技术领域technical field
本发明属于蛋白质组学领域，涉及一种儿童活动性结核病蛋白特征谱及其构建方法。The invention belongs to the field of proteomics, and relates to a protein signature spectrum of children with active tuberculosis and a construction method thereof.
背景技术Background technique
结核病是由结核分枝杆菌(Mycobacterium tuberculosis,MTB)感染引起的当今世界上最普遍的传染病之一，是当前导致发展中国家儿童死亡的重要疾病之一。据估计，结核病患儿已占全球结核病人总数的10％-15％，且该比率在结核病高发国家和地区如非洲更高。我国作为结核病高发国家之一，0-14岁儿童结核感染率为9.0％，活动性结核病患病率为91.8/10万。随着近年来耐药结核杆菌的流行以及HIV病毒感染的增多，儿童结核病出现明显回升趋势。因此，加强儿童结核病的早期诊断已经成为儿童结核病控制工作亟待解决的问题。Tuberculosis is one of the most common infectious diseases in the world today caused by Mycobacterium tuberculosis (MTB) infection, and it is one of the important diseases that cause the death of children in developing countries. It is estimated that children with tuberculosis have accounted for 10%-15% of the total number of tuberculosis patients in the world, and this rate is higher in countries and regions with high incidence of tuberculosis such as Africa. As one of the countries with high incidence of tuberculosis in my country, the tuberculosis infection rate of children aged 0-14 is 9.0%, and the prevalence rate of active tuberculosis is 91.8/100,000. With the prevalence of drug-resistant Mycobacterium tuberculosis and the increase of HIV virus infection in recent years, children's tuberculosis has shown a clear upward trend. Therefore, strengthening the early diagnosis of tuberculosis in children has become an urgent problem to be solved in the control of tuberculosis in children.
儿童结核病长期面临诊断困难的问题。首先，儿童结核病通常细菌载量较低，咳嗽反射和气管纤毛运动能力差，常常合并肺外结核而导致痰涂片阳性率很低。结核病患儿发病早期临床症状尚不明显时，实验室不能及时提供准确的病原学诊断结果，常常延误诊断，不能准确的选择有效药物，易引起病情恶化，失去抢救的最佳时机。免疫力低下、使用过免疫抑制剂以及患有严重结核病的患儿，由于机体免疫应答减弱，常规免疫学诊断方法常常出现假阴性，对早期诊断和预防治疗造成不良影响。Diagnosis of tuberculosis in children has long been difficult. First, children with tuberculosis usually have a low bacterial load, poor cough reflex and tracheal ciliary motility, and are often associated with extrapulmonary tuberculosis, resulting in a low sputum smear-positive rate. When the clinical symptoms of tuberculosis children are not obvious in the early stage, the laboratory cannot provide accurate etiological diagnosis results in time, often delays diagnosis, cannot accurately select effective drugs, easily leads to deterioration of the disease, and loses the best time for rescue. For children with low immunity, those who have used immunosuppressants, and children with severe tuberculosis, due to the weakened immune response of the body, routine immunological diagnosis methods often have false negatives, which has a negative impact on early diagnosis and preventive treatment.
结核菌素皮试(PPD试验)是目前诊断结核病的重要辅助手段，但由于PPD是多种抗原的混合物，所含抗原为致病性分枝杆菌、环境分枝杆菌及卡介苗菌株所共有，其结果受到卡介苗接种及其他分枝杆菌感染的影响，特异度较差，常可导致假阳性结果。当患儿出现免疫功能低下时可出现假阴性结果。PPD试验诊断的特异度和敏感度还受其阳性判断标准的影响，且目前国内PPD试验在儿童潜伏结核感染诊断中的标准尚不统一，从而给临床医师的诊断带来一定的困难。Tuberculin skin test (PPD test) is an important auxiliary means for diagnosing tuberculosis at present. However, since PPD is a mixture of multiple antigens, the antigens contained are common to pathogenic mycobacteria, environmental mycobacteria and BCG strains. The results are affected by BCG vaccination and other mycobacterial infections, and the specificity is poor, which can often lead to false positive results. False-negative results can occur when children are immunocompromised. The specificity and sensitivity of PPD test diagnosis are also affected by its positive judgment criteria, and the current domestic PPD test standards in the diagnosis of latent tuberculosis infection in children are not uniform, which brings certain difficulties to clinicians.
γ干扰素释放试验作为一种新型的免疫学诊断方法已在欧美等国广泛应用于结核病、结核性脑膜炎、结核病疗效评估等的常规诊断。由于该试验结果不受卡介苗接种的影响，因此尤其适用于卡介苗接种率较高的人群。但目前关于该实验在儿童结核病诊断中的准确性研究结论尚不一致。有研究认为γ干扰素释放试验不受小年龄的影响，在儿童中具有较好的敏感度。而有的研究则认为该试验即使在细菌培养阳性的儿童中，其敏感度也很低，不适用于在儿童中作为一种常规的诊断方法，因此，探索新的适于儿童的诊断方法，达到早期诊断早期治疗的目的势在必行。Interferon-γ release test, as a new type of immunological diagnostic method, has been widely used in the routine diagnosis of tuberculosis, tuberculous meningitis, tuberculosis efficacy evaluation, etc. in Europe and the United States. Since the results of this test are not affected by BCG vaccination, it is especially suitable for populations with high BCG vaccination rates. However, the current research conclusions about the accuracy of this test in the diagnosis of tuberculosis in children are not consistent. Studies have shown that interferon-γ release test is not affected by young age and has better sensitivity in children. However, some studies believe that even in children with positive bacterial culture, the test has low sensitivity and is not suitable for use as a routine diagnostic method in children. Therefore, to explore new diagnostic methods suitable for children, It is imperative to achieve the goal of early diagnosis and early treatment.
蛋白质组学是在1994年由Williams和Wilkins提出的，通过寻找特异性蛋白质，为研究疾病机制提供线索。近年来随着人类蛋白质组学计划的全面启动，蛋白质组学的研究也产生了巨大进展，蛋白质组学研究中应用的定量方法主要有两种，一种是基于传统双向凝胶电泳及染色基础上的定量，另外一种是基于质谱检测技术的定量，包括标记定量技术(iTRAQ)和非标记定量技术(Label free quantification)两种。非标记定量技术不需要昂贵的同位素标签做内部标准，实验耗费低；对样本的操作也最少，从而使其最接近原始状态；并且不受样品条件的限制，克服了标记定量技术在对多个样本进行定量方面的缺陷，因此它在定量蛋白质组学研究中受到了众多科学工作者的推崇，得到了越来越广泛的应用。Proteomics was proposed by Williams and Wilkins in 1994 to provide clues for the study of disease mechanisms by looking for specific proteins. In recent years, with the full launch of the Human Proteomics Project, proteomics research has also made great progress. There are two main quantitative methods used in proteomics research. One is based on traditional two-dimensional gel electrophoresis and staining. The other is quantification based on mass spectrometry detection technology, including two kinds of label quantification technology (iTRAQ) and non-label quantification technology (Label free quantification). Unlabeled quantitative technology does not require expensive isotope labels as internal standards, and the experimental cost is low; the operation of the sample is the least, so that it is closest to the original state; and it is not limited by the sample conditions, overcoming the label quantitative technology. Due to the defects in the quantification of samples, it has been praised by many scientists in quantitative proteomics research and has been more and more widely used.
近年来，蛋白质组学技术用于疾病诊断的研究已日趋广泛，该技术通过对机体病理改变引起的蛋白质产物的变化进行分析，运用化学计量学方法对疾病组和正常组进行辨识，寻找疾病相关的生物标志物，并用于临床诊断。该诊断技术克服了传统医学诊断模式的诊断准确率低、诊断指标单一、对专家诊疗水平依赖性强、治疗模式缺乏个体化等缺陷，充分利用蛋白质组学产物进行系统分析，对各个状态都作出更准确和客观的评价，大大提高诊断的科学化、定量化，并避免人为因素的误诊。In recent years, the research of proteomics technology for disease diagnosis has become more and more extensive. This technology analyzes the changes of protein products caused by pathological changes in the body, and uses chemometric methods to identify disease groups and normal groups to find disease-related factors. biomarkers for clinical diagnosis. This diagnostic technology overcomes the shortcomings of the traditional medical diagnostic model, such as low diagnostic accuracy, single diagnostic index, strong dependence on expert diagnosis and treatment, and lack of individualized treatment models. More accurate and objective evaluation greatly improves the scientific and quantitative diagnosis, and avoids misdiagnosis due to human factors.
发明内容Contents of the invention
本发明的一个目的是提供一种非诊断目的的儿童活动性结核病相关蛋白特征谱的构建方法。An object of the present invention is to provide a non-diagnostic method for constructing a protein profile associated with active tuberculosis in children.
本发明所提供的非诊断目的的儿童活动性结核病相关蛋白特征谱的构建方法，具体可包括如下步骤：分别从儿童活动性结核病患者组和健康对照儿童组的离体血浆中提取总蛋白，用胰蛋白酶进行酶解，对酶解所得多肽进行液相色谱-电喷雾电离串联质谱检测和相对定量分析，根据分析结果获得儿童活动性结核病相关蛋白特征谱；The method for constructing the protein profile associated with active tuberculosis in children for non-diagnostic purposes provided by the present invention may specifically include the following steps: extracting total protein from the isolated plasma of the active tuberculosis patient group in children and the healthy control children group respectively, and using Trypsin was used for enzymatic hydrolysis, and the polypeptide obtained from the enzymatic hydrolysis was subjected to liquid chromatography-electrospray ionization tandem mass spectrometry detection and relative quantitative analysis, and the characteristic spectrum of children's active tuberculosis-related proteins was obtained according to the analysis results;
所述儿童活动性结核病相关蛋白特征谱中，所述儿童活动性结核病患者组和所述健康对照儿童组中表达差异的蛋白质包括表达上调的蛋白质和表达下调的蛋白质；所述表达上调的蛋白质有实施例表2中的前242种蛋白(即如上所述PTCD3至所述TRPM5)；所述表达下调的蛋白质有实施例表2中的后137种蛋白(即如上所述KIAA1211至所述ZNF225)。In the children's active tuberculosis-associated protein profile, the proteins with differential expression in the children's active tuberculosis patient group and the healthy control children group include proteins with up-regulated expression and proteins with down-regulated expression; the proteins with up-regulated expression include: The first 242 proteins in Table 2 of the Examples (i.e. PTCD3 to the TRPM5 as described above); the down-regulated proteins have the last 137 proteins in Table 2 of the Examples (i.e. KIAA1211 to the ZNF225 as described above) .
其中，在提取总蛋白之后，还可包括去除高丰度蛋白、进行蛋白浓度测定的步骤。Wherein, after extracting the total protein, the steps of removing high-abundance protein and measuring protein concentration may also be included.
在所述方法中，所述相对定量分析过程中，利用Trans-Proteomic Pipeline软件提取一级质谱定量信息，利用ProfileAnalysis2.0软件进行定量，并利用ProteinScape2.1软件进行数据检索和整合，筛选出比值＞2的蛋白为表达上调的蛋白，比值<0.5的蛋白为表达下调的蛋白，所述比值为所述儿童活动性结核病患者组的蛋白表达量与所述健康对照儿童组的蛋白表达量的比值。In the method, in the relative quantitative analysis process, the Trans-Proteomic Pipeline software is used to extract the quantitative information of the primary mass spectrum, and the ProfileAnalysis2.0 software is used for quantification, and the ProteinScape2.1 software is used for data retrieval and integration, and the ratio is screened out. Proteins >2 are proteins whose expression is up-regulated, and proteins with a ratio <0.5 are proteins whose expression is down-regulated, and the ratio is the ratio of the protein expression level of the active tuberculosis patient group in children to the protein expression level of the healthy control children group .
其中，所述利用ProteinScape2.1软件进行数据检索和整合具体为：以Peptide FDR≤0.05对数据进行筛选过滤，导入Maxquant软件进行Label free分析。主要参数如下：Wherein, the use of ProteinScape2.1 software for data retrieval and integration specifically includes: screening and filtering the data with Peptide FDR≤0.05, and importing Maxquant software for Label free analysis. The main parameters are as follows:
Main search ppm：6Missed cleavage：2Main search ppm: 6 Missed cleavage: 2
MS/MS tolerance ppm：20De-Isotopic：TRUEMS/MS tolerance ppm: 20 De-Isotopic: TRUE
enzyme：Trypsin database：ipi.human.3.87.fastaenzyme: Trypsin database: ipi.human.3.87.fasta
Fixed modification：Carbamidomethyl(C)Fixed modification: Carbamidomethyl(C)
Variable modification：Oxidation(M)，Acetyl(Protein N-term)Variable modification: Oxidation (M), Acetyl (Protein N-term)
Decoy database pattern：reverseDecoy database pattern: reverse
Lable free quantification(LFQ)：TRUELable free quantification (LFQ): TRUE
LFQ min ratio count：2LFQ min ratio count: 2
Match between runs：2minMatch between runs: 2min
Peptide FDR：0.05Peptide FDR: 0.05
Protein FDR：0.05Protein FDR: 0.05
在所述方法中，所述液相色谱-电喷雾电离串联质谱检测过程中，质谱条件如下：分析时长为120min，检测方式为正离子，母离子扫描范围为300-1800m/z，多肽和多肽的碎片的质量电荷比按照每次全扫描后采集10个碎片图谱的方式采集，MS1在M/Z200时分辨率为70000，MS2在M/Z 200时分辨率为17500。In the method, during the liquid chromatography-electrospray ionization tandem mass spectrometry detection process, the mass spectrometry conditions are as follows: the analysis time is 120min, the detection mode is positive ion, the precursor ion scanning range is 300-1800m/z, and the polypeptide and polypeptide The mass-to-charge ratio of the fragments is collected in the manner of collecting 10 fragment maps after each full scan. MS1 has a resolution of 70,000 at M/Z 200, and MS2 has a resolution of 17,500 at M/Z 200.
在所述方法中，所述液相色谱-电喷雾电离串联质谱检测过程中，色谱为毛细管高效液相色谱，色谱条件如下：色谱柱的填料为反向碳18，内径为150μm，柱长100㎜(如Thermo EASY column SC200150μm*100mm(RP-C18))，流动相由A液和B液组成，所述A液为含有2％(体积分数)乙腈和0.1％(体积分数)甲酸的水溶液，B液为含有84％(体积分数)乙腈和0.1％(体积分数)甲酸的水溶液。采用所述A液和所述B液按照如下进行梯度洗脱：0min-100min，所述流动相中所述B液的体积百分含量由0％匀速增加至45％；100min-108min，所述流动相中所述B液的体积百分含量由45％匀速增加至100％；108min-120min，所述流动相中所述B液的体积百分含量维持在100％；流速为400nl/min。In the method, during the liquid chromatography-electrospray ionization tandem mass spectrometry detection process, the chromatography is capillary high performance liquid chromatography, and the chromatographic conditions are as follows: the packing of the chromatographic column is reverse carbon 18, the inner diameter is 150 μm, and the column length is 100 μm. ㎜ (such as Thermo EASY column SC200150μm*100mm (RP-C18)), the mobile phase is composed of liquid A and liquid B, the liquid A is an aqueous solution containing 2% (volume fraction) acetonitrile and 0.1% (volume fraction) formic acid, Liquid B is an aqueous solution containing 84% (volume fraction) of acetonitrile and 0.1% (volume fraction) of formic acid. Use the A liquid and the B liquid to carry out gradient elution as follows: 0min-100min, the volume percentage of the B liquid in the mobile phase increases from 0% to 45% at a constant speed; 100min-108min, the The volume percentage of the liquid B in the mobile phase is increased from 45% to 100% at a constant speed; 108min-120min, the volume percentage of the liquid B in the mobile phase is maintained at 100%; the flow rate is 400nl/min.
本发明的还一个目的是提供一种非诊断目的的儿童活动性结核病相关蛋白特征谱。Another object of the present invention is to provide a non-diagnostic active tuberculosis-associated protein profile in children.
本发明所提供的非诊断目的的儿童活动性结核病相关蛋白特征谱中差异表达的蛋白质为表达上调的蛋白质和表达下调的蛋白质；所述表达上调的蛋白质有实施例表2中的前242种蛋白(即如上所述PTCD3至所述TRPM5)；所述表达下调的蛋白质有实施例表2中的后137种蛋白(即如上所述KIAA1211至所述ZNF225)。The differentially expressed proteins in the children's active tuberculosis-associated protein profile for non-diagnostic purposes provided by the present invention are proteins with up-regulated expression and proteins with down-regulated expression; the proteins with up-regulated expression include the first 242 proteins in Table 2 of the Examples (i.e. PTCD3 to the TRPM5 as described above); the down-regulated proteins include the latter 137 proteins in Table 2 of the Examples (i.e. KIAA1211 to the ZNF225 as described above).
其中，所述表达上调的蛋白质为与健康对照儿童组相比，在儿童活动性结核病患者组中表达上调的蛋白质；所述表达下调的蛋白质为与健康对照儿童组相比，在儿童活动性结核病患者组中表达下调的蛋白质。Wherein, the protein whose expression is up-regulated is compared with the healthy control children group, the protein whose expression is up-regulated in the children's active tuberculosis patient group; Downregulated proteins expressed in the patient group.
本发明的再一个目的是提供一种用于检测如下379种蛋白表达量的成套产品在制备筛查儿童活动性结核病患者(即鉴定或辅助鉴定待测儿童是否为儿童活动性结核病患者)的系统中的应用：Another object of the present invention is to provide a system for detecting the following 379 kinds of protein expression levels in the preparation and screening of children with active tuberculosis (i.e. identifying or assisting in identifying whether the child to be tested is a child with active tuberculosis) Apps in:
PTCD3、ADAMTSL2、HTT、SNX4、VPS37B、RANBP17、UBE2O、VWDE、MCM4、ZC3H11A、WIZ、CCDC81、DSP、MMP2、GEMIN5、SPIRE2、PC、ADCY3、LIMS2、WWC1、RP1、ADAM18、AARSD1、PCSK6、ST3GAL4、ZNF584、ZMYM1、XB、LRRK2、SOCS5、RASAL3、SMC3、ATP10A、KDM1A、TTLL10、HERC6、PCCA、C1QTNF1、NUP88、SORBS1、NKD2、PIWIL4、AKAP2、SYNM、NPM1、HP、NOS1、NLRP14、SKIV2L、ICE1、ASAP2、ABAT、ORM1、CASK、SPECC1、NIPBL、DOCK11、CACTIN、ZNF526、SYNRG、FANCD2、PLXNC1、SPATA31D1、FBXO46、PSG3、BMPR2、DKFZp434A2017、KIF4A、COX10、ARHGDIG、RASAL2、MYH7、RFC1、DOCK5、ATP11A、ANKRD27、ZNF521、ZNF281、DLL1、TBL3、MYH14、EML4、CLTCL1、PRRC2C、TSTD2、C1orf94、NSD1、GPR137B、FRMPD2、ANKRD33B、SIN3A、TAS1R1、KIF3C、CYP26B1、HERC2、DCAF12L2、CCDC15、SP140、ABCC12、WDR91、SEC31A、GTPBP3、TLR4、CHMP4B、HECW1、CP、C16orf71、PPP2R5D、COL13A1、ZBTB10、ZNF311、PINLYP、AIM2、KCNMB3、HAL、MTHFD1、ALPP、PPP1R9A、IFT81、DDX60、CCDC71、RNF152、UBR4、ASH1L、CORO1C、ACAN、NARG1L、ZNF831、ABCA2、HCFC1、REXO1、TRIP12、PCNT、CAD、AK3、GUSB、SERPINA1、MTUS2、ZNF606、ARAP3、DZIP1、POLR3C、NLRP13、IFNA14、DNAH7、GPAM、SAMD9L、LMO3、USP11、RASGRF2、TATDN2、DCAF17、POFUT2、LAMA2、AXIN2、AP2A2、KIAA1033、HPS6、SORT1、TNPO3、NHLRC1、ADAM15、SLC25A41、CLEC14A、SERPINA3、DRG2、SYCP1、MRPS18B、ATP12A、SERPING1、MACF1、RSAD1、VPS13D、TTC3、RIMS1、GRK6、SP110、SOWAHC、HDGFRP3、POLR2A、C10orf76、INTS2、PROZ、MYO7B、TRMT1、ARHGEF1、LCE3A、SLC38A10、A2M、PAMR1、MCM5、ANK2、KLK8、PRG4、ZMYM3、PPP2R1B、TSSK4、INPP5B、SKIL、ZNF543、SLCO3A1、ATR、MID2、ZFP57、TICRR、XRCC4、SPTBN2、AHCTF1、C2CD3、ZC3H12B、LOX、PLOD3、ACACB、SDK2、C9orf85、CASP8、PLAA、KAT8、UTRN、MROH8、DMTN、SAA1、NRXN2、LEPREL4、SMC5、SPATA31C1、SAMHD1、PIAS3、L1CAM、JADE1、PKD1L1、MDC1、FAT2、PSD4、TAP1、CEP290、TTC28、ABCA7、LONP2、RGAG1、TAB2、TRPM5、KIAA1211、CGN、DDAH1、HHIP、CLIC3、PPP6C、PNN、PRPF8、KIT、GLMN、CCDC89、DNAH14、COL6A2、GFRA1、PRPF6、KIAA0319、ATP13A1、PHKG2、COL7A1、TRIP6、TRIM14、DNAJC1、PHLDB3、ICA1、PRPF4B、TFAP4、LACTB、CEP63、MYO19、ARHGAP19、ANKRD17、EYS、PRDM9、TIGD5、ECHDC3、RHBDF1、GLG1、TMEM141、TNS1、GDPD3、LYST、HRH3、HBD、MZF1、FLYWCH1、OVOL2、HSPA5、ZNF30、ZNF492、FRAT1、AHSG、PIK3R6、KANSL1、MUC12、ESCO1、MIEF1、CACNA1E、DPYSL3、CHD7、ADCY9、EIF5、DDX31、RIT1、TECTA、RAB3GAP2、APOC3、RNF144B、SF3B4、ZNF709、EVC2、RB1CC1、MYH13、TIAM2、SEMA4A、COBLL1、UHRF1BP1L、SPTAN1、BCOR、SMG1、DPYD、RNASE4、PCCB、WDR62、BRD4、RBM26、DOPEY2、ZMYM5、RAB11FIP1、INADL、ZNF19、WNK1、WDR46、DAOA、ADIPOQ、F11、GOLGA4、ALK、TANGO6、BCL9L、COL4A1、CLEC2B、PDLIM1、CD22、EFNB2、COL8A2、NXF2、RNF180、ADAMTSL3、TTI2、LRP4、CDC37、CHD6、OCRL、ANO7、GBA2、PCF11、AGO3、CPT2、KMT2A、RASA3、ZNF431、CHIC1、ISCA2、BRINP1、MTO1、CASP2、MKI67、CNTRL、RPP40、DOK3、KRT82、FNDC1、WFDC5、KRT12、PTGDR2、BEND2和ZNF225。所述379种蛋白在NCBI上的GI编号如实施例中表2所示。PTCD3, ADAMTSL2, HTT, SNX4, VPS37B, RANBP17, UBE2O, VWDE, MCM4, ZC3H11A, WIZ, CCDC81, DSP, MMP2, GEMIN5, SPIRE2, PC, ADCY3, LIMS2, WWC1, RP1, ADAM18, AARSD1, PCSK6, ST3GAL4, ZNF584, ZMYM1, XB, LRRK2, SOCS5, RASAL3, SMC3, ATP10A, KDM1A, TTLL10, HERC6, PCCA, C1QTNF1, NUP88, SORBS1, NKD2, PIWIL4, AKAP2, SYNM, NPM1, HP, NOS1, NLRP14, SKIV2L, ICE1, ASAP2, ABAT, ORM1, CASK, SPECC1, NIPBL, DOCK11, CACTIN, ZNF526, SYNRG, FANCD2, PLXNC1, SPATA31D1, FBXO46, PSG3, BMPR2, DKFZp434A2017, KIF4A, COX10, ARHGDIG, RASAL2, MYH7, RFC1, DO1ACK5, ATP ANKRD27, ZNF521, ZNF281, DLL1, TBL3, MYH14, EML4, CLTCL1, PRRC2C, TSTD2, C1orf94, NSD1, GPR137B, FRMPD2, ANKRD33B, SIN3A, TAS1R1, KIF3C, CYP26B1, HERC2, DCAF12L2, CCDC15, SP1240WAB9, DRCC SEC31A, GTPBP3, TLR4, CHMP4B, HECW1, CP, C16orf71, PPP2R5D, COL13A1, ZBTB10, ZNF311, PINLYP, AIM2, KCNMB3, HAL, MTHFD1, ALPP, PPP1R9A, IFT81, DDX60, CCDC71, RNF152, UBR4, ASH1L, CORO ACAN, NARG1L, ZNF831, ABCA2, HCFC1, REXO1, TRIP12, PCNT, CAD, AK3, GUSB, SERPINA1, MTUS2, ZNF606, ARAP3, DZIP1, POLR3C, NLRP13, IFNA14, DNAH7, GPAM, SAMD9L, LMO3, USP11, RASGRF2, TATDN2, DCAF17, POFUT2, LAMA2, AXIN2, AP2A2, KIAA1033, HPS6, S ORT1, TNPO3, NHLRC1, ADAM15, SLC25A41, CLEC14A, SERPINA3, DRG2, SYCP1, MRPS18B, ATP12A, SERPING1, MACF1, RSAD1, VPS13D, TTC3, RIMS1, GRK6, SP110, SOWAHC, HDGFRP3, POLR2A, C10orf76, INTS2, PROZ MYO7B, TRMT1, ARHGEF1, LCE3A, SLC38A10, A2M, PAMR1, MCM5, ANK2, KLK8, PRG4, ZMYM3, PPP2R1B, TSSK4, INPP5B, SKIL, ZNF543, SLCO3A1, ATR, MID2, ZFP57, TICRR, XRCC4, SPTBN2, AHCTF1, C2CD3, ZC3H12B, LOX, PLOD3, ACACB, SDK2, C9orf85, CASP8, PLAA, KAT8, UTRN, MROH8, DMTN, SAA1, NRXN2, LEPREL4, SMC5, SPATA31C1, SAMHD1, PIAS3, L1CAM, JADE1, PKD1L1, MDC1, FAT2, PSD4, TAP1, CEP290, TTC28, ABCA7, LONP2, RGAG1, TAB2, TRPM5, KIAA1211, CGN, DDAH1, HHIP, CLIC3, PPP6C, PNN, PRPF8, KIT, GLMN, CCDC89, DNAH14, COL6A2, GFRA1, PRPF6, KIAA0319, ATP13A1, PHKG2, COL7A1, TRIP6, TRIM14, DNAJC1, PHLDB3, ICA1, PRPF4B, TFAP4, LACTB, CEP63, MYO19, ARHGAP19, ANKRD17, EYS, PRDM9, TIGD5, ECHDC3, RHBDF1, GLG1, TMEM141, TNS1, GDPD3, LYST, HRH3, HBD, MZF1, FLYWCH1, OVOL2, HSPA5, ZNF30, ZNF492, FRAT1, AHSG, PIK3R6, KANSL1, MUC12, ESCO1, MIEF1, CACNA1E, DPYSL3, CHD7, ADCY9, EIF5, DDX31, RIT1, TECTA, RAB3GAP2, APOC3, RNF144B, SF3B4, ZNF709, EVC2, RB1CC1, MYH13, TIAM2, SEMA4A, COBLL1, UHRF1B P1L, SPTAN1, BCOR, SMG1, DPYD, RNASE4, PCCB, WDR62, BRD4, RBM26, DOPEY2, ZMYM5, RAB11FIP1, INADL, ZNF19, WNK1, WDR46, DAOA, ADIPOQ, F11, GOLGA4, ALK, TANGO6, BCL9L, COL4A1, CLEC2B, PDLIM1, CD22, EFNB2, COL8A2, NXF2, RNF180, ADAMTSL3, TTI2, LRP4, CDC37, CHD6, OCRL, ANO7, GBA2, PCF11, AGO3, CPT2, KMT2A, RASA3, ZNF431, CHIC1, ISCA2, BRINP1, MTO1, CASP2, MKI67, CNTRL, RPP40, DOK3, KRT82, FNDC1, WFDC5, KRT12, PTGDR2, BEND2, and ZNF225. The GI codes of the 379 proteins on NCBI are shown in Table 2 in the Examples.
本发明的另一个目的是提供一种筛查儿童活动性结核病患者的系统。Another object of the present invention is to provide a system for screening children with active tuberculosis.
本发明所提供的筛查儿童活动性结核病患者的系统，包括用于检测所述379种蛋白表达量的成套产品。The system for screening children with active tuberculosis provided by the present invention includes a complete set of products for detecting the expression levels of the 379 proteins.
在所述应用和所述系统中，所述成套产品包括用于从离体血浆中进行总蛋白抽提所需的试剂和/或仪器，以及胰蛋白酶和液相色谱-电喷雾电离串联质谱仪。根据需要，所述成套产品还可包括去除高丰度蛋白和/或进行蛋白浓度测定所需的试剂和/或仪器。其中，所述高丰度蛋白主要为白蛋白和IgG。In the application and the system, the complete set includes reagents and/or instruments required for total protein extraction from isolated plasma, as well as trypsin and liquid chromatography-electrospray ionization tandem mass spectrometer . According to needs, the complete set of products may also include reagents and/or instruments required for removing high-abundance proteins and/or performing protein concentration determination. Wherein, the high-abundance proteins are mainly albumin and IgG.
在所述应用和所述系统中，所述筛查儿童活动性结核病患者的系统包括蛋白质表达量数据处理装置，所述蛋白质表达量数据处理装置具有如下功能：分别比较取自待测儿童和健康对照儿童的离体血浆中的所述379种蛋白的表达量，根据比较结果确定待测儿童是否为儿童活动性结核病患者。In the application and the system, the system for screening children with active tuberculosis includes a protein expression data processing device, and the protein expression data processing device has the following functions: respectively compare the Control the expression levels of the 379 proteins in the isolated plasma of the child, and determine whether the child to be tested is a child with active tuberculosis according to the comparison result.
所述蛋白质表达量数据处理装置中内设模块；所述模块用于分别比较取自待测儿童和健康对照儿童的离体血浆中的所述379种蛋白的表达量；A module is built in the protein expression data processing device; the module is used to compare the expression of the 379 proteins in the isolated plasma of the child to be tested and the healthy control child respectively;
所述根据比较结果确定待测儿童是否为儿童活动性结核病患者，具体为：若所述待测儿童的离体血浆样本中前242种蛋白(即如上所述PTCD3至所述TRPM5)与所述健康对照儿童相比上调，且所述待测儿童的离体血浆样本中后137种蛋白(即如上所述KIAA1211至所述ZNF225)与所述健康对照儿童相比下调，则所述待测儿童为或候选为儿童活动性结核病患者；反之，则所述待测儿童不为或候选不为儿童活动性结核病患者。其中，“上调”体现为比值＞2，“下调”体现为比值<0.5，所述比值为某一蛋白在所述儿童活动性结核病患者中的表达量与在所述健康对照儿童组中的表达量的比值。According to the comparison result, it is determined whether the child to be tested is a child with active tuberculosis, specifically: if the first 242 proteins (ie, PTCD3 to the TRPM5 as described above) in the isolated plasma sample of the child to be tested are consistent with the Up-regulated compared with healthy control children, and the latter 137 proteins (i.e. KIAA1211 to ZNF225 as described above) are down-regulated compared with the healthy control children in the isolated plasma samples of the tested children, then the tested children is or is a candidate for active tuberculosis in children; otherwise, the child to be tested is not or is not a candidate for active tuberculosis in children. Wherein, "up-regulation" is reflected in the ratio > 2, and "down-regulation" is reflected in the ratio <0.5, and the ratio is the expression amount of a certain protein in the children with active tuberculosis patients and the expression in the healthy control children group Quantity ratio.
在本发明中，所述儿童的年龄为3个月-16岁。In the present invention, the age of the child is from 3 months to 16 years old.
本发明检测儿童活动性结核病蛋白质组学产物，可用于建立血浆特征代谢产物模型以及应用于在儿童活动性结核病早期检测和筛查，本发明的模型，可用于儿童活动性结核病的早期检测和筛查。本发明与其它儿童活动性结核病的检测方法比较，具有以下优点：The invention detects proteomic products of active tuberculosis in children, which can be used to establish a model of plasma characteristic metabolites and be applied to early detection and screening of active tuberculosis in children. The model of the present invention can be used for early detection and screening of active tuberculosis in children check. Compared with other detection methods for active tuberculosis in children, the present invention has the following advantages:
第一，本发明采用非标记定量蛋白质组学方法对儿童活动性结核病患者与正常人血浆的检测，并采用了传统统计学与现代生物信息学方法相结合的方法进行数据处理，从而得到儿童活动性结核病患者和健康儿童血浆蛋白质质谱检测模型，并且所发现的一系列蛋白质为寻找新的更理想的标志物提供了基础和资源。First, the present invention uses the non-labeled quantitative proteomics method to detect the blood plasma of children with active tuberculosis patients and normal people, and uses a method combining traditional statistics and modern bioinformatics methods for data processing, thereby obtaining children's activity The plasma protein mass spectrometry detection model of tuberculosis patients and healthy children, and a series of proteins discovered provide the basis and resources for finding new and more ideal markers.
第二，与以往的血浆学检测方法比较具有较高的敏感性和特异性，并能用于筛选儿童活动性结核病的药物中。Second, it has higher sensitivity and specificity compared with previous plasma detection methods, and can be used to screen drugs for active tuberculosis in children.
第三，本发明模型的构建方法设计合理可行，为提供儿童活动性结核病的临床治愈率提供了新的筛查方法，同时也为探索疾病发生发展的机制提供了新的思路。Thirdly, the design of the construction method of the model of the present invention is reasonable and feasible, which provides a new screening method for the clinical cure rate of active tuberculosis in children, and also provides a new idea for exploring the mechanism of the occurrence and development of the disease.
第四，本发明检测方法结果准确，可对儿童活动性结核病做诊断，尽早对病人治疗。Fourth, the result of the detection method of the present invention is accurate, which can diagnose active tuberculosis in children and treat patients as early as possible.
附图说明Description of drawings
图1为蛋白质含量测定标准曲线图。Figure 1 is a standard curve diagram for the determination of protein content.
具体实施方式Detailed ways
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。The experimental methods used in the following examples are conventional methods unless otherwise specified.
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.
实施例1、儿童活动性结核病蛋白特征谱的建立Embodiment 1, the establishment of active tuberculosis protein profile in children
1、样本和仪器1. Samples and instruments
39例选自年龄为3个月-16岁的儿童血浆样本，其中19例为儿童活动性结核病患者血浆，另外20例为健康儿童血浆，儿童活动性结核病患者均有实验室及临床诊断报告确定。所有的血浆样本均在清晨空腹下抽取，分离血浆后储存在-80低温冰箱中。39 cases were selected from children's plasma samples aged 3 months to 16 years, of which 19 cases were plasma samples from children with active tuberculosis, and the other 20 cases were plasma samples from healthy children. All children with active tuberculosis were confirmed by laboratory and clinical diagnosis reports . All plasma samples were drawn in the morning on an empty stomach, and the plasma was separated and stored in a -80 low-temperature refrigerator.
实验用仪器是液相色谱-电喷雾电离串联质谱(Q-Exactive Thermo Finnigan)，电泳仪(Bio-rad,美国)，MultiSkans酶标仪(Thermo,美国)，白蛋白/IgG去除试剂盒购自德国Merck公司。The experimental instruments were liquid chromatography-electrospray ionization tandem mass spectrometry (Q-Exactive Thermo Finnigan), electrophoresis instrument (Bio-rad, USA), MultiSkans microplate reader (Thermo, USA), albumin/IgG removal kit was purchased from German Merck company.
2、蛋白质提取2. Protein extraction
采用默克公司的ProteoExtract Albumin/IgG Removal Kit去除样本中的高丰度蛋白。去高速离心后的血浆样本50μL，在冰上按使用说明书操作处理，处理后的样本体积1800μL。The ProteoExtract Albumin/IgG Removal Kit from Merck was used to remove high-abundance proteins in the samples. Remove 50 μL of the plasma sample after high-speed centrifugation, and process it on ice according to the instruction manual. The volume of the processed sample is 1800 μL.
由于样本经过去除高丰度蛋白处理后，浓度被稀释了300倍，因此需要对样本进行浓缩处理。1800μL处理后的样本加入8mL-20℃预冷丙酮，沉淀过夜。6000g离心后，弃去上清，晾干丙酮后，用100μL溶解液(7M尿素和2M硫脲)溶解沉淀。溶解后的样本4℃下40000g离心30min，待用。Since the concentration of the sample was diluted 300 times after removing the high-abundance protein, the sample needs to be concentrated. Add 8mL-20°C pre-cooled acetone to 1800μL of the treated sample, and precipitate overnight. After centrifugation at 6000g, the supernatant was discarded, and after the acetone was dried, the precipitate was dissolved with 100 μL of dissolving solution (7M urea and 2M thiourea). The dissolved samples were centrifuged at 40,000 g for 30 min at 4°C and set aside.
3、蛋白质定量3. Protein quantification
A.方法A. method
(1)开分光光度计预热20min，选择“光度测量”，调节λ＝595nm。(1) Turn on the spectrophotometer to warm up for 20 minutes, select "photometric measurement", and adjust λ=595nm.
(2)用CK(2μL裂解缓冲液+18μL ddH2O+1ml Bradford)进行调零三次。(要点：第一次按“Zero”即可，观察第二、三次的OD值，如果都很小且相差不大即可。调零完毕后，显示“-0.301Abs”。)(2) Zero adjustment was performed three times with CK (2 μL lysis buffer + 18 μL ddH2 O + 1 ml Bradford). (Key points: just press "Zero" for the first time, and observe the OD values of the second and third times, if they are all small and not much different. After the zero adjustment is completed, "-0.301Abs" will be displayed.)
(3)配置表1中的各牛血清标准蛋白溶液并测定OD595值，绘制标准曲线。(3) Prepare each bovine serum standard protein solution in Table 1 and measure the OD595 value, and draw a standard curve.
(4)测B2(2μL BSA+18μL ddH2O+1ml Bradford)、B5(5μL BSA+15μL ddH2O+1ml Bradford)、B8(8μL BSA+12μL ddH2O+1ml Bradford)各三管，用于确定校正系数，校正蛋白浓度。其中，标准曲线计算方法为：在excel表格中，一列蛋白质浓度，一列对应OD值，插入图标，选择折线图，选项中选择显示R值和公式。(4) Measure three tubes each of B2 (2μL BSA+18μL ddH2 O+1ml Bradford), B5 (5μL BSA+15μL ddH2 O+1ml Bradford), B8 (8μL BSA+12μL ddH2 O+1ml Bradford), and use To determine the correction factor, correct the protein concentration. Among them, the calculation method of the standard curve is: in the excel table, one column of protein concentration, one column corresponding to OD value, insert the icon, select the line chart, and select the option to display R value and formula.
注：BSA为牛血清标准蛋白；要点：所有管加好BSA和ddH2O后，加一管Bradford，测一管；动作要领“三摇一拍”；读值为放入比色皿1s后的第一个值；如有数值异常可舍弃读值或多做一次重复。Note: BSA is bovine serum standard protein; key points: after adding BSA and ddH2 O to all tubes, add one tube of Bradford and measure one tube; the action essentials are "three shakes and one beat"; the reading value is 1 second after putting into the cuvette The first value; if the value is abnormal, the reading value can be discarded or repeated one more time.
(5)测样品蛋白OD595值。操作同上。将OD595值代入标准曲线方程，得到蛋白浓度测定值，进而计算实际蛋白浓度，实际蛋白浓度＝蛋白浓度测定值×校正系数。(5) Measure the OD595 value of the sample protein. The operation is the same as above. Substitute the OD595 value into the standard curve equation to obtain the measured value of protein concentration, and then calculate the actual protein concentration, actual protein concentration=measured value of protein concentration×correction coefficient.
表1牛血清标准蛋白溶液的设置及OD595值测定结果Table 1 The settings of bovine serum standard protein solution and the results of OD595 value determination
注：OD1-OD5为五个重复。Note: OD1-OD5 are five replicates.
B.结果B. Results
根据表1测定结果，得到标准曲线方程为y＝8.7949x-0.1539，R2＝0.9985。其中，x为OD595，y为蛋白浓度。具体见图1。According to the measurement results in Table 1, the equation of the standard curve was obtained as y=8.7949x-0.1539, R2 =0.9985. Among them, x is OD595, y is protein concentration. See Figure 1 for details.
根据上述步骤(4)计算得到校正系数为0.9985。The correction coefficient calculated according to the above step (4) is 0.9985.
进一步，测定步骤2所得的儿童活动性结核病患者血浆处理后样品中的蛋白浓度为7.8μg/μl，健康儿童血浆处理后样品中的蛋白浓度为7.1μg/μl。Further, the protein concentration in the processed plasma sample of children with active tuberculosis obtained in step 2 was 7.8 μg/μl, and the protein concentration in the processed plasma sample of healthy children was 7.1 μg/μl.
4、胰蛋白酶解样本4. Trypsinized samples
(1)蛋白质定量后取200μg蛋白溶液置于离心管中，用8M尿素将体系定至125μl；(1) After protein quantification, take 200 μg of protein solution and place it in a centrifuge tube, and set the system to 125 μl with 8M urea;
(2)加入现配的5μl 1M DTT溶液至体系中，混匀后，37℃孵育1h；(2) Add 5 μl of 1M DTT solution to the system, mix well, and incubate at 37°C for 1 hour;
(3)加入现配的20μl 1M IAA溶液至体系中，混匀后，避光，室温(25℃)反应1h；(3) Add 20 μl of 1M IAA solution to the system, mix well, keep away from light, and react at room temperature (25°C) for 1 hour;
(4)吸取所有样品加入到10kD超滤管中，12000rpm(不超过14000g)离心20min，弃掉收集管底部溶液；(4) Draw all the samples into a 10kD ultrafiltration tube, centrifuge at 12000rpm (not exceeding 14000g) for 20min, and discard the solution at the bottom of the collection tube;
(5)加入100μl尿素(8M)至超滤管中，12000rpm离心10min，重复2次。(5) Add 100 μl of urea (8M) to the ultrafiltration tube, centrifuge at 12000 rpm for 10 min, and repeat twice.
(6)在超滤管中加入胰蛋白酶(100U/μg)2-4μg(与蛋白质量比1:50-100)，体积50μl，37℃反应过夜20h；次日，12000rpm离心20min，酶解消化后的肽段溶液离心于收集管底部。(6) Add trypsin (100U/μg) 2-4μg (to protein ratio 1:50-100) into the ultrafiltration tube, volume 50μl, react overnight at 37℃ for 20h; the next day, centrifuge at 12000rpm for 20min, enzymatic digestion The final peptide solution was centrifuged at the bottom of the collection tube.
5、液相分级以及蛋白质质谱检测5. Liquid phase fractionation and protein mass spectrometry detection
取酶解产物，按照定量结果取10μg进行LCMSMS分析。采用纳升流速HPLC液相系统EASY-nLC1000进行分离。液相A液为含有2％(体积分数)乙腈和0.1％(体积分数)甲酸的水溶液(即以100ml计算组成如下：2ml乙腈，0.1ml甲酸，余量为水)，B液为含有84％(体积分数)乙腈和0.1％(体积分数)甲酸的水溶液(即以100ml计算组成如下：84ml乙腈，0.1ml甲酸，余量为水)。色谱柱Thermo EASY column SC200150μm×100mm(RP-C18)以100％的A液平衡。样品由自动进样器上样到ThermoEASY column SC001traps 150μm×20mm(RP-C18)(Thermo)，再经色谱柱分离，流速为400nl/min。相关液相梯度如下：0min-100min，B液线性梯度从0％到45％(体积百分比含量，下同)；100min-108min，B液线性梯度从45％到100％；108min-120min，B液维持在100％。酶解产物经毛细管高效液相色谱分离后用Q-Exactive质谱仪(Thermo Finnigan)进行质谱分析。分析时长：120min，检测方式：正离子，母离子扫描范围：300-1800m/z，多肽和多肽的碎片的质量电荷比按照下列方法采集：每次全扫描(full scan)后采集10个碎片图谱(MS2scan，HCD)。MS1在M/Z 200时分辨率为70000，MS2在M/Z 200时分辨率为17500。Take the enzymatic hydrolysis product, and take 10 μg according to the quantitative results for LCMSMS analysis. The nanoliter flow rate HPLC liquid phase system EASY-nLC1000 was used for separation. Liquid phase A liquid is an aqueous solution containing 2% (volume fraction) acetonitrile and 0.1% (volume fraction) formic acid (that is, the composition is as follows in 100ml: 2ml acetonitrile, 0.1ml formic acid, and the balance is water), and B liquid contains 84% (volume fraction) an aqueous solution of acetonitrile and 0.1% (volume fraction) formic acid (that is, the composition is as follows in 100ml: 84ml acetonitrile, 0.1ml formic acid, and the balance is water). The chromatographic column Thermo EASY column SC200 150μm×100mm (RP-C18) is balanced with 100% A solution. The sample was loaded from the autosampler to ThermoEASY column SC001traps 150μm×20mm (RP-C18) (Thermo), and then separated by the chromatographic column at a flow rate of 400nl/min. The relevant liquid phase gradient is as follows: 0min-100min, the linear gradient of B liquid is from 0% to 45% (volume percentage content, the same below); 100min-108min, the linear gradient of B liquid is from 45% to 100%; 108min-120min, B liquid Maintained at 100%. The enzymatic hydrolysis products were separated by capillary high-performance liquid chromatography and then analyzed by Q-Exactive mass spectrometer (Thermo Finnigan). Analysis time: 120min, detection method: positive ion, precursor ion scanning range: 300-1800m/z, the mass-to-charge ratio of peptides and peptide fragments is collected according to the following method: 10 fragment spectra are collected after each full scan (MS2scan, HCD). MS1 has a resolution of 70,000 at M/Z 200, and MS2 has a resolution of 17,500 at M/Z 200.
6、质谱鉴定6. Identification by mass spectrometry
先利用Trans-Proteomic Pipeline软件提取一级质谱定量信息(采用软件默认参数)，利用ProfileAnalysis2.0软件进行定量(采用软件默认参数)。然后利用ProteinScape2.1软件进行数据检索和整合，具体如下：First, the Trans-Proteomic Pipeline software was used to extract the quantitative information of the primary mass spectrometer (using software default parameters), and the ProfileAnalysis2.0 software was used for quantification (using software default parameters). Then use ProteinScape2.1 software for data retrieval and integration, as follows:
以Peptide FDR≤0.05对数据进行筛选过滤。导入Maxquant软件(属于ProteinScape2.1软件的一部分)进行Label-free分析。主要参数如下：Filter the data with Peptide FDR≤0.05. Import Maxquant software (part of ProteinScape2.1 software) for Label-free analysis. The main parameters are as follows:
Main search ppm：6Missed cleavage：2Main search ppm: 6 Missed cleavage: 2
MS/MS tolerance ppm：20De-Isotopic：TRUEMS/MS tolerance ppm: 20 De-Isotopic: TRUE
enzyme：Trypsin database：ipi.human.3.87.fastaenzyme: Trypsin database: ipi.human.3.87.fasta
Fixed modification：Carbamidomethyl(C)Fixed modification: Carbamidomethyl(C)
Variable modification：Oxidation(M)，Acetyl(Protein N-term)Variable modification: Oxidation (M), Acetyl (Protein N-term)
Decoy database pattern：reverseDecoy database pattern: reverse
Lable free quantification(LFQ)：TRUELable free quantification (LFQ): TRUE
LFQ min ratio count：2LFQ min ratio count: 2
Match between runs：2minMatch between runs: 2min
Peptide FDR：0.05Peptide FDR: 0.05
Protein FDR：0.05Protein FDR: 0.05
筛选出379个fold change(比值)＞2或者<0.5的差异蛋白(见表2)，从而获得儿童活动性结核病蛋白特征谱。其中，比值＞2的蛋白为表达上调的蛋白，比值<0.5的蛋白为表达下调的蛋白，所述比值为所述儿童活动性结核病患者组的表达量与所述健康对照儿童组的表达量的比值。379 differential proteins with fold change (ratio) > 2 or < 0.5 were screened out (see Table 2), so as to obtain the protein profile of children with active tuberculosis. Wherein, proteins with a ratio>2 are proteins whose expression is up-regulated, proteins with a ratio<0.5 are proteins whose expression is down-regulated, and the ratio is the expression level of the active tuberculosis patient group in children and the expression level in the healthy control children group ratio.
最终所得儿童活动性结核病蛋白特征谱中含有儿童活动性结核病患者组和健康对照儿童组表达差异的379种蛋白质，所述儿童活动性结核病患者组和所述健康对照儿童组中表达差异的蛋白质包括表达上调的蛋白质和表达下调的蛋白质；所述表达上调的蛋白质有PTCD3、ADAMTSL2、HTT、SNX4、VPS37B、RANBP17、UBE2O、VWDE、MCM4、ZC3H11A、WIZ、CCDC81、DSP、MMP2、GEMIN5、SPIRE2、PC、ADCY3、LIMS2、WWC1、RP1、ADAM18、AARSD1、PCSK6、ST3GAL4、ZNF584、ZMYM1、XB、LRRK2、SOCS5、RASAL3、SMC3、ATP10A、KDM1A、TTLL10、HERC6、PCCA、C1QTNF1、NUP88、SORBS1、NKD2、PIWIL4、AKAP2、SYNM、NPM1、HP、NOS1、NLRP14、SKIV2L、ICE1、ASAP2、ABAT、ORM1、CASK、SPECC1、NIPBL、DOCK11、CACTIN、ZNF526、SYNRG、FANCD2、PLXNC1、SPATA31D1、FBXO46、PSG3、BMPR2、DKFZp434A2017、KIF4A、COX10、ARHGDIG、RASAL2、MYH7、RFC1、DOCK5、ATP11A、ANKRD27、ZNF521、ZNF281、DLL1、TBL3、MYH14、EML4、CLTCL1、PRRC2C、TSTD2、C1orf94、NSD1、GPR137B、FRMPD2、ANKRD33B、SIN3A、TAS1R1、KIF3C、CYP26B1、HERC2、DCAF12L2、CCDC15、SP140、ABCC12、WDR91、SEC31A、GTPBP3、TLR4、CHMP4B、HECW1、CP、C16orf71、PPP2R5D、COL13A1、ZBTB10、ZNF311、PINLYP、AIM2、KCNMB3、HAL、MTHFD1、ALPP、PPP1R9A、IFT81、DDX60、CCDC71、RNF152、UBR4、ASH1L、CORO1C、ACAN、NARG1L、ZNF831、ABCA2、HCFC1、REXO1、TRIP12、PCNT、CAD、AK3、GUSB、SERPINA1、MTUS2、ZNF606、ARAP3、DZIP1、POLR3C、NLRP13、IFNA14、DNAH7、GPAM、SAMD9L、LMO3、USP11、RASGRF2、TATDN2、DCAF17、POFUT2、LAMA2、AXIN2、AP2A2、KIAA1033、HPS6、SORT1、TNPO3、NHLRC1、ADAM15、SLC25A41、CLEC14A、SERPINA3、DRG2、SYCP1、MRPS18B、ATP12A、SERPING1、MACF1、RSAD1、VPS13D、TTC3、RIMS1、GRK6、SP110、SOWAHC、HDGFRP3、POLR2A、C10orf76、INTS2、PROZ、MYO7B、TRMT1、ARHGEF1、LCE3A、SLC38A10、A2M、PAMR1、MCM5、ANK2、KLK8、PRG4、ZMYM3、PPP2R1B、TSSK4、INPP5B、SKIL、ZNF543、SLCO3A1、ATR、MID2、ZFP57、TICRR、XRCC4、SPTBN2、AHCTF1、C2CD3、ZC3H12B、LOX、PLOD3、ACACB、SDK2、C9orf85、CASP8、PLAA、KAT8、UTRN、MROH8、DMTN、SAA1、NRXN2、LEPREL4、SMC5、SPATA31C1、SAMHD1、PIAS3、L1CAM、JADE1、PKD1L1、MDC1、FAT2、PSD4、TAP1、CEP290、TTC28、ABCA7、LONP2、RGAG1、TAB2和TRPM5(即表2中的前242种蛋白)；所述表达下调的蛋白质有KIAA1211、CGN、DDAH1、HHIP、CLIC3、PPP6C、PNN、PRPF8、KIT、GLMN、CCDC89、DNAH14、COL6A2、GFRA1、PRPF6、KIAA0319、ATP13A1、PHKG2、COL7A1、TRIP6、TRIM14、DNAJC1、PHLDB3、ICA1、PRPF4B、TFAP4、LACTB、CEP63、MYO19、ARHGAP19、ANKRD17、EYS、PRDM9、TIGD5、ECHDC3、RHBDF1、GLG1、TMEM141、TNS1、GDPD3、LYST、HRH3、HBD、MZF1、FLYWCH1、OVOL2、HSPA5、ZNF30、ZNF492、FRAT1、AHSG、PIK3R6、KANSL1、MUC12、ESCO1、MIEF1、CACNA1E、DPYSL3、CHD7、ADCY9、EIF5、DDX31、RIT1、TECTA、RAB3GAP2、APOC3、RNF144B、SF3B4、ZNF709、EVC2、RB1CC1、MYH13、TIAM2、SEMA4A、COBLL1、UHRF1BP1L、SPTAN1、BCOR、SMG1、DPYD、RNASE4、PCCB、WDR62、BRD4、RBM26、DOPEY2、ZMYM5、RAB11FIP1、INADL、ZNF19、WNK1、WDR46、DAOA、ADIPOQ、F11、GOLGA4、ALK、TANGO6、BCL9L、COL4A1、CLEC2B、PDLIM1、CD22、EFNB2、COL8A2、NXF2、RNF180、ADAMTSL3、TTI2、LRP4、CDC37、CHD6、OCRL、ANO7、GBA2、PCF11、AGO3、CPT2、KMT2A、RASA3、ZNF431、CHIC1、ISCA2、BRINP1、MTO1、CASP2、MKI67、CNTRL、RPP40、DOK3、KRT82、FNDC1、WFDC5、KRT12、PTGDR2、BEND2和ZNF225(即表2中的后137种蛋白)。各蛋白在所述儿童活动性结核病患者组的表达量与所述健康对照儿童组的表达量的比值具体见表2中的“差异倍数”一栏。The final protein profile of active tuberculosis in children contains 379 proteins with differential expression between the active tuberculosis patient group in children and the healthy control children group, and the proteins with differential expression in the active tuberculosis patient group in children and the healthy control children group include Up-regulated proteins and down-regulated proteins; the up-regulated proteins include PTCD3, ADAMTSL2, HTT, SNX4, VPS37B, RANBP17, UBE2O, VWDE, MCM4, ZC3H11A, WIZ, CCDC81, DSP, MMP2, GEMIN5, SPIRE2, PC , ADCY3, LIMS2, WWC1, RP1, ADAM18, AARSD1, PCSK6, ST3GAL4, ZNF584, ZMYM1, XB, LRRK2, SOCS5, RASAL3, SMC3, ATP10A, KDM1A, TTLL10, HERC6, PCCA, C1QTNF1, NUP88, SORBS1, NKD2, PIWIL4 , AKAP2, SYNM, NPM1, HP, NOS1, NLRP14, SKIV2L, ICE1, ASAP2, ABAT, ORM1, CASK, SPECC1, NIPBL, DOCK11, CACTIN, ZNF526, SYNRG, FANCD2, PLXNC1, SPATA31D1, FBXO46, PSG3, BMPR2, DKFZp434A2017 , KIF4A, COX10, ARHGDIG, RASAL2, MYH7, RFC1, DOCK5, ATP11A, ANKRD27, ZNF521, ZNF281, DLL1, TBL3, MYH14, EML4, CLTCL1, PRRC2C, TSTD2, C1orf94, NSD1, GPR137B, FRMPD2, ANKRD33B, SIN3A, , KIF3C, CYP26B1, HERC2, DCAF12L2, CCDC15, SP140, ABCC12, WDR91, SEC31A, GTPBP3, TLR4, CHMP4B, HECW1, CP, C16orf71, PPP2R5D, COL13A1, ZBTB10, ZNF311, PINLYP, AIM2, KCNMB3, D1, AL, MTH , PPP1R9A, IFT81, DDX60, CCDC71, RNF152, UBR4, ASH1L, CORO1C, ACAN, NARG1L, ZNF831, ABCA2, HCFC1, REXO1, TRIP12, PCNT, CAD, AK3, GUSB, SERPINA1, MTUS2, ZNF606, ARAP3, DZI P1, POLR3C, NLRP13, IFNA14, DNAH7, GPAM, SAMD9L, LMO3, USP11, RASGRF2, TATDN2, DCAF17, POFUT2, LAMA2, AXIN2, AP2A2, KIAA1033, HPS6, SORT1, TNPO3, NHLRC1, ADAM15, SLC25A41, CLEC14A, SERPINA3 DRG2, SYCP1, MRPS18B, ATP12A, SERPING1, MACF1, RSAD1, VPS13D, TTC3, RIMS1, GRK6, SP110, SOWAHC, HDGFRP3, POLR2A, C10orf76, INTS2, PROZ, MYO7B, TRMT1, ARHGEF1, LCE3A, SLC38A10, A2M, PAMR1, MCM5, ANK2, KLK8, PRG4, ZMYM3, PPP2R1B, TSSK4, INPP5B, SKIL, ZNF543, SLCO3A1, ATR, MID2, ZFP57, TICRR, XRCC4, SPTBN2, AHCTF1, C2CD3, ZC3H12B, LOX, PLOD3, ACACB, SDK2, C9orf85, CASP8, PLAA, KAT8, UTRN, MROH8, DMTN, SAA1, NRXN2, LEPREL4, SMC5, SPATA31C1, SAMHD1, PIAS3, L1CAM, JADE1, PKD1L1, MDC1, FAT2, PSD4, TAP1, CEP290, TTC28, ABCA7, LONP2, RGAG1, TAB2 and TRPM5 (i.e. the first 242 proteins in Table 2); the down-regulated proteins include KIAA1211, CGN, DDAH1, HHIP, CLIC3, PPP6C, PNN, PRPF8, KIT, GLMN, CCDC89, DNAH14, COL6A2, GFRA1, PRPF6, KIAA0319, ATP13A1, PHKG2, COL7A1, TRIP6, TRIM14, DNAJC1, PHLDB3, ICA1, PRPF4B, TFAP4, LACTB, CEP63, MYO19, ARHGAP19, ANKRD17, EYS, PRDM9, TINSGD5, ECHDC3, RHBDF1, GLG1, TMEM141, T GDPD3, LYST, HRH3, HBD, MZF1, FLYWCH1, OVOL2, HSPA5, ZNF30, ZNF492, FRAT1, AHSG, PIK3R6, KANSL1, MUC12, ESCO1, MIEF1, CACNA1E, DPYSL3, CHD7, ADCY9, EIF5, DDX31, RIT1, TECTA, RAB3GAP2, APOC3, RNF144B, SF3B4, ZNF709, EVC2, RB1CC1, MYH13, TIAM2, SEMA4A, COBLL1, UHRF1BP1L, SPTAN1, BCOR, SMG1, DPYD, RNASE4, PCCB, WDR62, BRD4, RBM26, DOPEY2, ZMYM5, RAB11FIP1, INADL, ZNF19, WNK1, WDR46, DAOA, ADIPOQ, F11, GOLGA4, ALK, TANGO6, BCL9L, COL4A1, CLEC2B, PDLIM1, CD22, EFNB2, COL8A2, NXF2, RNF180, ADAMTSL3, TTI2, LRP4, CDC37, CHD6, OCRL, ANO7, GBA2, PCF11, AGO3, CPT2, KMT2A, RASA3, ZNF431, CHIC1, ISCA2, BRINP1, MTO1, CASP2, MKI67, CNTRL, RPP40, DOK3, KRT82, FNDC1, WFDC5, KRT12, PTGDR2, BEND2 and ZNF225 (ie the last 137 proteins in Table 2). The ratio of the expression level of each protein in the active tuberculosis patient group of children to the expression level of the healthy control children group is specifically shown in the column "Difference Multiple" in Table 2.
表2 379种蛋白在NCBI数据库中的编号及对应基因号Table 2 Numbers and corresponding gene numbers of 379 proteins in NCBI database
7、数据分析7. Data Analysis
(1)样本检测总离子流强度一致，说明样本仪器检测上样量基本一致，具有定量分析意义。(1) The total ion current intensities detected by the samples are consistent, indicating that the amount of sample loaded by the sample instrument is basically the same, which has the significance of quantitative analysis.
(2)样本检测TIC离子流峰型基本一致，说明样本酶解较完全，且各样本无蛋白损失，这与SDS-PAGE图谱也相符合。(2) The peak patterns of the TIC ion currents detected by the samples were basically the same, indicating that the samples were hydrolyzed relatively completely, and there was no protein loss in each sample, which was also consistent with the SDS-PAGE pattern.
(3)样本肽段的串联质谱峰分布比较平均，且大部分出现在有效梯度内，说明液相色谱条件较适合样本分析。(3) The tandem mass spectrum peak distribution of sample peptides is relatively average, and most of them appear in the effective gradient, indicating that the liquid chromatography conditions are more suitable for sample analysis.
(4)样本的离子流峰保留时间比较一致，说明色谱比较稳定且重复性好，适合比较同一保留时间内的质谱峰，适合定量分析。(4) The retention times of the ion current peaks of the samples are relatively consistent, indicating that the chromatogram is relatively stable and has good repeatability, and is suitable for comparing mass spectrum peaks within the same retention time and for quantitative analysis.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105181837A (en)* | 2015-08-28 | 2015-12-23 | 程永娟 | Mycobacterium tuberculosis detection method |
| CN105241986A (en)* | 2015-09-10 | 2016-01-13 | 首都医科大学附属北京儿童医院 | Protein characteristic spectrum for distinguishing latent tuberculosis children infectors and active tuberculosis children patients |
| CN105588944A (en)* | 2016-02-25 | 2016-05-18 | 首都医科大学附属北京胸科医院 | Application of AGP1, SERPINA3 and CDH1 content detection system in screening active tuberculosis patients |
| CN106636443A (en)* | 2017-02-28 | 2017-05-10 | 北京泱深生物信息技术有限公司 | Application of DNAH14 gene in tumor diagnosis and treatment |
| CN107449916A (en)* | 2017-07-27 | 2017-12-08 | 北京邦菲生物科技有限公司 | Lung cancer and pulmonary nodule protein specificity spectrum and its construction method and application |
| WO2018013883A1 (en)* | 2016-07-14 | 2018-01-18 | Caprion Proteomics Inc. | Biomarkers of latent tuberculosis infection |
| CN108956791A (en)* | 2017-05-23 | 2018-12-07 | 湖北普罗金科技有限公司 | A kind of method of Large-scale Screening protein biomarkers |
| CN108982868A (en)* | 2018-06-25 | 2018-12-11 | 哈尔滨医科大学 | The application of nucleome Protein S P110 and kit containing the albumen in preparation alcoholic myocardiopathy early diagnosis reagent |
| CN110244048A (en)* | 2019-06-19 | 2019-09-17 | 中国人民解放军总医院第八医学中心 | Application of the SERPING1 albumen as marker in exploitation diagnostic activities reagent lungy |
| CN110261611A (en)* | 2019-06-14 | 2019-09-20 | 上海四核生物科技有限公司 | Application and its kit of the ZNF709 albumen as gastric cancer serum biomarker |
| CN110283905A (en)* | 2019-08-05 | 2019-09-27 | 中国人民解放军总医院第八医学中心 | Based on ABCA2 quantitative fluorescent PCR diagnostic activities kit lungy |
| CN110596397A (en)* | 2019-08-09 | 2019-12-20 | 佛山市第四人民医院(佛山市结核病防治所) | A serum protein marker for the diagnosis of tuberculosis drug resistance and its application |
| WO2020198990A1 (en)* | 2019-03-29 | 2020-10-08 | 西南大学 | Use of tuberculosis markers in tuberculosis diagnosis and efficacy evaluation |
| CN112063725A (en)* | 2020-09-14 | 2020-12-11 | 华南农业大学 | Molecular markers and applications of AGO3 gene related to shank length and shank diameter in chickens |
| CN114264818A (en)* | 2021-12-01 | 2022-04-01 | 陕西脉元生物科技有限公司 | Autoimmune system disease marker anti-ASAP 2 autoantibody and application thereof |
| CN114577959A (en)* | 2022-03-04 | 2022-06-03 | 深圳华大基因科技服务有限公司 | A method for analyzing multiple protein modifications in biological samples |
| CN117706013A (en)* | 2023-12-20 | 2024-03-15 | 首都医科大学附属北京朝阳医院 | New coronavirus adverse prognostic protein signature spectrum and its construction methods, systems and applications |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103018358A (en)* | 2012-11-18 | 2013-04-03 | 浙江大学 | Kit for detecting pulmonary tuberculosis syndrome characteristic protein and application thereof |
| CN103065065A (en)* | 2012-11-18 | 2013-04-24 | 浙江大学 | Active tuberculosis differential expression protein profile model and building method thereof |
| WO2014059336A1 (en)* | 2012-10-12 | 2014-04-17 | University Of Notre Dame Du Lac | Exosomes and diagnostic biomarkers |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014059336A1 (en)* | 2012-10-12 | 2014-04-17 | University Of Notre Dame Du Lac | Exosomes and diagnostic biomarkers |
| CN103018358A (en)* | 2012-11-18 | 2013-04-03 | 浙江大学 | Kit for detecting pulmonary tuberculosis syndrome characteristic protein and application thereof |
| CN103065065A (en)* | 2012-11-18 | 2013-04-24 | 浙江大学 | Active tuberculosis differential expression protein profile model and building method thereof |
| Title |
|---|
| LU ZHANG等: "Identification of putative biomarkers for the serodiagnosis of drug-resistant Mycobacterium tuberculosis", 《PROTEOME SCIENCE》, vol. 10, no. 12, 31 December 2012 (2012-12-31), pages 1 - 10* |
| QI LIU等: "Serum Protein Profiling of Smear-Positive and Smear-Negative Pulmonary Tuberculosis Using SELDI-TOF Mass Spectrometry", 《LUNG》, vol. 188, 31 December 2010 (2010-12-31), pages 15 - 23, XP 019780944* |
| 方素芳等: "耐多药肺结核患者血清蛋白指纹图谱测定初步研究", 《中国防痨杂志》, vol. 34, no. 1, 31 January 2012 (2012-01-31), pages 40 - 44* |
| 翁丽珍等: "肺结核蛋白指纹图谱诊断技术研究", 《中国人兽共患病学报》, vol. 26, no. 11, 31 December 2010 (2010-12-31), pages 1048 - 1051* |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105181837A (en)* | 2015-08-28 | 2015-12-23 | 程永娟 | Mycobacterium tuberculosis detection method |
| CN105241986A (en)* | 2015-09-10 | 2016-01-13 | 首都医科大学附属北京儿童医院 | Protein characteristic spectrum for distinguishing latent tuberculosis children infectors and active tuberculosis children patients |
| CN105588944A (en)* | 2016-02-25 | 2016-05-18 | 首都医科大学附属北京胸科医院 | Application of AGP1, SERPINA3 and CDH1 content detection system in screening active tuberculosis patients |
| US10935549B2 (en) | 2016-07-14 | 2021-03-02 | Caption Biosciences Inc. | Biomarkers of latent tuberculosis infection |
| WO2018013883A1 (en)* | 2016-07-14 | 2018-01-18 | Caprion Proteomics Inc. | Biomarkers of latent tuberculosis infection |
| CN106636443A (en)* | 2017-02-28 | 2017-05-10 | 北京泱深生物信息技术有限公司 | Application of DNAH14 gene in tumor diagnosis and treatment |
| CN106636443B (en)* | 2017-02-28 | 2019-12-10 | 北京泱深生物信息技术有限公司 | Application of DNAH14 gene in preparation of tumor diagnosis and treatment products |
| CN108956791A (en)* | 2017-05-23 | 2018-12-07 | 湖北普罗金科技有限公司 | A kind of method of Large-scale Screening protein biomarkers |
| CN107449916A (en)* | 2017-07-27 | 2017-12-08 | 北京邦菲生物科技有限公司 | Lung cancer and pulmonary nodule protein specificity spectrum and its construction method and application |
| CN108982868A (en)* | 2018-06-25 | 2018-12-11 | 哈尔滨医科大学 | The application of nucleome Protein S P110 and kit containing the albumen in preparation alcoholic myocardiopathy early diagnosis reagent |
| CN113631723B (en)* | 2019-03-29 | 2023-11-14 | 西南大学 | Application of tuberculosis markers in tuberculosis diagnosis and efficacy evaluation |
| CN113631723A (en)* | 2019-03-29 | 2021-11-09 | 西南大学 | Application of tuberculosis markers in tuberculosis diagnosis and efficacy evaluation |
| WO2020198990A1 (en)* | 2019-03-29 | 2020-10-08 | 西南大学 | Use of tuberculosis markers in tuberculosis diagnosis and efficacy evaluation |
| CN110261611A (en)* | 2019-06-14 | 2019-09-20 | 上海四核生物科技有限公司 | Application and its kit of the ZNF709 albumen as gastric cancer serum biomarker |
| CN110261611B (en)* | 2019-06-14 | 2021-06-22 | 上海四核生物科技有限公司 | Application of ZNF709 protein as gastric cancer serum biomarker and kit thereof |
| CN110244048A (en)* | 2019-06-19 | 2019-09-17 | 中国人民解放军总医院第八医学中心 | Application of the SERPING1 albumen as marker in exploitation diagnostic activities reagent lungy |
| CN110283905A (en)* | 2019-08-05 | 2019-09-27 | 中国人民解放军总医院第八医学中心 | Based on ABCA2 quantitative fluorescent PCR diagnostic activities kit lungy |
| CN110596397A (en)* | 2019-08-09 | 2019-12-20 | 佛山市第四人民医院(佛山市结核病防治所) | A serum protein marker for the diagnosis of tuberculosis drug resistance and its application |
| CN112063725A (en)* | 2020-09-14 | 2020-12-11 | 华南农业大学 | Molecular markers and applications of AGO3 gene related to shank length and shank diameter in chickens |
| CN114264818A (en)* | 2021-12-01 | 2022-04-01 | 陕西脉元生物科技有限公司 | Autoimmune system disease marker anti-ASAP 2 autoantibody and application thereof |
| CN114264818B (en)* | 2021-12-01 | 2023-02-03 | 陕西脉元生物科技有限公司 | Autoimmune system disease marker anti-ASAP 2 autoantibody and application thereof |
| CN114577959A (en)* | 2022-03-04 | 2022-06-03 | 深圳华大基因科技服务有限公司 | A method for analyzing multiple protein modifications in biological samples |
| CN117706013A (en)* | 2023-12-20 | 2024-03-15 | 首都医科大学附属北京朝阳医院 | New coronavirus adverse prognostic protein signature spectrum and its construction methods, systems and applications |
| Publication number | Publication date |
|---|---|
| CN104792894B (en) | 2016-09-07 |
| Publication | Publication Date | Title |
|---|---|---|
| CN104792894B (en) | Children's activity tuberculosis protein specificity spectrum and construction method thereof | |
| US20050064516A1 (en) | Biological markers for diagnosing multiple sclerosis | |
| Fu et al. | Proteomic analysis of sputum in patients with active pulmonary tuberculosis | |
| KR102700025B1 (en) | Biomarkers for Diagnosis of Tuberculosis by Metabolomics | |
| Laila et al. | Role of proteomics in the discovery of autism biomarkers | |
| Gonzalez et al. | Identification of novel candidate protein biomarkers for the post-polio syndrome—implications for diagnosis, neurodegeneration and neuroinflammation | |
| Piras et al. | Serum protein profiling of early and advanced stage Crohn's disease | |
| Stubendorff et al. | Urine protein profiling identified alpha-1-microglobulin and haptoglobin as biomarkers for early diagnosis of acute allograft rejection following kidney transplantation | |
| Santos et al. | Evaluation of MALDI-TOF MS in the microbiology laboratory | |
| Osbak et al. | Candidate Treponema pallidum biomarkers uncovered in urine from individuals with syphilis using mass spectrometry | |
| Liu et al. | Urinary proteomic analysis to identify a potential protein biomarker panel for the diagnosis of tuberculosis | |
| US12247977B2 (en) | Nasopharyngeal protein biomarkers of acute respiratory virus infection and methods of using same | |
| JP7173495B2 (en) | A mass spectrometry-based method for detecting circulating histones H3 and H2B in plasma from patients with sepsis or septic shock (SS) | |
| Kawasaki et al. | Next-generation proteomics of serum extracellular vesicles combined with single-cell RNA sequencing identifies MACROH2A1 associated with refractory COVID-19 | |
| Chen et al. | Several potential serum proteomic biomarkers for diagnosis of osteoarticular tuberculosis based on mass spectrometry | |
| EP3044594A1 (en) | Identification of novel biomarkers of flares of systemic lupus erythematosus | |
| Collins et al. | A plasma metabolic signature to diagnose pulmonary tuberculosis and monitor treatment response | |
| CN105241986B (en) | Protein characteristic spectrum for distinguishing latent tuberculosis children infectors and active tuberculosis children patients | |
| Wu et al. | Identification and validation of argininosuccinate synthase as a candidate urinary biomarker for major depressive disorder | |
| Haenen et al. | Biomarker discovery in asthma and COPD: Application of proteomics techniques in human and mice | |
| He et al. | Quantitative proteomics reveals plasma protein profile and potential pathways in pulmonary tuberculosis patients with and without diabetes | |
| Zhang et al. | Proteomic pilot study of tuberculosis pleural effusion | |
| WO2021209575A2 (en) | Biomarker for monitoring coronavirus disease 2019 | |
| Wang et al. | Serum proteomics identifies biomarkers for predicting non-survivors in elderly COVID-19 patients | |
| Bai et al. | Proteome analysis of haptoglobin in cerebrospinal fluid of neuromyelitis optica |
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