iTRAQ-based quantitative proteomic analysis of transgenic and non-transgenic maize seeds

Proteomics provides a powerful approach to evaluate the unintended effects of transgenic crops. In this study, iTRAQ (isobaric tags for relative and absolute quantitation)-based quantitative proteomics was applied to estimate the differences in the proteomic profiles of maize seeds from 3 natural genotypic varieties and 4 genetically modified (GM) lines. Compared with their isogenic controls, there were 108, 69, 180 and 204 differentially expressed proteins (DEPs) in GM maize C0030.2.4, C0030.3.5, C0010.1.1 and C0010.3.1 seeds. Molecular functional classification showed that these DEPs were mainly involved in catalytic activity and binding. KEGG pathway annotation showed that most of these DEPs participated in metabolic pathways, the biosynthesis of secondary metabolites and microbial metabolism in diverse environments. In addition to the foreign protein EPSPS, 4 maize proteins were simultaneously identified as being differentially expressed among the 4 GM maize lines. Nevertheless, the regulation trends of these DEPs were not exactly consistent among the different GM maize lines. The data also showed that the differences in protein expression among the different natural genotypic varieties were greater than those caused by genetic modification. These results provide new information regarding the unintended effects of GM maize seeds through iTRAQ-based quantitative proteomic analysis.     

iTRAQ技术筛选人黑素瘤A375细胞系let-7a靶标蛋白

目的 同位素标记相对和绝对定量（iTRAQ）技术在人黑素瘤A375细胞系中筛选let-7a潜在的靶标蛋白,探讨let-7a的抑癌机制。 方法 100 nmol/L let-7a模拟物及其对照转染A375细胞,提取细胞总蛋白,运用同位素标记的iTRAQ技术分析和鉴定差异表达的蛋白质,运用生物信息学分析let-7a靶标蛋白及其功能,采用双荧光素酶报告系统验证靶标。 结果 let-7a模拟物组和对照组相比,质谱共分析鉴定到327个显著差异蛋白,其中表达量上调 > 1.2倍的蛋白有151种,下调 < 0.8倍的蛋白有176种。下调的176种蛋白中,软件预测到结合靶标有47个,双荧光素酶报告系统验证表明,let-7a模拟物组与对照组相比,HMGA2和THOC2野生型3′UTR比值分别降低64.3%和46.4%。 结论 iTRAQ技术和生物信息学方法在人黑素瘤A375细胞系中筛选出47个let-7a候选靶标蛋白,其中HMGA2和THOC2分别为let-7a的靶标。     

应用iTRAQ多重化学标记串联质谱技术筛选晚期卵巢浆液性腺癌组织中卡铂耐药相关蛋白

目的 利用同位素标记相对和绝对定量(iTRAQ) 技术筛选晚期卵巢癌组织中卡铂耐药相关差异表达蛋白,为临床个体化治疗奠定实验基础。方法 收集Ⅲ期低分化卵巢浆液性腺癌标本并通过ATP-TCA药敏试验检测卡铂敏感度。取卡铂敏感和耐药标本各15例, iTRAQ试剂标记,被标记的肽段进行高效液相色谱（HPLC）分离及质谱检测（MS）。结果 共鉴定出iTRAQ标记定量信息有755个显著差异表达的蛋白。卡铂耐药组与卡铂敏感组相比,上调1.2倍以上的蛋白429个;下调0.83倍以下的蛋白326个。上调蛋白中有osteopontin（OPN）、clusterin（CLU）、5'-nucleotidase（CD73）和tissue inhititor of metalloproteinases 1（TIMP1）等18个蛋白与肿瘤恶性行为及化疗耐药相关。结论 应用iTRAQ技术能筛选出多种与晚期卵巢癌卡铂耐药相关的差异表达蛋白,该技术对于肿瘤组织蛋白质组学的研究有很好的应用前景。     

应用iTRAQ技术筛选乳腺癌白苔和黄苔患者血清差异表达蛋白

目的应用同位素标记相对和绝对定量（iTRAQ）技术筛选乳腺癌白苔和黄苔患者与正常组之间的血清差异表达蛋白,以寻找乳腺癌早期诊断的生物标记物和探索中医舌苔形成的机制和原理。方法临床收集乳腺癌患者中具有典型的白厚苔和黄厚苔者各10例,并以正常薄白苔者10例作为对照,采血取血清提取蛋白,然后进行蛋白变性、还原及酶解,最后进行iTRAQ标记和ESI-QTOF-QⅡ质谱仪鉴定,得到峰图用DATA ANALYSIS、MASCOTT 2.2搜库和Scaffold软件分析,得到表达量差异结果。结果共鉴定出335个蛋白,其中达到严格定量标准的蛋白数59个,与正常组比较,乳腺癌白苔组和黄苔组共筛选出11个血清差异表达蛋白,乳腺癌白苔组和黄苔组之间筛选出6个差异蛋白。结论 iTRAQ结合LC-MS/MS技术能够快速、有效地进行差异蛋白质组学研究,同时发现一些蛋白与乳腺癌的发生以及中医舌苔形成机制密切相关。     

益胃汤对初老雌性大鼠下丘脑作用的差异蛋白分析

[目的]研究益胃汤对初老雌性大鼠下丘脑产生调节作用的相关蛋白质,在分子水平上探索益胃汤的作用机制。[方法]将20只4～6月龄雌性大鼠随机分成正常组、去势组,每组10只,10只10～12月龄雌性大鼠作为初老组。给药结束后,取3组大鼠下丘脑组织,运用同位素标记相对和绝对定量技术(iTRAQ)分离、分析、鉴定大鼠下丘脑差异蛋白。[结果]分析比较初老组、去势组、正常组3组大鼠下丘脑蛋白质,通过多组数据韦恩图分析发现初老组与去势组有17种共同表达的差异蛋白。通过火山图(Volcano Plot)过滤,确定显著差异蛋白[差异倍数(FC)1.5或2/3,P0.05];筛选出了7种与衰老相关的显著差异蛋白及其相关KEGG通路,它们分别为:plasminogen、α-crystallin B chain、Hsp70 binding protein 1、serum transferrin、unconventional myosin-Id、myelin oligodendrocyte glycoprotein、protein Fbxo44。[结论]益胃汤对这7种显著差异蛋白起到上调或下调的作用,为研究益胃汤延缓衰老作用的靶蛋白奠定了基础。     

Investigation into the pulmonary inflammopathology of exposure to nickel oxide nanoparticles in mice

We investigated the effects of nickel oxide nanoparticles (NiONPs) on the pulmonary inflammopathology. NiONPs were intratracheally installed into mice, and lung injury and inflammation were evaluated between 1 and 28 days. NiONPs caused significant increases in LDH, total protein, and IL-6 and a decrease in IL-10 in the BALF and increases in 8-OHdG and caspase-3 in lung tissues at 24 h. Airway inflammation was present in a dose-dependent manner from the upper to lower airways at 24 h of exposure as analyzed by SPECT. Lung parenchyma inflammation and small airway inflammation were observed by CT after NiONP exposure. 8-OHdG in lung tissues had increased with formation of fibrosis at 28 days. Focal adhesion was the most important pathways identified at 24 h as determined by protemics, whereas glutathione metabolism was the most important identified at 28 days. Our results demonstrated the pulmonary inflammopathology caused by NiONPs based on image-to-biochemical approaches.     

稳定同位素iTRAQ标记联合液相色谱-串联质谱法定量分析药物性肝损伤患者血清氨基酸的变化

目的探索药物性肝损伤(DILI)患者治疗的不同阶段血清氨基酸水平的变化差异,探讨其临床意义及可用于DILI早期诊断及疗效评价的氨基酸类生物标志物。方法收集21例健康志愿者(健康组)及24例DILI患者不同阶段(初诊、治疗、转归)的血清标本,采用稳定同位素iTRAQ标记方法联合液相色谱串联质谱技术检测血清氨基酸进行定量并分析其差异。结果在血清中共定量氨基酸28种,与健康组比较,DILI患者共有14种氨基酸浓度在诊疗不同阶段发生显著性改变;其中异亮氨酸、亮氨酸、苯丙氨酸及丝氨酸的浓度在初诊时显著升高,随治疗进程降低,逐渐接近于正常水平。结论 4种氨基酸中,苯丙氨酸与DILI的相关性更高,可能为DILI早期诊断及治疗效果评价的潜在生物标志物。     

基于iTRAQ标记技术研究慢性浅表性胃炎脾虚证的唾液差异表达蛋白

目的 运用iTRAQ定量蛋白质组学技术筛选慢性浅表性胃炎(chronic superficial gastritis,CSG)不同中医证候患者唾液中的潜在特异性生物标志物,探索一种客观化、规范化的中医辨证方法 .方法采用iTRAQ定量蛋白质组学技术及后期生物信息学方法,筛选CSG脾虚证28例与CSG湿热证28例对比以及与正常对照组28例对比的唾液差异蛋白质分析.结果 CSG脾虚证对比CSG湿热证,Ratio≥1.5的共有128个蛋白,Ratio≤0.667的共有39个蛋白;CSG脾虚证对比正常对照组Ratio≥1.5的共有113个蛋白,Ratio≤0.667的共有72个蛋白.筛选出多个重要蛋白,如Keratin家族、ANX家族和apolipoprotein家族中的多种亚型以及γ-谷氨酸转移酶等,可能与CSG不同中医证候的发生、发展有密切的关系.结论 iTRAQ唾液蛋白质组技术筛选出了多个具有潜在价值的CSG中医证候无创诊断的重要蛋白,值得进一步深入研究.     

Successful scleral buckling for long-standing retinal detachment with subretinal proliferation 4-year after strabismus surgery

AIM: To proliminarily test proteomics in aqueous humor in patients with dry age-related macular degeneration (AMD) by using the proteomic technology. METHODS: Aqueous humor samples were collected from patients with or without dry AMD, who underwent cataract surgery. The aqueous samples were analyzed with isobaric tags for relative and absolute quantitation (iTRAQ) combined with liquid chromatography tandem mass spectrometry (LC-MS/MS) technology. The differential expressed proteins were analyzed with gene ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) and protein-protein interaction (PPI) network analysis. The data were partly validated by ELISA and Western blot. False discovery rate was used for statistical analysis. RESULTS: A total of 244 proteins were detected, in which 38 proteins were up-regulated and 51 were down-regulated significantly in patients with dry AMD compared with that in control groups (FDR value <1.0%). Several proteins, e.g., protein S100-A8 (S10A8), dystroglycan (DAG1), Ig alpha-1 chain C region (IGHA1), carbonic anhydrase 3 (CAH3) and alpha-1-acid glycoprotein (A1AG1) were increased more than 5 times of that in control group. The bioinformatics analysis showed that dry AMD is closely associated with inflammation or immune reaction, oxidative stress, blood coagulation and remodeling of extracellular matrix. CONCLUSION: iTRAQ-based proteomic analysis of aqueous humor demonstrated the differential expressions of proteins between dry AMD and control groups, providing the clues to understand the mechanisms and possible treatments of dry AMD.     

Proteomic analysis using isobaric tags for relative and absolute quantification technology reveals mechanisms of toxic effects of tris (1,3-dichloro-2-propyl) phosphate on RAW264.7 macrophage cells

Tris (1,3-dichloro-2-propyl) phosphate (TDCIPP) is one of the most commonly used organophosphorus flame retardants. Immuno-toxicity induced by TDCIPP is becoming of increasing concern. However, effects of TDCIPP on immune cells and mechanisms resulting in those effects are poorly understood. In this study, it was determined, for the first time, by use of isobaric tags for relative and absolute quantification (iTRAQ) based proteomic techniques expression of global proteins in RAW264.7 cells exposed to 10 μM TDCIPP. A total of 180 significantly differentially expressed proteins (DEPs) were identified. Of these, 127 were up-regulated and 53 were down-regulated. The DEPs associated with toxic effects of TDCIPP were then screened by use of Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes for enrichment analysis. Results showed that these DEPs were involved in a number of pathways including apoptosis, DNA damage, cell cycle arrest, immune-toxicity, and signaling pathways, such as the Toll-like receptor, PPAR and p53 signaling pathways. The complex regulatory relationships between different DEPs, which might play an important role in cell death were also observed in the form of a protein–protein interaction network. Meanwhile, mitochondrial membrane potential (MMP) in RAW264.7 cells after TDCIPP treatment was also analyzed, the collapse of the MMP was speculated to play an important role in TDCIPP induced apoptosis. Moreover, some of the important regulator proteins discovered in this study, such as Chk1, Aurora A, would provide novel insight into the molecular mechanisms involved in toxic responses to TDCIPP.