受体2、钙粘附相关家族成员5、免疫球蛋白重链恒定区γ4、天冬氨酸蛋白酶A或其组合；
    更优选地，所述蛋白组合由血浆蛋白酶C1抑制剂、激肽释放酶1和血小板反应蛋白解整合素金属肽酶1组成；
    优选地，所述芯片的固体基质包括聚苯乙烯、聚氯乙烯、琼脂糖、脂质体、膜载体、高分子磁微粒。
  8. 一组诊断遗传性血管水肿的系统，所述系统中包括根据以下任意一种或多种蛋白的检测结果计算受试者是否患有遗传性血管水肿的计算模块：
    血浆蛋白酶C1抑制剂、己酮糖激酶、非那嗪类生物合成结构域蛋白、收缩蛋白α链、前纤维蛋白1、组织蛋白酶H前体、IST1同族体、苹果酸脱氢酶、Cochlin、含有崩解素和金属蛋白酶结构域的蛋白10、基底膜特异性硫酸肝素蛋白聚糖核心蛋白、激肽原1蛋白、含v-集域的t细胞活化抑制剂1、表皮生长因子前体、连接粘附分子A、N-乙酰化α-联酸二肽酶2、跨膜糖蛋白NMB、前胶原c内肽酶增强剂2、尿激酶纤溶酶原激活物表面受体、血小板反应蛋白解整合素金属肽酶1、组织蛋白酶Z、组织蛋白酶L1、内皮细胞选择性粘附分子、激肽释放酶1、补体成分C9、补体成分C1、钙结合蛋白、补体成分C7、水通道蛋白2、促卵泡激素β亚基、原钙粘附蛋白α-C2、血管内皮生长因子受体2、钙粘附相关家族成员5、免疫球蛋白重链恒定区γ4和天冬氨酸蛋白酶A；
    优选地，所述蛋白选自以下任意一种或多种：
    苹果酸脱氢酶、激肽释放酶1、血浆蛋白酶C1抑制剂、表皮生长因子前体、激肽原1、天冬氨酸蛋白酶A、血前纤维蛋白1、血小板反应蛋白解整合素金属肽酶1；
    更优选地，所述蛋白包括血浆蛋白酶C1抑制剂、激肽释放酶1和血小板反应蛋白解整合素金属肽酶1的组合。
  9. 如权利要求8所述系统，所述系统依次包括以下装置：
    1)数据收集模块，用于收集对受试者样品进行检测的结果，具体地，对以上所述蛋白标志物的表达量进行检测；
    2)计算模块，用于根据所述数据收集模块中收集的检测结果计算受试者是否患有遗传性血管水肿；
    3)诊断结果输出模块，用于输出计算模块的诊断结果。
  10. 一种存储计算机可读指令的计算机可读存储介质，所述计算机可读指令在被一个或多个处理器执行时，执行前述诊断受试者是否患有遗传性血管水肿的方法，所述方法包括以下步骤：
    1)获得受试者或对照的尿液样本，
    2)确定所述受试者的尿液样本中以下任意一种或多种蛋白的表达水平：
    血浆蛋白酶C1抑制剂、己酮糖激酶、非那嗪类生物合成结构域蛋白、收缩蛋白α链、前纤维蛋白1、组织蛋白酶H前体、IST1同族体、苹果酸脱氢酶、Cochlin、含有崩解素和金属蛋白酶结构域的蛋白10、基底膜特异性硫酸肝素蛋白聚糖核心蛋白、激肽原1、含v-集域的t细胞活化抑制剂1、表皮生长因子前体、连接粘附分子A、N-乙酰化α-联酸二肽酶2、跨膜糖蛋白NMB、前胶原c内肽酶增强剂2、尿激酶纤溶酶原激活物表面受体、血小板反应蛋白解整合素金属肽酶1、组织蛋白酶Z、组织蛋白酶L1、内皮细胞选择性粘附分子、激肽释放酶-1、补体成分C9、补体成分C1、钙结合蛋白、补体成分C7、水通道蛋白2、促卵泡激素β亚基、原钙粘附蛋白α-C2、血管内皮生长因子受体2、钙粘附相关家族成员5、免疫球蛋白重链恒定区γ4、天冬氨酸蛋白酶A或其组合；
    3)将步骤2)检测得到的结果和标准值比较，根据比较结果给出诊断结果，也即判断受试者是否是遗传性血管水肿患者；
    优选地，所述标准值是获自对照的样本中所述蛋白表达水平；
    优选地，所述对照是指未患有遗传性血管水肿的个体。
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