水稻稻瘟菌抗性相关蛋白的双向电泳分析

建立抗感水稻品种受稻瘟菌侵染和未侵染蛋白质的双向凝胶电泳图谱,分析其差异表达的蛋白,寻找稻瘟病的抗性相关蛋白,以阐明稻瘟病的发病机制。采用TCA/丙酮沉淀法提取四种愈伤组织材料的总蛋白并采用固相pH梯度( immobilized pH gradient, IPG)双向凝胶电泳( two-dimensional gel electrophshiya, oresis, 2-DE)分离四种材料总蛋白质, 凝胶经银染显色后,用PDQuest图像分析软件进行比较分析、识别差异表达的蛋白质。成功获得抗感水稻品种受稻瘟菌侵染和未侵染蛋白质的双向凝胶电泳图谱。获得未侵染内香优2号平均蛋白点数为447个,汕优63平均蛋白点数为440个;侵染后抗性品种内香优2号平均蛋白数为523个,感性品种汕优63平均蛋白质点数为326个。内香优2号未经侵染和侵染后图谱匹配率达 89%和 87%,汕优63未经侵染和侵染后图谱匹配率达86％和85％。内香优2号的差异表达蛋白点数为76个,汕优63的差异表达蛋白点数为114个。两者间存在一些差异表达的蛋白质,为阐明稻瘟病的发病机制打下了坚实的基础。     

Cerebrospinal Fluid Proteins Studied by Two-Dimensional Gel Electrophoresis and Immunoblotting Technique

The proteins of lumbar CSF have been investigated by two-dimensional gel electrophoresis, and their patterns have been compared with the corresponding serum protein patterns. Serum proteins in CSF have been identified by electroblotting and immunoreaction with antiserum against total human serum proteins. Proteins derived from brain have been identified with antiserum against human brain proteins. The most prominent CSF protein group has been identified as a multiple form of apolipoprotein E. The correct position of the glial fibrillary acidic protein has also been determined. The prefractionation of CSF proteins by size exclusion chromatography or by affinity chromatography followed by two-dimensional electrophoresis has facilitated the detection of trace components in CSF and the corresponding serum.     

小麦幼穗蛋白质双向电泳条件的优化

本研究以温光敏小麦为试材,用TCA/丙酮和酚提取法提取小麦幼穗蛋白样品,进行了双向电泳优化分析,并对双向电泳过程中出现的问题进行了讨论。结果表明,用TCA/丙酮法提取小麦幼穗蛋白质其产率(浓度)高于酚提取法。SDS-PAGE电泳显示,用TCA/丙酮提取法提取的蛋白质能获得较清晰条带,分辨率较高,而酚提取法提取的蛋白质其条带模糊,分辨率低。对蛋白质纯化除盐可以提高分辨率,减少横竖纹,获得背景清晰的圆形蛋白点。通过ImageMasterTM 2D Platinum5.0软件分析凝胶图谱,结果显示纯化后可降低噪点,纯化后蛋白点数可从未纯化蛋白点数的216增加到583。显然,采用TCA/丙酮法可获得高浓度高质量的蛋白质,而进一步纯化、除盐离子可进一步获得背景清晰可高重复性的电泳图谱。在双向电泳实验过程中,观察到一些异常缺陷胶的出现,如双向电泳图谱中蛋白点扩散,蛋白聚集形成斑点串,没有点或点很少,出现纵纹横纹及图谱扭曲等影响图谱质量的严重问题,本研究对这些问题做了分析并提出了解决方案。     

水稻红莲型细胞质雄性不育系小孢子不同发育时期花药总蛋白质比较分析

采用双向凝胶电泳对水稻红莲型细胞质雄性不育的不育系小孢子发育单核期和二核期花药总蛋白进行了分离,通过银染显色,获得了分辨率和重复性较好的双向电泳图谱,且单核期和二核期花药总蛋白质在双向电泳胶上分布的图谱十分相似。PDQuest 2DE图像分析软件在等电点(pI)3.0~10.0、分子量(M.W.)9.0~98.0 kD之间可识别约1 800个蛋白质点。比较分析发现单核期和二核期花药中共有241个差异表达的蛋白质点,其中仅在单核期中表达的点数为125,仅在二核期中表达的为13点;表现为表达量差异的105点,其中在二核期表达下调的点数为70点,表达上调的为33点。还对蛋白质点集中的区域(pI 4.5~8.0,M.W.25.0~70.0 kD)中的41个差异蛋白质点进行了分子量和等电点分析。     

乌龙岭’龙眼胚胎发育时期特异性蛋白质的变化

应用IEF-SDS-PAGE技术分析龙眼胚胎分化发育过程中蛋白质组分的变化。结果表明,在各发育阶段大多数蛋白质组分的电泳图谱基本一致,但也有变化。其中花后38d存在TE1(27．1kD、p,7．3),TE2(17．5kD、pI8．2)2个特异蛋白,45d存在TE3(11．4kD、pI7．6),TE4(13．2kD、pI9．9)2个特异蛋白,52d存在TE5(22．6kD、pI7．2),TE6(18．6kD、pI8．3),TE,(23．5kD、pI3．6)3个特异蛋白。31d胚胎电泳图谱中的蛋白质点数相对较多,表明此时蛋白质旺盛合成与积累,这与蛋白含量的变化基本一致。龙眼胚胎发育过程中特异蛋白的出现或消失．对胚胎的分化发育具有重要作用。     

油松雌性不育系球果蛋白质双向电泳技术的建立

本文建立了油松雌性不育系雌球果蛋白质组研究中的双向电泳技术.第一向采用固定pH值梯度(IPG)胶条在IPGphorTM等电聚焦仪上进行等电聚焦,第二向在恒功率且恒温条件下于Ettan-DALTTMⅡ高通量电泳仪上进行SDS-PAGE电泳,以银染和考马斯亮蓝两种方法染色.通过对全蛋白的提取、胶条pH值和胶条肿胀等技术环节的优化和比较,得到了重复性很高,分离效果良好的蛋白质双向图谱.     

小鼠晶体蛋白质组的双向电泳和质谱鉴定研究

目的应用双向电泳和质谱技术研究5周龄小鼠晶体蛋白质组。方法提取小鼠晶体总蛋白,进行固相pH梯度（IPG）等电聚焦双向电泳,胶体考马斯亮蓝R-250染色,使用PDQuest7．30图像分析软件分析电泳图像。选择主要蛋白点胶上酶解,应用基质辅助激光解析电离飞行时间／飞行时间（MALDI—TOF／TOF）仪器进行串联质谱（MS／MS）鉴定。结果上样量为882μg和190μg时,分别检测370±41蛋白点（n=3）和57±5个蛋白点（n=3）。高上样量能够较好地分离晶体低丰度蛋白,如念珠状纤维结构蛋白BFSP;低上样量可很好地分离高丰度蛋白-晶体蛋白（包括αA、αB;βA1～βA4;βB1～βB3;γA～γF和γS等）。质谱鉴定得到1种细胞骨架蛋白和16种高丰度晶体蛋白。结论双向电泳和质谱技术有效考察了晶体总蛋白质,为分析白内障形成过程中蛋白质的表达改变提供了新的方法和途径。     

不同上样方式对蛋白质组双向电泳图谱质量的影响比较

蛋白质组技术难点之一是如何获取尽可能多的细胞或组织的蛋白信息.双向电泳蛋白斑点数目直接反映了实验蛋白质组信息的完整性.除样品制备外,蛋白上样方式对双向电泳图谱的质量和完整性有直接的影响.实验论文从以下3个方面考察不同的蛋白上样方式对双向电泳图谱的影响;即：水化上样与杯上样;一次上样与重复上样;以及酸性端加样与碱性端上样.实验结果发现;蛋白上样量较大时,杯上样方式的图谱斑点数目较水化上样方式明显增多;样品蛋白浓度较高时,稀释多次上样明显优于一次性浓缩蛋白上样;蛋白裂解液(MCF7乳腺癌细胞)在酸性端加样对偏碱性蛋白的分离未发现明显优势.相反,在等电聚焦伏小时(Vh) 足够的前提下,碱性端加样对偏碱性端蛋白反而有利,表现为斑点数目较多,而且等电点方向拖尾减轻.实验结果对提高双向电泳的质量以及相关蛋白质组信息的完整性提供了有益的技术参考.     

衫木叶片蛋白质组的双向电泳技术优化

为建立适用于杉木(Cunninghaimia lanceolata)叶片蛋白质组研究的双向电泳技术,对杉木叶片蛋白质的溶解方法、上样量、IEF及SDS-PAGE电泳等关键步骤进行了优化。结果表明,杉木叶片蛋白质主要分布在pH4-7范围;裂解液中含有硫脲(2mmol/L)才能较充分地溶解蛋白,DTT浓度为60mmol/L、上样量1.5mg时得到的图谱分辨率较好且蛋白斑点分布均匀、清晰,拖尾现象明显减少,平衡液Ⅱ中碘代乙酰胺浓度为450mg(15ml)-1时能提高图谱分辨率;采用与质谱兼容的考马斯亮兰进行染色,得到近700个蛋白点。     

采用亲和层析结合制备凝胶电泳获得高纯度的重组抗原蛋白用于制备蛋白质芯片

为了得到制备抗原芯片所需的高纯度重组抗原蛋白,需要建立一套适合于多种重组抗原表达和纯化的技术路线．采用了亲和层析结合制备胶电泳的方法,对16种用于构建蛋白质芯片的食管癌相关抗原基因进行了克隆重组并在大肠杆菌中进行了表达．对高表达的重组蛋白首先制备包涵体,然后采用Ni-Sepharose亲和层析得到初步纯化的蛋白质,最后使用SDS-PAGE制备胶电泳作进一步纯化．经过透析复性后,用于制备蛋白质芯片．采用亲和层析纯化重组蛋白,得率为71% ,纯度约为70%;在SDS-PAGE制备胶进一步纯化后,得率为32%,纯度为95%,经过透析和复性后,最终得率为21%,纯度为95%．得到的重组蛋白RPS4在ELISA检测中可以和血清中识别RPS4 的自身抗体起反应,并且,采用精纯抗原制备的蛋白质芯片,在检测抗原与抗体这一对反应中也具有较高的敏感性和特异性,适合大规模血清抗体的检测．研究表明,采用亲和层析结合制备凝胶电泳纯化抗原蛋白,是一条简便快捷,适合需要量不大,但对纯度要求比较高的蛋白质芯片制备的技术路线．     

The Postsynaptic Density: Constituent and Associated Proteins Characterized by Electrophoresis, Immunoblotting, and Peptide Sequencing

The proteins of the postsynaptic density (PSD) fraction of cerebral cortex were resolved by two-dimensional electrophoresis (2DE) and more than 30 proteins identified by characteristic 2DE mobility, immunoblotting with specific antibodies, and N-terminal and peptide sequencing. The PSD fraction is enriched for spectrin, actin, tublin and microtubule associated protein II, myosin, enzymes of glycolysis, creatine kinase, elongation factor 1 alpha, and receptor protein. The three neurofilament proteins are detected but a 58-kDa protein is prominent and is, by peptide sequencing, the bovine homolog of the recently cloned 66-kDa neurofilament protein; in contrast to the latter, however, it is enriched in cerebrum compared with spinal cord. A 68-kDa protein is identified as a member of the hsp70/BiP family of proteins. A protein, designated dynamin, indicating its putative role as a microtubule motor, is identified as a major protein, is found, however, greatly enriched in the particulate fraction, and is significantly denaturant and detergent insoluble. A protein designated N-ethylmaleimide-sensitive factor is also detected. Thus, two proteins implicated in vesicular transport are present in the PSD fraction. Seven polyclonal antibodies were produced to 2DE separated and electroeluted proteins of the PSD and were identified by peptide sequence analysis and 2DE profile as the hsp70/BiP homologous protein, the novel neurofilament protein synapsin IIa, pyruvate kinase, dynamin, aconitase and an unknown contaminating protein, and a 115-kDa protein that by subcellular fractionation and immunoblotting is a diagnostic PSD molecule. In addition, peptide sequences are obtained for four additional higher molecular weight proteins of the PSD that are not related at the level of primary structure to any known proteins.     

蓝色温和凝胶双向电泳用于分析嗜盐菌质膜蛋白复合物

为了揭示细胞对盐胁迫渗透适应的分子机制,以新鉴定的中度嗜盐芽孢杆菌Bacillussp.I121为实验材料,分析了该嗜盐菌质膜上的盐胁迫响应蛋白.为此,通过蓝色温和凝胶双向电泳(BN/SDS-PAGE)对纯化的质膜组分进行了差异蛋白质组学研究.经MALDI-TOF/TOF质谱分析,鉴定了8个盐胁迫响应蛋白.盐胁迫诱导上调表达的蛋白质包括ABC型转运蛋白、3-磷酸甘油透性酶、嘧啶核苷转运蛋白和甲酸脱氢酶,下调表达的蛋白质包括琥珀酸脱氢酶(succinate dehydrogenase)铁硫亚基、黄素蛋白亚基、细胞色素b556亚基以及分子伴侣DnaJ的同源蛋白;酶活力测定结果表明胁迫条件下上述蛋白质的活性变化与表达量变化相一致.这些蛋白质中绝大多数属于高度疏水的跨膜蛋白,主要负责物质跨膜运输及能量代谢.上述结果表明,中度嗜盐菌Bacillus sp.I121可通过加快跨膜物质运输,同时抑制TCA循环完成盐胁迫条件下相容性溶质脯氨酸和四氢嘧啶的合成与积累.也进一步证明,蓝色温和凝胶双向电泳不仅可用于线粒体、叶绿体中蛋白质复合物的分析,也同样适用于细胞质膜上高度疏水蛋白复合物的比较研究.     

蛋白质与核酸凝胶电泳分子量测定方法的探讨

文中介绍了一种在凝胶电泳中,当凝胶两端损坏、指示剂染料模糊不清或标记物丢失而无法确定电泳的起点与前缘时,进行正确的分量测定的补救方法,并从分子量测定的算法上进行了推导证明,用实际事例作了验证．     

A Novel Gaussian Extrapolation Approach for 2D Gel Electrophoresis Saturated Protein Spots

Analysis of images obtained from two-dimensional gel electrophoresis (2D-GE) is a topic of utmost importance in bioinformatics research, since commercial and academic software available currently has proven to be neither completely effective nor fully automatic, often requiring manual revision and refinement of computer generated matches. In this work, we present an effective technique for the detection and the reconstruction of over-saturated protein spots. Firstly, the algorithm reveals overexposed areas, where spots may be truncated, and plateau regions caused by smeared and overlapping spots. Next, it reconstructs the correct distribution of pixel values in these overexposed areas and plateau regions, using a two-dimensional least-squares fitting based on a generalized Gaussian distribution. Pixel correction in saturated and smeared spots allows more accurate quantification, providing more reliable image analysis results. The method is validated for processing highly exposed 2D-GE images, comparing reconstructed spots with the corresponding non-saturated image, demonstrating that the algorithm enables correct spot quantification.     

口虾蛄性腺组织蛋白质双向电泳体系的建立及优化

旨在通过口虾蛄雄性、雌性性腺组织蛋白质双向电泳技术体系的优化,获得雄性、雌性口虾蛄性腺蛋白质的表达图谱。结果表明,不同的蛋白提取方法,上样前处理方法,上样量,聚焦时间及雌、雄性口虾蛄性腺蛋白表达图谱存在一定的差异。雌性、雄性口虾蛄用Tris-HCl提取后用丙酮沉淀方法提取蛋白质、不经上样缓冲液处理、上样量为10μg时,得到较好图谱。对图谱分析发现在pH4-6.5范围内,雌性口虾蛄性腺可溶性蛋白质种类多于雄性。雄性口虾蛄蛋白质点相对于雌性较少,且蛋白质大部分分布于酸性端。经过蛋白质双向电泳体系的优化,能显著提高双向电泳图谱的分辨率,为进一步研究口虾蛄性别差异表达蛋白的筛选,后续的口虾蛄蛋白质组学研究提供技术保障。     

Proteins of BrdU-Dependent Hamster Cell Lines as Characterized by Two-Dimensional Gel Electrophoresis

Cell lines which exhibit the BrdU-dependent phenotype (B4 and HAB) were studied with respect to BrdU-induced alterations in genetic expression by two-dimensional gel electrophoresis. A comparison of the proteins from the HAB cells, in which the DNA is 100% substituted by BrdU, to those of the unsubstituted parent line (3460) showed 55 protein alterations; the synthesis of 15 increased while that of the other 40 decreased. When 3460 cells were grown in BrdU such that their DNA was > 50% substituted, 27 protein changes could be detected; of these, the synthesis of 10 increased while that of 17 decreased. A comparison of all these changes in the various cell lines showed six which were common to the BrdU-substituted cell lines. The proteins from another Syrian hamster cell line, BHK.-21 (C-13) and those of HAB cells grown in thymidine or BrdC were also examined on two-dimensional gels.
Although BrdU has a dramatic effect on many cellular functions, relatively few changes in the pattern of protein synthesis could be detected in these cell lines, perhaps reflecting the specialized action of this analogue on particular cellular functions.     

水稻根质膜蛋白质组双向电泳水化液的优化

以水稻(Oryza sativa L.)苗期幼嫩根尖作为材料,利用葡聚糖-聚乙二醇两相分配法纯化得到纯度达90%的质膜组分,使用4种不同的水化液溶解质膜蛋白,进行IEF/SDS-PAGE双向电泳和MALDI-TOF/TOF质谱分析.结果显示,4种水化液中,以7 mol/L Urea2、mol/L Thiourea、4%CHAPS、20 mmol/L DTE、1%ASB14的条件对膜蛋白的溶解效果和双向电泳分离效果最好;16个被鉴定蛋白中有9个为质膜相关蛋白,5个为未知蛋白,来自其它细胞器的蛋白仅有2个.研究表明,在常用水化液中添加磺基甘氨酸三甲内盐ASB14有利于植物细胞质膜蛋白质组的分析,并且该优化条件下的双向电泳适合分离水稻质膜中亲水性相对较高的膜附着蛋白.     

双向凝胶电泳缺陷胶及其原因

双向凝胶电泳是蛋白质组学研究中的关键技术之一,涉及较多的实验步骤和试剂,常出现缺陷胶而导致实验失败。从数千张电泳胶图中选取了21张典型的缺陷胶图,对它们进行了归类和总结,分析和讨论了形成缺陷胶的多种原因,提出了实验改进的方法和注意事项。     

一种分析叶绿体类囊体膜色素蛋白复合物的蓝绿温和胶电泳系统

采用蓝绿温和胶电泳系统可以非常有效地分离叶绿体蛋白质复合物,包括PSⅠ, PSⅡ, ATP合酶,细胞色素b₆f复合物,捕光色素复合物和1,5-二磷酸核酮糖羧化酶.还结合SDS-聚丙烯酰胺凝胶电泳将叶绿体多亚基复合物的50多种蛋白质分开,利用免疫印迹对蛋白质复合物进行了初步鉴定,同时还应用蓝色温和胶电泳分析基质、基粒类囊体复合物的组成.     

Proteomic Analysis of Human Plasma Proteins by Two-Dimensional Gel Electrophoresis and by Antibody Arrays Following Depletion of High-Abundance Proteins

Detecting proteins that are present at lower levels in human plasma, for the identification of potential disease biomarkers, is complicated by a few highly abundant proteins. One promising strategy is the removal of these abundant proteins interfering with the analysis of plasma content by proteomic techniques. This study compared three affinity-based methods to remove the most abundant proteins in human plasma. Two of them, based on antibodies, which depletes the six or the 12 most abundant proteins, demonstrated the highest efficiency in enriching less abundant plasma proteins. Comparison of two anticoagulant treatments for plasma preparation, EDTA and CTAD, showed that this treatment influenced the patterns of lower-abundance proteins visible on 2-dimensional (2-D) gels. Several staining procedures including two fluorescent dyes, Sypro Ruby and Deep Purple, were also compared with a very sensitive silver staining method for the visualization of lower-abundance proteins on 2-D gels. Furthermore, treatments of lower-abundance plasma proteins with hydroxyethyl disulfide enhanced protein sharpness and resolution. The purpose of all these systematic comparisons was to select the most reliable methods in different steps of plasma preparation and handling as well as in analysis of proteins by 2-D gels to obtain highly reproducible patterns of lower-abundance plasma proteins. Importantly, the lower-abundance plasma proteins enriched by these optimized conditions were further analyzed by antibody microarrays allowing the identification of 61 proteins using 350 antibodies directed against signalling proteins suggesting that this proteomic strategy is a valuable approach for detecting potential plasma biomarkers. Richard R. Desrosiers and édith Beaulieu contributed equally to this work.