iTRAQ蛋白质组学方法发现高同型半胱氨酸诱导的鸡胚神经管畸形中存在氧化磷酸化通路异常

目的筛选同型半胱氨酸硫代内酯(HTL)诱导鸡胚神经管畸形差异蛋白以明确高同型半胱氨酸(Hcy)导致神经管畸形的发病机制。方法采用白来航鸡种蛋于孵化28 h进行神经沟注射HTL处理,于第5天(E5)收集胚胎脑组织,采用iTRAQ联合二维液相色谱-串联质谱技术(2D-LC-MS/MS)对差异蛋白质进行筛选,并进一步用蛋白质非标记(1abel-free)定量技术进行差异蛋白的验证。结果在鸡脑组织中共鉴定出369种差异蛋白质;KEGG通路分析提示差异蛋白富集于代谢通路和氧化磷酸化通路;1abel-free(PRM)验证氧化磷酸化通路蛋白ATP5H和ATP5MF在NTDs中表达异常。结论 HTL诱导鸡胚神经管畸形脑中氧化磷酸化通路蛋白异常表达。     

双向电泳-质谱技术寻找原发性高尿酸血症血清差异蛋白初探

目的 通过对比分析正常人和高尿酸血症患者血清蛋白双向电泳图谱,初步分析高尿酸血症血清差异蛋白.方法 采用双向凝胶电泳(2-dimentional gel electrophoresis,2-DE)和基质辅助激光解吸飞行时间质谱(matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry,MALDI-TOF-MS)对高尿酸血症患者和对照组健康人血清进行分析,寻找高尿酸血症血清差异蛋白.结果 差异表达蛋白质点数为10个,质谱成功鉴定出4个差异蛋白质,其中补体C3、触珠蛋白、α1-抗胰蛋白酶呈上调表达,载脂蛋白L1呈下调表达.结论 补体C3、触珠蛋白、o1-抗胰蛋白酶、载脂蛋白L1在高尿酸血症组和正常对照组血清中存在差异,但结果有待运用其他生物学的方法进一步验证.     

应用iTRAQ技术筛选房间隔缺损血清差异表达蛋白研究

目的:应用同位素标记相对和绝对定量(i TRAQ)技术结合LC-MALDI-TOF/TOF MS方法筛选潜在的房间隔缺损血清标志蛋白,探索房间隔缺损的发病机制。方法:收集房间隔缺损患者和与之相匹配的室间隔缺损患者、健康人血清各20例,等量混合后使用多重亲和去除系统(MARS)去除14种高丰度蛋白,经i TRAQ试剂标记后应用二维液相色谱分离,然后利用质谱进行相对定量分析和生物信息学分析。结果:质谱鉴定出置信度>95%的蛋白质共256种,其中表达量差异有统计学意义(P<0.05)的蛋白质有22种,房间隔缺损组较室间隔缺损组和健康对照组表达量均上调>1.3倍的蛋白有8种,下调<0.77倍的蛋白有14种。结论:应用i TRAQ技术筛选出22种房间隔缺损相关差异性蛋白,这些蛋白可作为房间隔缺损血清蛋白标志物的重要候选因子。     

血清TMT标记定量蛋白质组学对多囊卵巢综合征病因研究

目的 通过应用串联质谱标签(tandem mass tag,TMT)联合液相色谱-串联质谱(liquid chromatography/tandem mass spectrometry,LC-MS/MS)技术、差异表达蛋白及其相关生物通路对多囊卵巢综合征(polycystic ovarian syndrome,PCOS)患者病因研究。 方法 本研究纳入20例PCOS患者作为PCOS组,18例正常女性作为对照组。通过TMT联合LC-MS／MS检测所有研究对象的空腹血清样本,对数据进行统计学和生物信息学分析。 结果 鉴定了38个差异表达蛋白质,其中21个蛋白在PCOS中表达上调(FC≥1.2,P＜0.05),17个在PCOS中表达下调(FC≤0.83,P＜0.05)。采用京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)和Reactome对差异表达蛋白进行富集分析,分别富集到18个和78个通路。将两个数据库富集分析结果整合,均发现补体级联反应的富集通路,共同涉及的差异表达蛋白为甘露糖结合蛋白(mannose-binding protein,MBL)。 结论 PCOS患者体内可能存在补体系统的过度激活,差异蛋白MBL可能是其中的关键致病因子。     

基于iTRAQ技术筛选室间隔缺损患儿血清蛋白标志物

目的:应用同位素标记相对和绝对定量(iTRAQ)技术结合LC-MALDI-TOF/TOF方法比较室间隔缺损(VSD)患儿和健康对照组儿童血清中的差异表达蛋白质,筛选并鉴定潜在的VSD血清蛋白标志物。方法:收集VSD患儿和与之相匹配的健康儿童血清各30例,组内等量混合后利用多重免疫亲和层析柱(MARS)去除14种高丰度蛋白,iTRAQ试剂标记后的样本应用二维液相色谱分离,采用MALDI-TOF/TOF质谱鉴定及相对定量。结果:质谱鉴定出置信度>95%的蛋白质共166种,其中差异表达蛋白质21种,VSD组较健康对照组表达量上调≥1.5倍的蛋白质有13种,下调≤0.67倍的蛋白质有8种。结论:筛选出多种与VSD相关的差异表达蛋白质,为进一步验证VSD血清蛋白标志物奠定了基础。     

职业性三氯乙烯药疹样皮炎患者血清蛋白质差异表达分析

目的 比较职业性三氯乙烯药疹样皮炎(OMDTE)患者发病急性期与治愈后的血清蛋白质表达谱.方法 患者血清经前处理后,双向凝胶电泳分离蛋白质,软件分析凝胶图像,基质辅助激光解吸电离飞行时间串联质谱鉴定差异表达蛋白斑点.结果 与治愈后的血清蛋白质表达谱比较,在发病急性期发现41个明显差异表达的蛋白斑点,鉴定出31个蛋白.上调的蛋白有S100钙结合蛋白A8、钙网蛋白、血清淀粉样蛋白A、亮氨酸氨基肽酶、谷胱甘肤过氧化物酶等;下调的蛋白有视黄醇结合蛋白、乙酰辅酶A羧化酶、羧肽酶N等;涉及的功能通路包括炎症反应、氧化应激、视黄醇代谢、脂肪酸代谢、激肤和过敏毒素的调节等.结论 OMDTE患者在发病急性期血清蛋白质存在明显的差异表达,鉴定的差异表达蛋白质不仅为其发病机制研究提供候选靶标,并且可能作为生物标志物应用于早期诊治.     

四氯化碳诱发大鼠肝脏蛋白质表达变化的分析

目的分析四氯化碳(CCl4)诱发肝纤维化大鼠肝脏细胞蛋白表达的变化,为寻找CCl4致肝纤维化早期生物效应的相关蛋白提供实验基础。方法采用皮下注射CCl4诱发实验性大鼠肝损伤模型,测定大鼠血清生化指标和观察肝组织病理学的变化,提取大鼠肝细胞总蛋白并采用二维凝胶电泳(2-DE)进行分离,Image Master 2D Platinum软件分析电泳图谱,选择表达有显著差异的蛋白点进行基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)测定。结果大鼠血清生化指标和病理学结果提示肝纤维化造模成功,CCl4损伤组肝细胞蛋白质2-DE图谱表达发生改变,差异点经MALDI-TOF-MS分析和数据库搜索,鉴定出4个已知序列蛋白。结论应用蛋白质组学技术筛选和鉴定出的蛋白可能与肝纤维化的发生发展有关,为进一步研究CCl4诱发肝纤维化机制提供依据。     

肾衰患者肾纤维化发展的关键候选基因和通路的生物信息学分析

目的肾脏纤维化是各种原发或继发性肾脏病持续进展,导致正常肾脏组织结构被细胞外基质所取代,伴肾功能进行性不可逆性损害的病理过程,是引起终末期肾衰竭的主要原因和病理基础之一。ESRD患者需要接受RRT以延长生命,消耗大量医学资源。近年来对肾脏纤维化机制的有关研究很多,但涉及的信号通路和驱动基因在很大程度上还不清楚。利用生物信息学方法分析影响肾脏纤维化发病的关键基因和通路,为进一步动物实验研究其功能和参与的调控机制提供一定的理论依据。方法在公共基因芯片数据库(GEO)中下载肾脏纤维化相关基因表达谱数据(GSE66494)。该芯片包括53例肾脏纤维化患者组织样本及8例正常人肾脏组织。我们利用GEO2R工具对肾纤维化患者组织与正常组织中表达的基因进行差异分析,并利用DAVID工具对差异表达基因进行功能和通路富集分析(GO、KEGG)。我们还通过生物信息学工具STRING v10.5构建差异基因的蛋白质-蛋白质相互作用网络,并利用Cytoscape对蛋白质互作网络进行可视化并筛选核心基因。结果通过对53例肾纤维化组织与正常组织基因表达谱数据进行差异分析,我们从GSE66494数据集中共筛选出514个差异表达的基因,其中138个在肾纤维化组织中上调,376个在肾纤维化组织中下调。GO分析结果显示,差异表达基因主要富集在细胞外组成成分,急性期反应以及物质运输相关生物学过程等。KEGG结果显示差异表达的基因参与调控的显著的信号通路主要包括:胰腺分泌,蛋白质的消化和吸收,非洲锥虫病,PPAR信号通路,PI3K-Akt信号通路以及疟疾通路。通过构建差异表达基因的蛋白质-蛋白质相互作用网络,我们从差异表达基因中筛选出了10个核心基因包括ALB,TOP2A,MYC,FOS,PLG,IL10,CCNB2,REN,PTGS2,TAC1。结论我们通过生物信息学方法发现了在正常组织与肾纤维化组织中差异表达的514个基因,并发现了其调控的重要的信号通路。以上这些差异基因尤其是核心基因,和信号通路,可能是参与肾纤维化发生,发展的关键基因和通路,这为肾脏纤维化的诊断和治疗提供了新的思路或新靶点。     

甲苯职业接触者血清蛋白质谱差异的研究

目的研究甲苯接触工人与健康人群蛋白质图谱差异。方法收集甲苯接触工人(6例)和健康人群(6例)的血清,采用蛋白质组学非标记Label free质谱分析技术进行蛋白鉴定和定量,对甲苯接触组与健康对照组样本差异表达的蛋白质进行分析,应用生物信息学工具分析筛选到的蛋白质功能及其参与生物过程。结果蛋白质组学结果显示甲苯接触工人组与健康对照组之间差异表达超过2倍以上有39个蛋白,其中24个在甲苯接触工人组血清中表达上调,15个蛋白质在甲苯接触工人组表达下调。筛选到的这些蛋白质主要涉及信号转导、丝氨酸内肽酶活力、炎性反应、蛋白修饰、应激反应、凝血反应等过程。结论差异表达的蛋白质为甲苯接触人群的健康评估和早期疾病诊断提供了潜在蛋白标志物,有助于对甲苯早期中毒的分子机制的理解。     

应用iTRAQ标记结合2D LC-MS/MS分析慢性苯中毒患者血清蛋白质组

目的 比较慢性苯中毒患者与健康人的血清蛋白质组,筛选差异表达蛋白质,为阐明其发病机制和寻找生物标志物提供靶标.方法 免疫层析柱去除14种高丰度血清蛋白质后,应用8标iTRAQ技术结合2D LC-MS/MS分析并鉴定慢性苯中毒病例与健康人血清的差异表达蛋白质.结果 对9例慢性苯中毒患者和9名健康人的血清蛋白样品的iTRAQ标记,分析并成功鉴定159种蛋白质,其中差异表达有统计学意义(P＜0.05)的蛋白质有纤维蛋白溶酶原(PLG)、载脂蛋白B100( APOB100)、血小板碱性蛋白(PBP).这些差异表达蛋白质具有结合、催化、酶调节和转运活性等分子功能,参与的生物过程包括免疫、代谢、应激、转运和凋亡等.结论 PLG、APOB100和PBP可能作为慢性苯中毒发病机制研究的候选靶标.     

Proteomic Analysis of Cellular and Membrane Proteins in Fluconazole-Resistant Candida glabrata

ObjectivesCandida glabrata is one of the most common causes of Candida bloodstream infections worldwide. Some isolates of C glabrata may be intermediately resistant to azoles, with some strains developing resistance during therapy or prophylaxis with fluconazole. In this study, we used a proteomic approach to identify differentially expressed proteins between fluconazole-resistant and -susceptible strains.MethodsMembrane and cellular proteins were extracted from fluconazole-susceptible and fluconazole-resistant C glabrata strains. Differentially expressed proteins were compared using two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins with >1.5-fold difference in expression were identified by liquid chromatography tandem mass spectrometry (LC-MS/MS).ResultsA total of 65 proteins were differentially expressed in the cellular and membrane fractions. Among the 39 cellular proteins, 11 were upregulated and 28 were downregulated in fluconazole-resistant strains in comparison with fluconazole-susceptible strains. In the membrane fraction, a total of 26 proteins were found, of which 19 were upregulated and seven were downregulated. A total of 31 proteins were identified by LC-MS/MS that are involved in glycolysis, carbohydrate transport, energy transfer, and other metabolic pathways. Heat shock proteins were identified in various spots.ConclusionHeat shock and stress response proteins were upregulated in the membrane fraction of the fluconazole-resistant C glabrata strain. Compared with susceptible strains, fluconazole-resistant strains showed increased expression of membrane proteins and decreased expression of cellular proteins.     

120例特发性肺间质纤维化患者血流变、凝血功能的研究

[目的]通过对特发性肺间质纤维化患者的凝血功能、血液流变学、D-2聚体等指标进行深入的观察和检测,总结并分析该病的诊治方法与效果. [方法]采用问卷调查表形式,收集120例特发性肺间质纤维化患者为病例组,128例正常人为对照组,检测其凝血功能、血液流变学、D-2聚体等指标.对所收集的数据以统计学处理,以P＜0.05作为有统计学意义的标准. [结果]全血黏度1、全血黏度5、全血黏度200、血浆黏度、红细胞压积与此病的分期呈正相关;PT、TT、APTT与挟感发作期、慢性迁延期、重症多变期分期呈负相关,FIB与分期呈正相关;PT、TT、APTT与急性、亚急性、慢性证型呈负相关,FIB与急性、亚急性、慢性证型证型呈正相关;D-二聚体与分期及分型呈正相关. [结论]特发性肺间质纤维化病情呈现高血凝,高黏滞变化特点;高龄和男性是危险因素.     

Comparative proteomic exploration of whey proteins in human and bovine colostrum and mature milk using iTRAQ-coupled LC-MS/MS

Whey, an essential source of dietary nutrients, is widely used in dairy foods for infants. A total of 584 whey proteins in human and bovine colostrum and mature milk were identified and quantified by the isobaric tag for relative and absolute quantification (iTRAQ) proteomic method. The 424 differentially expressed whey proteins were identified and analyzed according to gene ontology (GO) annotation, Kyoto encyclopedia of genes and genomes (KEGG) pathway, and multivariate statistical analysis. Biological processes principally involved biological regulation and response to stimulus. Major cellular components were extracellular region part and extracellular space. The most prevalent molecular function was protein binding. Twenty immune-related proteins and 13 proteins related to enzyme regulatory activity were differentially expressed in human and bovine milk. Differentially expressed whey proteins participated in many KEGG pathways, including major complement and coagulation cascades and in phagosomes. Whey proteins show obvious differences in expression in human and bovine colostrum and mature milk, with consequences for biological function. The results here increase our understanding of different whey proteomes, which could provide useful information for the development and manufacture of dairy products and nutrient food for infants.

The advanced iTRAQ proteomic approach was used to analyze differentially expressed whey proteins in human and bovine colostrum and mature milk.     

Response to pneumococcal vaccine in interstitial lung disease patients: Influence of systemic immunosuppressive treatment

BackgroundInterstitial lung diseases (ILD) are severe respiratory diseases, and ILD patients are treated with corticosteroid and immunosuppressive agents. However, it is unclear whether these medications influence the response of pneumococcal vaccine.ObjectivesWe examined the immunogenicity of pneumococcal vaccines (PPSV23 and PCV13) in ILD patients undergoing immunosuppressive treatment.MethodsILD patients who were regularly followed at the outpatient clinic were enrolled. Sera were collected before and 4–8 weeks after vaccination. Serotype-specific immunoglobulin G (IgG) concentrations against pneumococcal serotype 19F were measured by ELISA.ResultsIgG concentrations to serotype 19F were increased in all groups in response to the vaccine. Both PCV13 and PPSV23 induced IgG concentrations in patients immunized for the first time. Response rates for the ILD group were comparable with those for the ILD group undergoing corticosteroid therapy. Only idiopathic pulmonary fibrosis patients undergoing immunosuppressive therapy had a significantly lower response.     

Proteomic Analysis of Intracellular and Membrane Proteins From Voriconazole-Resistant Candida glabrata

ObjectivesThe proteomic analysis of voriconazole resistant Candida glabrata strain has not yet been investigated. In this study, differentially expressed proteins of intracellular and membrane fraction from voriconazole-susceptible, susceptible dose-dependent (S-DD), resistant C. glabrata strains were compared with each other and several proteins were identified.MethodsThe proteins of intracellular and membrane were isolated by disrupting cells with glass bead and centrifugation from voriconazole susceptible, S-DD, and resistant C. glabrata strains. The abundance of expressed proteins was compared using two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis and proteins showing continuous twofold or more increase or reduction of expression in resistant strains compared to susceptible and S-DD strain were analyzed by liquid chromatography/mass spectrometry-mass spectrometry method.ResultsOf 34 intracellular proteins, 15 proteins showed expression increase or reduction (twofold or more). The identified proteins included regulation, energy production, carbohydrate transport, amino acid transport, and various metabolism related proteins. The increase of expression of heat shock protein 70 was found. Among membrane proteins, 12, 31 proteins showed expression increase or decrease in the order of susceptible, S-DD, and resistant strains. This expression included carbohydrate metabolism, amino acid synthesis, and response to stress-related proteins. In membrane fractions, the change of expression of 10 heat shock proteins was observed, and 9 heat shock protein 70 (Hsp70) showed the reduction of expression.ConclusionThe expression of Hsp70 protein in membrane fraction is related to voriconazole resistant C. glabrata strains.     

Identification of differentially expressed proteins associated with recurrence in ovarian endometriotic cysts

ABSTRACT

The objective of this study was to identify proteins that are differentially expressed in the cystic wall tissues of ovarian endometriotic cysts, simple ovarian cysts, and in normal ovarian tissues. Specimens of ovarian endometriotic cyst wall tissue, simple ovarian cyst wall tissue, and normal ovarian tissue (six specimens per group) were collected from patients who received gynecologic surgery, respectively. Differentially expressed proteins related to the ovarian endometriotic cysts were screened by use of isobaric tags for relative and absolute quantitation (iTRAQ) combined with functional annotation and bioinformatics analyses. All differentially expressed proteins related to cysts were validated using immunohistochemistry methods in recurrent and non-recurrent ovarian endometriotic cyst. A total of 359 proteins were identified as up-regulated in ovarian endometriotic cyst groups when compared with both the normal ovary and simple ovarian cyst groups. The levels of 27 proteins were >two-fold higher in the ovarian endometriotic cyst group than that in the other two groups. Of note, the five most significantly upregulated proteins were Charcot-Leyden Crystal Galectin (CLC), Defensin, alpha 1 (DEFA1), S100 calcium-binding protein A9 (S100A9), S100 calcium-binding protein A8 (S100A8), and Ferritin Light Chain (FTL). Immunohistochemistry results showed that the changes of S100A9 and S100A8 were consistent with the results shown by iTRAQ. However, no similarity of CLC, DEFA1, and FTL proteins was found between iTRAQ and immunohistochemistry. The ratio of patients with abnormally high S100A9 and S100A8 expression in the recurrent ovarian endometriotic cyst group was significantly higher than that in the non-recurrent group (P < 0.05). Our data identify differentially expressed proteins S100A9 and S100A8, and suggest they may serve as novel molecular markers to predict postoperative recurrence of an ovarian endometriotic cysts.

Abbreviations: iTRAQ: isobaric tags for relative and absolute quantitation; HPRD: Human Protein Reference Database; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; EM: Endometriosis; COX-2: cyclooxyenase-2; NF-kB: nuclear factor kappa-B; PR-B: progesterone receptor type B.     

盆腔器官脱垂患者阴道组织比较蛋白质组学研究

目的:建立盆腔器官脱垂(pelvic organ prolapse,POP)患者及非脱垂对照者阴道组织蛋白质双向凝胶电泳图谱,并结合质谱技术分析其差异表达蛋白质,寻找POP疾病相关蛋白,以进一步阐明POP的发病机制。方法:收集绝经后年龄相匹配的POP组和对照组尿道周围阴道壁组织标本各10份,提取组织总蛋白,进行双向凝胶电泳(two-dimensional gel electro-phoresis,2-DE)得到凝胶图谱,采用Image Master 5.0 2D图象分析软件进行匹配和差异分析,识别两组间差异表达蛋白。将部分差异蛋白点胶内原位酶解后进行MALDI-TOF-MS分析,获取肽质量指纹图谱并进行生物信息学检索鉴定蛋白质。结果:得到了分辨率高,重复性好的POP组和对照组阴道壁组织蛋白双向凝胶电泳图谱,POP组的平均蛋白质点数为(871±96)个,正常对照组为(856±89)个。通过比较分析,差异表达蛋白质点数为27个。选取其中8个具有明显差异的蛋白点进行质谱鉴定,初步鉴定出5种蛋白质,其中2个蛋白质在POP组表达上调,3个蛋白质表达下调。结论:两者间存在一些差异表达的蛋白质,为阐明POP发病机制打下了坚实的基础。     

Characterization and identification of differentially expressed microRNAs during the process of the peribiliary fibrosis induced by Clonorchis sinensis

Clonorchis sinensis (C. sinensis) infection can lead to biliary fibrosis. MicroRNAs (miRNAs) play important roles in regulation of genes expression in the liver diseases. However, the differential expression of miRNAs that probably regulates the portal fibrogenesis caused by C. sinensis has not yet been investigated. Hepatic miRNAs expression profiles from C. sinensis-infected mice at different time-points were analyzed by miRNA microarray and validated by quantitative real-time PCR (qRT-PCR). 349 miRNAs were differentially expressed in the liver of the C. sinensis-infected mice at 2, 8 or 16 weeks post infection (p.i.), compared with those at 0 week p.i., and there were 143 down-regulated and 206 up-regulated miRNAs among them. These all dysregulated miRNAs were potentially involved in the pathological processes of clonorchiasis by regulation of cancer-related signaling pathway, TGF-β signaling pathway, MAPK signaling pathway, Toll-like receptor signaling pathway, PI3K /AKT signaling pathway, etc. 169 of these dysregulated miRNAs were predicted to be involved in the TGF/Smads signaling pathway which plays an important role in the biliary fibrosis caused by C. sinensis. Additionally, miRNA-32, miRNA-34a, miRNA-125b and miRNA-497 were negatively correlated with Smad7 expression, indicating these miRNAs may specifically down-regulate Smad7 expression and participate in regulation of biliary fibrosis caused by C. sinensis. The results of the present study for the first time demonstrated that miRNAs were differentially expressed in the liver of mice infected by C. sinensis, and these miRNAs may play important roles in regulation of peribiliary fibrosis caused by C. sinensis, which may provide possible therapeutic targets for clonorchiasis.