Serum proteins differentially expressed in gestational diabetes mellitus assessed using isobaric tag for relative and absolute quantitation proteomics

BACKGROUND As a well-known fact to the public,gestational diabetes mellitus(GDM)could bring serious risks for both pregnant women and infants.During this important investigation into the linkage between GDM patients and their altered expression in the serum,proteomics techniques were deployed to detect the differentially expressed proteins(DEPs)of in the serum of GDM patients to further explore its pathogenesis,and find out possible biomarkers to forecast GDM occurrence.METHODS Subjects were divided into GDM and normal control groups according to the IADPSG diagnostic criteria.Serum samples were randomly selected from four cases in each group at 24-28 wk of gestation,and the blood samples were identified by applying iTRAQ technology combined with liquid chromatography-tandem mass spectrometry.Key proteins and signaling pathways associated with GDM were identified by bioinformatics analysis,and the expression of key proteins in serum from 12 wk to 16 wk of gestation was further verified using enzyme-linked immunosorbent assay (ELISA).RESULTS Forty-seven proteins were significantly differentially expressed by analyzing the serum samples between the GDMgravidas as well as the healthy ones. Among them, 31 proteins were found to be upregulated notably and the rest16 proteins were downregulated remarkably. Bioinformatic data report revealed abnormal expression of proteinsassociated with lipid metabolism, coagulation cascade activation, complement system and inflammatory responsein the GDM group. ELISA results showed that the contents of RBP4, as well as ANGPTL8, increased in the serumof GDM gravidas compared with the healthy ones, and this change was found to initiate from 12 wk to 16 wk ofgestation.CONCLUSION GDM symptoms may involve abnormalities in lipid metabolism, coagulation cascade activation, complementsystem and inflammatory response. RBP4 and ANGPTL8 are expected to be early predictors of GDM.

Identification of protein targets for the antidepressant effects of Kai-Xin-San in Chinese medicine using isobaric tags for relative and absolute quantitation

Kai-Xin-San consists of Ginseng Radix, Polygalae Radix, Acori Tatarinowii Rhizoma, and Poria at a ratio of 3:3:2:2. Kai-Xin-San has been widely used for the treatment of emotional disorders in China. However, no studies have identified the key proteins implicated in response to Kai-Xin-San treatment. In this study, rat models of chronic mild stress were established using different stress methods over 28 days. After 14 days of stress stimulation, rats received daily intragastric administrations of 600 mg/kg Kai-Xin-San. The sucrose preference test was used to determine depression-like behavior in rats, while isobaric tags were used for relative and absolute quantitation-based proteomics to identify altered proteins following Kai-Xin-San treatment. Kai-Xin-San treatment for 2 weeks noticeably improved depression-like behaviors in rats with chronic mild stress. We identified 33 differentially expressed proteins: 7 were upregulated and 26 were downregulated. Functional analysis showed that these differentially expressed proteins participate in synaptic plasticity, neurodevelopment, and neurogenesis. Our results indicate that Kai-Xin-San has an important role in regulating the key node proteins in the synaptic signaling network, and are helpful to better understand the mechanism of the antidepressive effects of Kai-Xin-San and to provide objective theoretical support for its clinical application. The study was approved by the Ethics Committee for Animal Research from the Chinese PLA General Hospital(approval No. X5-2016-07) on March 5, 2016.

Different protein expression patterns in rat spinal nerves during wallerian degeneration assessed using isobaric tags for relative and absolute quantitation proteomics profiling

Sensory and motor nerve fibers of peripheral nerves have different anatomies and regeneration functions after injury. To gain a clear understanding of the biological processes behind these differences, we used a labeling technique termed isobaric tags for relative and absolute quantitation to investigate the protein profiles of spinal nerve tissues from Sprague-Dawley rats. In response to Wallerian degeneration, a total of 626 proteins were screened in sensory nerves, of which 368 were upregulated and 258 were downregulated. In addition, 637 proteins were screened in motor nerves, of which 372 were upregulated and 265 were downregulated. All identified proteins were analyzed using the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis of bioinformatics, and the presence of several key proteins closely related to Wallerian degeneration were tested and verified using quantitative real-time polymerase chain reaction analyses. The differentially expressed proteins only identified in the sensory nerves were mainly relevant to various biological processes that included cell-cell adhesion, carbohydrate metabolic processes and cell adhesion, whereas differentially expressed proteins only identified in the motor nerves were mainly relevant to biological processes associated with the glycolytic process, cell redox homeostasis, and protein folding. In the aspect of the cellular component, the differentially expressed proteins in the sensory and motor nerves were commonly related to extracellular exosomes, the myelin sheath, and focal adhesion. According to the Kyoto Encyclopedia of Genes and Genomes, the differentially expressed proteins identified are primarily related to various types of metabolic pathways. In conclusion, the present study screened differentially expressed proteins to reveal more about the differences and similarities between sensory and motor nerves during Wallerian degeneration. The present findings could provide a reference point for a future investigation into the differences between sensory and motor nerves in Wallerian degeneration and the characteristics of peripheral nerve regeneration. The study was approved by the Ethics Committee of the Chinese PLA General Hospital, China(approval No. 2016-x9-07) in September 2016.

Isobaric Tag for Relative and Absolute Quantitation-Based Proteomics for Investigating the Effect of Guasha on Lumbar Disc Herniation in Rats

Objective:The objective of the study is to examine the possible mechanism by which Guasha(scraping therapy)affects the expression profiles of proteins in a lumbar disc herniation(LDH)rat model using isobaric tags for relative and absolute quantitation(iTRAQ)-based proteomics.Methods:Thirty-six rats were used in this study.LDH rats were subjected to noncompressive LDH surgeries.Rats were randomly divided into the model and Guasha groups.Guasha was applied on alternate days for a total of nine times(three courses).At the end of each course,six rats were randomly selected from each group and their blood samples were collected.iTRAQ labeling was used to examine the mechanism of action of Guasha against LDH.The molecular functions,cellular components,and biological processes were analyzed using gene ontology analysis.The Ingenuity Pathway Analysis database was used to identify canonical pathways involving these proteins.Results:Compared to the model group,198,182,and 170 proteins were identified as differentially expressed at the three respective Guasha treatment courses.Pathways,including focal adhesion kinase signaling,acute-phase response signaling,and the LXR/RXR activation pathway,were closely related to the effects of Guasha in LDH rats.Furthermore,Rac1,Orm1,and Hpx were validated by western blotting,and the results were consistent with the protein expression levels observed using the iTRAQ method.Conclusion:Guasha could not only regulate the pathological changes related to LDH,but also achieve therapeutic effects by stimulating physiological changes.Our results offer a better understanding of the effects of Guasha on LDH.

大理野茄M239响应黄萎病病菌侵染的差异表达蛋白分析

为了探索植物响应黄萎病病原菌侵染的作用机制,以黄萎病高感野茄材料大理野茄(M239)为研究对象,通过人工接种黄萎病病原菌大丽轮枝菌的方法,首先测定M239在接种大丽轮枝菌后0,6,12,24,48 h的根部4个关键生理指标,即过氧化物酶(POD)、过氧化氢酶(CAT)、苯丙氨酸解氨酶(PAL)的活性和可溶性蛋白(SP)含量的变化。结果确定接种大丽轮枝菌后12,24 h为蛋白组分析取样的关键时间点;在此基础上,利用iTRAQ技术分析M239在接种大丽轮枝菌后根部蛋白的变化。结果发现,与清水接种(CK)相比,M239接种大丽轮枝菌后12 h有463个差异表达蛋白(DEPs),其中305个下调,158个上调;接种后24 h,有456个DEPs被病原菌诱导表达,包括296个下调,160个上调。这些蛋白主要富集在碳代谢、乙醛酸和二羧酸代谢、次生代谢物的生物合成、丙酮酸代谢、新陈代谢等途径。通过与已报道的黄萎病抗性材料的研究结果进行对比分析发现,M239在响应大丽轮枝菌的侵染过程中,参与防御响应的蛋白数量和代谢通路较少,未能形成有效的防御网络,最终表现为感病。

基于iTRAQ技术对低级别胶质瘤与正常脑组织差异性表达蛋白的初步筛选研究

目的应用同位素标记相对和绝对定量(iTRAQ)技术筛选低级别胶质瘤与正常脑组织的差异表达蛋白质。方法将6例低级别胶质瘤实体组织标本与2例正常脑组织标本各自混合后提取总蛋白,进行定量分析和酶解。iTRAQ标记后进行液相色谱-串联质谱(LC-MS/MS)分析。通过Mascot2.9软件进行相关图谱分析。结果有效鉴定出低级别胶质瘤与正常脑组织差异表达蛋白质162个,其中34个上调蛋白质,128个下调蛋白质,这些差异表达蛋白质具有多种生物学活性,参与不同的代谢以及信号通路。结论通过iTRAQ技术可有效鉴定出众多低级别胶质瘤与正常脑组织的差异表达蛋白质,为后续寻找低级别胶质瘤的生物标记物奠定基础。

iTRAQ-based quantitative proteome characterization of wheat grains during filling stages

Using isobaric tags for relative and absolute quantification （iTRAQ） and associated analytic technologies, we have cataloged and compared 7 069 unique wheat proteins expressed during four substages of the filling stage. Among them, 859 are differentially expressed, showing at least a 2-fold difference in concentration across substages. Differentially expressed proteins （DEPs） includind high-molecular weight giutenin subunit （W5AIU1）, low-molecular weight glutenin subunit （QSW3V4）, gliadin/avenin-like seed protein （D2KFG9）, and avenin-like protein （W5DVL2）, all of which have previously been identified as important for nutritional quality and bread-making properties, and all of which were found to increase at the latter stages of development. We have applied statistical techniques to group the proteins into hierarchical clusters, and have consulted databases to infer functional and other relationships among the identified proteins.

Inflammation and apoptosis accelerate progression to irreversible atrophy in denervated intrinsic muscles of the hand compared with biceps: proteomic analysis of a rat model of obstetric brachial plexus palsy

In treating patients with obstetric brachial plexus palsy,we noticed that denervated intrinsic muscles of the hand become irreversibly atrophic at a faster than denervated biceps.In a rat model of obstetric brachial plexus palsy,denervated intrinsic musculature of the forepaw entered the irreversible atrophy far earlier than denervated biceps.In this study,isobaric tags for relative and absolute quantitation were examined in the intrinsic musculature of forepaw and biceps on denervated and normal sides at 3 and 5 weeks to identify dysregulated proteins.Enrichment of pathways mapped by those proteins was analyzed by Kyoto Encyclopedia of Genes and Genomes analysis.At 3 weeks,119 dysregulated proteins in denervated intrinsic musculature of the forepaw were mapped to nine pathways for muscle regulation,while 67 dysregulated proteins were mapped to three such pathways at 5 weeks.At 3 weeks,27 upregulated proteins were mapped to five pathways involving inflammation and apoptosis,while two upregulated proteins were mapped to one such pathway at 5 weeks.At 3 and 5 weeks,53 proteins from pathways involving regrowth and differentiation were downregulated.At 3 weeks,64 dysregulated proteins in denervated biceps were mapped to five pathways involving muscle regulation,while,five dysregulated proteins were mapped to three such pathways at 5 weeks.One protein mapped to inflammation and apoptotic pathways was upregulated from one pathway at 3 weeks,while three proteins were downregulated from two other pathways at 5 weeks.Four proteins mapped to regrowth and differentiation pathways were upregulated from three pathways at 3 weeks,while two proteins were downregulated in another pathway at 5 weeks.These results implicated inflammation and apoptosis as critical factors aggravating atrophy of denervated intrinsic muscles of the hand during obstetric brachial plexus palsy.All experimental procedures and protocols were approved by the Experimental Animal Ethics Committee of Fudan University,China(approval No.DF-325)in January 2015.

同位素标记相对和绝对定量评价非创伤性股骨头坏死患者的血清蛋白组学分析

背景:非创伤性股骨头坏死是一类以髋关节疼痛、功能障碍进行性加重为主要症状的疾病,严重危害人体健康。它的发病机制、早期诊断和治疗仍是临床骨科医生所面临的重大挑战。目的:采用同位素标记相对和绝对定量(isobaric tags for relative and absolute quantitation,iTRAQ)技术,用蛋白质组学筛选可能与非创伤性股骨头坏死相关的关键蛋白。通过分析这些蛋白的表达特性及潜在的生物学功能,为进一步研究非创伤性股骨头坏死病因及分子机制奠定基础。方法:提取非创伤性股骨头坏死患者和健康人血清蛋白,应用iTRAQ技术标记蛋白,联合液相色谱和串联质谱分离和分析肽段。采用Proteome Discoverer 2.1软件对两组人群蛋白进行鉴定和定量分析,获得差异蛋白,再对这些表达差异蛋白进行GO注释、KEGG通路注释和功能富集聚类分析。结果与结论:①质谱鉴定共得到1006个蛋白,通过分析得到137个差异表达蛋白,其中54个蛋白表达上调,83个蛋白表达下调;②与非创伤性股骨头坏死发生相关的表达差异显著的蛋白质包括载脂蛋白B(ApoB)、载脂蛋白A2(ApoA2)、载脂蛋白E(ApoE)、补体C4和C7等;③KEGG信号通路富集分析结果显示,关键靶点的主要信号通路为p53信号通路、转录生长因子β信号通路、扩张型心肌病信号通路;④该结果提示,载脂蛋白与非创伤性股骨头坏死发生密切相关,并发现了与非创伤性股骨头坏死相关的活跃信号通路,为非创伤性股骨头坏死发生及分子机制奠定一定的实验基础。

基于iTRAQ技术对戊四氮点燃癫痫大鼠海马组织的蛋白质组学分析

目的:筛选癫痫大鼠海马组织差异表达蛋白(DEPs),为进一步探索癫痫的发病机制和药物治疗靶点提供思路。方法:采用戊四氮(PTZ)诱导建立SD大鼠癫痫模型(PTZ组),利用同位素标记相对和绝对定量(iTRAQ)联合LC-MS/MS技术检测大鼠海马组织蛋白质谱,以PTZ组对control组蛋白表达量变化倍数>1.5或<0.67且P<0.05为标准筛选DEPs,并对DEPs进行基因本体(GO)功能注释和京都基因与基因百科全书(KEGG)通路富集等生物信息学分析。结果:共筛选出80个显著DEPs,其中上调39个,下调41个。GO分析显示:上调的DEPs主要涉及细胞对神经生长因子刺激的反应、轴突发育、细胞表面受体信号通路、神经元迁移、肌动蛋白细丝解聚和信号转导等生物过程;下调的DEPs主要涉及三羧酸循环、柠檬酸盐代谢过程、丙酮酸-乙酰辅酶A生物合成过程、草酰乙酸代谢过程、粘附聚集体的调节等生物过程。KEGG通路富集分析显示:上调的DEPs主要参与腺苷酸活化蛋白激酶(AMPK)信号通路、肌动蛋白细胞骨架的调节、鞘脂信号通路、苯丙氨酸代谢和胰岛素信号通路等5条信号通路;下调的DEPs主要参与柠檬酸循环、碳代谢、乙醛酸和二羧酸代谢、代谢途径、氨基酸的生物合成和肌萎缩侧索硬化症等6条信号通路。结论:本研究通过iTRAQ蛋白质组学方法筛选出了癫痫海马组织的DEPs,对DEPs进行生物信息学分析所富集的代谢通路可能与癫痫的发病密切相关。

Lactobacillus gasseri LA39 promotes hepatic primary bile acid biosynthesis and intestinal secondary bile acid biotransformation

A growing body of evidence has linked the gut microbiota to liver metabolism.The manipulation of intestinal microflora has been considered as a promising avenue to promote liver health.However,the effects of Lactobacillus gasseri LA39,a potential probiotic,on liver metabolism remain unclear.Accumulating studies have investigated the proteomic profile for mining the host biological events affected by microbes,and used the germ-free(GF)mouse model to evaluate host-microbe interaction.Here,we explored the effects of L.gasseri LA39 gavage on the protein expression profiles of the liver of GF mice.Our results showed that a total of 128 proteins were upregulated,whereas a total of 123 proteins were downregulated by treatment with L.gasseri LA39.Further bioinformatics analyses suggested that the primary bile acid(BA)biosynthesis pathway in the liver was activated by L.gasseri LA39.Three differentially expressed proteins(cytochrome P450 family 27 subfamily A member 1(CYP27A1),cytochrome P450 family 7 subfamily B member 1(CYP7B1),and cytochrome P450 family 8 subfamily B member 1(CYP8B1))involved in the primary BA biosynthesis pathway were further validated by western blot assay.In addition,targeted metabolomic analyses demonstrated that serum and fecalβ-muricholic acid(a primary BA),dehydrolithocholic acid(a secondary BA),and glycolithocholic acid-3-sulfate(a secondary BA)were significantly increased by L.gasseri LA39.Thus,our data revealed that L.gasseri LA39 activates the hepatic primary BA biosynthesis and promotes the intestinal secondary BA biotransformation.Based on these findings,we suggest that L.gasseri LA39 confers an important function in the gut‒liver axis through regulating BA metabolism.

百草枯中毒大鼠的GO及KEGG分析

目的探讨百草枯中毒后机体蛋白质表达变化及受影响的信号通路。方法采用同位素标记相对和绝对定量技术检测百草枯中毒大鼠肺组织中差异表达蛋白,对差异表达蛋白进行基因本体论(gene ontology,GO)功能富集分析和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)分析。结果百草枯中毒大鼠肺组织中发现84种差异蛋白,其中48种上调,36种下调。在百草枯中毒早期,差异蛋白主要存在于细胞外区域,主要涉及代谢和应激,补体和凝血级联通路、2-氧代羧酸代谢途径、神经活性配体-受体相互作用途径和金黄色葡萄球菌感染途径4条信号通路受到明显影响。结论差异蛋白和信号转导通路在百草枯致肺损伤早期即可产生变化,可能与炎症损伤和肺纤维化有关。

应用iTRAQ质谱分析技术对小鼠精原干细胞标志物进行动态研究与筛选

目的:利用i TRAQ蛋白质质谱分析技术检测各年龄阶段小鼠精原干细胞(SSCs)蛋白的表达,进一步分析SSCs标志物表达的动态变化,运用生物信息学蛋白质组数据库筛选并发现新的标志物。方法:以健康雄性C57BL/6小鼠为研究对象,按照鼠龄不同分为8组,无菌提取各组小鼠睾丸组织,采用复合酶消化、免疫磁珠分选(MACS)的方法纯化SSCs后提取蛋白,利用双向电泳及蛋白质质谱分析技术并结合蛋白质数据库对各组蛋白质进行分析鉴定。结果:质谱实验共得到谱图248 510张,通过Mascot软件进行分析,共鉴定到1 132种蛋白。以蛋白丰度差异倍数达到1.2倍以上,且经统计学检验P<0.05视为存在差异性蛋白,8组共检测到差异蛋白298种。鉴定到目前已知的SSCs标志物9种(PCNA、GFRα1、CDH1、Annexin A7、UCHL1、VASA、CD49f、CD29、PLZf),通过各组对比获得在不同鼠龄段内表达变化趋势,其中GFRα1、CD49f、CD29在SSCs中的变化趋势与以往文献一致,并筛选出10种蛋白质(P63、CD71、CD98、K19、ACE、K18、K15、K17、SH2、SH3)作为SSCs标志物进一步研究的对象。结论:SSCs内的蛋白随着小鼠鼠龄的变化表达存在差异,i TRAQ蛋白质质谱分析技术可以对SSCs蛋白质组学信息进行分析与比较,获得各鼠龄阶段差异性表达蛋白,为进一步分析和研究这些差异性蛋白的功能与作用提供了新的方法。

应用iTRAQ技术筛选先天性心脏畸形胎儿母亲血清差异表达蛋白

目的筛选先天性心脏畸形(CHD)母体血清差异表达蛋白质,明确和CHD相关的候选标志物。方法收集40例CHD胎儿(实验组)母亲血清和10例正常胎儿(对照组)母亲血清,其中实验组包括法洛氏四联征10例,室间隔缺损10例,共同动脉干10例,其他少见先天性心脏畸形10例。每组10例标本等体积混合后,应用同位素标记相对和绝对定量技术(iTRAQ)对蛋白质进行鉴定和相对定量。结果孕妇外周血血清中共鉴定到蛋白质606个,实验组与对照组差异达到1.5倍以上的蛋白质47个,其中23个在实验组中上调,24个在实验组中下调。结论基于i TRAQ技术的蛋白质组学方法能鉴定出多种CHD相关的差异蛋白,CHD发生是一个多种蛋白质分子参与的结果。

空间环境诱导褪色沙雷菌LCT-SM166的蛋白质组学分析

目的分析和鉴定神舟八号飞船搭载褪色沙雷菌的差异表达蛋白质。方法对神州八号飞船搭载的褪色沙雷菌LCT-SM166及地面对照组LCT-SM213进行分离并进行16sRNA鉴定,采用同重同位素相对与绝对定量(isobaric tags for relativeand absolute quantitation,iTRAQ)方法对褪色沙雷菌LCT-SM166和LCT-SM213进行蛋白质组质谱检测,分析两株菌的差异表达蛋白。结果共鉴定到1 713个蛋白质,LCT-SM166与LCT-SM213的差异蛋白组学分析发现了111个蛋白质表达的改变,其中29个蛋白表达上调,91个蛋白表达下调。基因本体论(gene ontology,GO)功能富集分析显示大多数差异蛋白质主要与能量代谢有关。结论空间环境可影响褪色沙雷菌大量蛋白质的表达,差异表达蛋白主要分布在代谢相关过程。空间诱变菌株的蛋白质组学研究为后续贯穿组学的研究奠定基础。

基于iTRAQ蛋白质组学技术对前列腺癌血清标志物的筛查

目的:应用同位素标记相对和绝对定量(iTRAQ)蛋白质组学技术筛选前列腺癌血清中的差异表达蛋白质,提供新的候选标志物。方法:收集4组患者的外周血清标本:良性前列腺增生(BPH)(n=10)、高级别前列腺上皮内瘤变(HGPIN)(n=10)、局限性前列腺癌(localized PCa)(n=10)以及伴有远处转移的前列腺癌(metastatic PCa)(n=10)。每组10例患者的血清样本进行等体积混合后,应用iTRAQ技术联合液相串联质谱分析(LC-ESI-MS/MS)对蛋白质进行鉴定和相对定量。结果:在患者外周血清中共鉴定到蛋白质825个。相对于良性前列腺增生,在前列腺癌组中表达差异在1.2倍以上的蛋白质13个,其中9个表达上调,4个表达下调。结论:基于iTRAQ技术的蛋白质组学方法有助于鉴定出前列腺癌相关的差异蛋白质,为进一步探索前列腺癌肿瘤标志物提供了新的思路和线索。

基于iTRAQ技术的血清蛋白质组学研究进展

随着分子生物学的快速发展,血清蛋白质组学成为研究的热点。相对和绝对定量同位素标记(i TRAQ)技术没有出现之前大部分研究主要在双向凝胶电泳技术的支持下完成,但是该技术具有费时、费力、重复性差等缺点,不能完全避免和消除,人们就注入了新的研究方法—i TRAQ技术,该技术具有灵敏度高、分辨率高、重复性好、易自动化和检测范围广等诸多优点,使该技术在医学和生物制药领域受到高度关注和广泛应用。该文就最近几年基于i TRAQ技术的血清蛋白质组学研究进展、标志物的探索等相关问题进行综述。

iTRAQ技术鉴定小鼠睾丸发育差异表达蛋白的研究

目的用同量异位素标记的相对和绝对定量(isobaric tags for relative and absolute quantitation,iTRAQ)技术结合液相色谱质谱技术构建3周龄小鼠与成年小鼠睾丸蛋白差异表达谱系。方法分别抽提3周龄小鼠与成年小鼠睾丸总蛋白质,iTRAQ标记,液相色谱(LC)分离蛋白质,LTQ OrbitrapTM质谱仪进行蛋白质鉴定。结果鉴定出20个差异表达蛋白质,其中18个为3周龄小鼠睾丸高表达蛋白质,2个为成年睾丸高表达蛋白质。差异表达蛋白质主要参与细胞的有丝分裂、减数分裂、细胞增殖、分化、凋亡及精子发生等重要细胞事件。结论用目前最新的定量蛋白质组技术,构建的3周龄小鼠睾丸与成年睾丸差异表达蛋白谱系对研究睾丸功能、精子发生有一定理论价值,为定量蛋白组学技术在雄性生殖领域的应用奠定了基础。

应用同位素标记相对和绝对定量技术筛选白厚苔和黄厚苔乳腺癌患者唾液差异表达蛋白

目的:筛选乳腺癌白苔和黄苔患者与健康对照组之间的唾液差异表达蛋白,以寻找乳腺癌早期诊断的生物标记物,探索中医舌苔形成的机制。方法:纳入具有典型的白厚苔或黄厚苔的乳腺癌患者以及薄白苔健康人各10例。采集唾液提取蛋白,然后进行蛋白变性、还原及酶解,最后进行同位素标记相对和绝对定量(isobaric tags forrelative and absolute quantitation,i TRAQ)标记和液相色谱串联质谱联用技术鉴定,得到的峰图用Data Analysis4.0,Mascott2.2软件和Scaffold软件分析,得出表达量差异结果。鉴定蛋白的组间比值>1.5或<0.6被认为存在表达差异。结果:共鉴定出464个蛋白,其中达到严格定量标准的蛋白125个。与健康对照组比较,乳腺癌白苔组和黄苔组共筛选出9个唾液差异表达蛋白,乳腺癌白苔组和黄苔组之间筛选出16个差异蛋白。结论:本研究证实i TRAQ结合液相色谱串联质谱联用技术能够快速、有效地进行差异蛋白质组学研究,发现一些蛋白与乳腺癌的发生以及中医舌苔形成机制相关。

利用转录组及iTRAQ技术筛选高油酸油菜抗病相关基因

高油酸油菜和低油酸油菜对菌核病的抗性存在显著差异,为了弄清其分子机理,以一组高油酸油菜近等基因系自交授粉后20-35 d的种子为材料,分别进行转录组和同位素相对标记与绝对定量技术分析。分析了与抗病相关的氧化磷酸化、植物激素信号转导通路和植物病原体互作3个分类,将注释的基因和蛋白进行关联,并对其中可能与抗病相关的基因进行定量PCR验证。结合前人研究发现,基因表达或蛋白表达发生显著变化的基因gi｜260505503（多聚半乳糖醛酸酶抑制蛋白）、gi｜226346102（HSR203J类蛋白）、gi｜470103214（钙调蛋白类）与抗病相关;而gi｜297843222（结合蛋白）、gi｜18397961（2-铁,2-硫-铁氧化还原类蛋白）、gi｜196052306（还原型烟酰胺腺嘌呤二核苷酸脱氢酶亚基）、gi｜18423437（还原型烟酰胺腺嘌呤二核苷酸脱氢酶（辅酶）1α-复形5）和gi｜297794581（激酶家族蛋白）等基因差异显著。