突触蛋白质组学分析中双向电泳技术的优化

背景：双向电泳是突触蛋白质组学分析中最流行最通用的蛋白质分离方法之一。但文献中突触蛋白双向电泳对线性固相pH梯度(immobilized pH gradients,IPG)胶条和十二烷基磺酸钠聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electroph-oresis,SDS-PAGE)浓度的选择很多,尚未见统一标准。 目的：对突触蛋白双向电泳的IPG胶条和SDS-PAGE凝胶浓度进行优化,以获得高质量的突触蛋白双向电泳图谱。 方法：以大鼠海马突触蛋白为试材,比较pH 5.0-8.0与pH 3.0-10.0线性IPG胶条,线性与非线性pH 3.0-10.0 IPG胶条,以及单一浓度10%与12% SDS-PAGE对双向电泳的影响。此外,还使用计算机检索了1989至2013年中国知网及PubMed数据库中关于突触蛋白双向电泳的文献,对文献中选择的IPG胶条、SDS-PAGE凝胶浓度进行了统计和评价,并总结了突触蛋白在不同pH值IPG胶条和不同浓度SDS-PAGE凝胶的双向电泳图谱上的分布特征。 结果与结论：结果表明,使用pH 3.0-10.0的非线性胶条及单一浓度10% SDS-PAGE凝胶进行突触蛋白双向电泳较为适宜,电泳图谱质量好,实验操作便利;同时还推荐合并使用pH 4.0-7.0和pH 6.0-11.0的IPG胶条,以及线性梯度浓度9%-16% SDS-PAGE凝胶,也适用于突触蛋白双向电泳分析。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

人晶状体上皮细胞蛋白质组的双向电泳和质谱鉴定

目的： 建立人晶状体上皮细胞蛋白质组研究体系，探讨双向电泳和质谱鉴定技术在人晶状体上皮细胞蛋白质组研究中的作用。 方法: 体外培养人晶状体上皮细胞株，用两种不同方法提取总蛋白，进行固相pH梯度(IPG)等电聚焦双向凝胶电泳，凝胶通过GS-800扫描仪（Bio-Rad）获取图像并使用PDQuest专业图像分析软件分析。在此基础之上，胰酶消化蛋白质斑点并进行质谱分析。 结果: 获得了重复性较好的人晶状体上皮细胞蛋白质组电泳图谱。晶状体蛋白质斑点在等电点pH值为4-7、相对分子质量为17-72 kD之间均有分布。其中高丰度蛋白点主要分布于分子量19-50 kD、PI 5-7范围内。2个蛋白点通过质谱分析和数据库的检索得到了初步的鉴定。 结论: 建立起了一个稳定的分析人晶状体上皮细胞蛋白质组学实验体系；为进一步研究人类晶状体在生理状态及白内障等病理条件下的改变提供了蛋白质组学的研究方法和途径。     

发热大鼠下丘脑组织中蛋白质组双向电泳图谱初步分析

目的：初步观察酵母性发热大鼠下丘脑组织中蛋白质的差异表达，为进一步探讨发热的分子机制提供依据。方法：应用双向电泳技术，对正常大鼠和酵母发热大鼠下丘脑组织中蛋白质表达进行比较，观察其差异表达。结果：发热模型大鼠与正常大鼠比较，下丘脑蛋白表达有明显差异，差异蛋白数量约占可分辨蛋白点的3.8%。其中9种蛋白表达强于对照，4种蛋白表达低于对照，1种蛋白等电点发生了改变，1种蛋白仅见于酵母发热大鼠。结论：发热与正常大鼠下丘脑组织中所表达的差异蛋白，是否是酵母介导大鼠体温升高的相关蛋白或特异蛋白，值得验证。     

SV40转化的人胃黏膜上皮细胞与胃癌细胞间的比较蛋白质组学研究

目的　研究猴病毒 4 0 (SV4 0 )转化的人胃黏膜上皮细胞GES 1与人胃癌细胞MGC 80 3的差异蛋白质组。方法　运用固相pH梯度双向凝胶电泳技术比较两者差异蛋白 ,差异蛋白用胰酶进行胶内酶切 ,肽混合物使用基质辅助激光解吸 /电离飞行时间质谱仪 (MALDI TOF MS)进行质谱分析 ,将肽质量指纹谱数据输入互联网上的蛋白质数据库进行检索。结果　获得 17个GES 1和MGC 80 3细胞间表达差异蛋白质的明确信息 ,涉及细胞骨架、细胞调控、细胞凋亡和生物氧化等种类蛋白质。结论　SV4 0转化的人胃黏膜上皮细胞GES 1与人胃癌细胞MGC 80 3间的表达蛋白具有差异 ,这些差异蛋白分析有助于深入研究胃癌发生发展机制 ,进而为胃癌的早期诊断和防治提供依据     

The apoE polymorphism studied by two-dimensional, high-resolution gel electrophoresis of serum

The apoE polymorphism of human very low density lipoprotein (VLDL) was studied by the two-dimensional high-resolution gel electrophoresis technique of O'Farrell, which combines isoelectric focusing and SDS-polyacrylamide gel electrophoresis. With whole serum, six major patterns and two "sub-variants" of apoE were observed. A series of twin pairs (21 monozygotic (MZ) and 13 dizygotic (DZ) pairs) as well as unrelated people were analyzed. Both members of MZ pairs always exhibited the same pattern, whereas DZ twins were often discordant. The patterns observed would be consistent with the hypothesis that three apoE isopeptides are coded for by three different alleles at one single locus. In this small series, all three postulated homozygous patterns, namely apoE-II, apoE-III and apoE-IV as well as the three heterozygous patterns apoE-11, 111; apoE-III, IV and apoE-ll, IV were seen.     

恶性胶质瘤细胞SHG-44诱导分化后的差异蛋白质组双向电泳分析

目的用双向电泳分析诺帝诱导胶质瘤细胞SHG-44分化后的差异蛋白质组,为进一步了解这些差异蛋白质的作用打下基础。方法将诺帝诱导胶质瘤细胞SHG-44分化后的总蛋白及其相应的空白对照组细胞总蛋白进行双向电泳分离,重复3次后用PDQuest7.1软件比较分析蛋白质表达差异并获得差异蛋白质的相对分子质量、等电点等信息。结果诺帝诱导胶质瘤细胞SHG-44分化后有23个差异蛋白点,其中21个蛋白点表达下调,2个蛋白点表达上调。结论诺帝诱导胶质瘤细胞SHG-44分化后大部分蛋白质表达下调,推测诺帝诱导胶质瘤细胞SHG-44分化时蛋白表达以抑制作用为主。     

Application of two-dimensional electrophoresis in the research of retinal proteins of diabetic rat

Diabetes mellitus （DM） is a chronic disease which is associated with numerous serious health complications such as diabetic retinopathy, and is the leading cause of new cases of blindness in adults at the age of 20-74 years old. The aim of the study was to establish and optimize a two-dimensional polyacrylamide gel electrophoresis （2-DE） technique for retina proteomics to improve the resolution and reproducibility, and to observe the proteomic changes of retinal tissues in diabetic and normal rats. Proteins were extracted from retinal tissues of normal and 8 weeks diabetic SD rats and used in two-dimensional electrophoresis. Various conditions of retina proteomic 2-DE were adjusted, optimized and protein spots of differential expression were obtained through analysis of 2-DE images with PDQuest software. By choosing appropriate sample amount, using pre-cast IPG dry strips （pH 5-8） and casting 12% equal gel, satisfactory 2-DE images of retina were obtained and a steady 2-DE technique was established. In this way, we found 36 spots in 2-DE gel of diabetic retinas that exhibited statistically significant variations, including up-regulation of 5 proteins in diabetic rat retinas, down-regulation of 23, and disappearance of 8, in comparison with normal tissues. The differences of protein expression were observed in retinas between diabetic and normal rats. Our established 2-DE technique of retina proteins could be effectively applied in proteomics of retina diseases. Cellular ＆ Molecular Immunology. 2007;4（1）：65-70.     

Immunohistochemical studies on human gastric mucosa

Summary Two different fixatives were applied to human gastric mucosa for the study of antigenic marker substances. The first consists of 96% ethanol and 1% acetic acid (EA method), the second of 4% formaldehyde, 0.5% picric acid and 0.25% glutaraldehyde (FPG method). Samples of resected gastric specimens were fixed, dehydrated and cleared in benzene and embedded in paraplast. The morphology of gastric tissue was well preserved by both methods and permitted the simultaneous application of classical staining procedures and the immunoenzyme peroxidase technique for the demonstration of antigenic substances. The following marker substances could be demonstrated: Pepsinogen I and II group, surface epithelial antigen, parietal cell antigen, chief cell antigen, antral mucous cell antigen, carcinoembryonic antigen, goblet cell antigen and common site antigen of leucocytes. Various factors responsible for nonspecific reactions, such as endogeneous peroxidase activity and protein-protein interactions were studied. The latter were circumvented by the use of highly purified antibodies or immunoglobulin fractions. The EA method proved to be the method of choice for future routine application of combined classical histology and immunoenzyme histology in gastric and intestinal diseases.Supported by grants of the Ministry of Research and Technology, Bonn, Germany     

A two-dimensional electrophoresis reference map of human ovary

The ovary plays a central role in oogenesis and gonadal hormone secretion. Proteomic analysis is a valuable approach for gaining an increased understanding of the molecular nature of the ovary. In this work, two-dimensional electrophoresis for protein separation followed by matrix-assisted laser desorption/ionization mass spectrometry and database searches, identified 231 protein spots corresponding to 138 individual proteins that were found in gels representing both the follicular and luteal phases. The data were used to construct a database online (). The identified proteins were functionally classified into seven groups: (1) cell signaling/communication, (2) cell division, (3) gene/protein expression, (4) metabolism, (5) cell structure and motility, (6) cell/organism defense, and (7) unclassified. Among the proteins identified, 47% had not been previously reported in the human ovary. In addition, a number of disease-related proteins were identified in this protein map, including some cancer- and polycystic ovarian syndrome-related proteins. Two proteins with phosphorylation were verified by Western blot analysis. Comparison of protein abundance between follicular and luteal stages produced seven protein spots that had been identified in our database. This study provides a preliminary reference map of normal human ovary that will form a basis for comparative studies on normal and pathological conditions of the human ovary and may serve as a potential tool for clinical diagnosis, therapeutics, and prognosis.Electronic Supplementary Material Supplementary material is available in the online version of this article at L. Wang and Y.-F. Zhu contributed equally to this work     

双向凝胶电泳脑脊液蛋白质提取方法的比较

 目的 对 3 种常用的双向凝胶电泳脑脊液蛋白质提取方法进行比较，优选脑脊液蛋白质提取方法。 方法 收集新诊断尚未经药物治疗的 16 例帕金森病患者和 7 例头痛患者的脑脊液，分别以 3 种不同的方法提取脑脊液中的蛋白质。①透析法：用 PluseOne 微透析试剂盒处理脑脊液样品。②丙酮沉淀法：以冷丙酮溶液处理脑脊液样品，丙酮的终浓度为 80%。③三氯醋酸（TCA）/丙酮沉淀法：以 4 倍体积的 10% TCA/丙酮溶液处理脑脊液样品，TCA 的终浓度为 8%，丙酮的终浓度为 80%。取沉淀物进行双向凝胶电泳，用银染法对电泳后的凝胶进行染色，比较经 3 种不同方法提取的脑脊液蛋白质的双向凝胶电泳图谱。 结果 经透析法处理样品的电泳图谱显示的蛋白点较清晰、较圆，条纹较少，但所见几乎都是高丰度蛋白，低丰度蛋白很难显现；经丙酮沉淀法处理样品的电泳图谱显示横竖条纹均较多，大部分蛋白堆积在凝胶的上部，下面的点也显示不清晰，蛋白点出现横向漂移；经 TCA/丙酮沉淀法处理样品的电泳图谱显示横条纹相对较少，低丰度蛋白能较清晰显现，样品中所含高丰度蛋白明显少于以上两种方法处理的样品。 结论 以 TCA/丙酮沉淀法提取蛋白质进行双向凝胶电泳时聚焦效果较好，而且同时去除了大部分白蛋白，使低丰度蛋白能很好地显现出来，优于丙酮沉淀法和透析法。