叶绿体蛋白质组研究进展

亚细胞蛋白质组学是近年来蛋白组学研究中的一个热点。通过细胞器的纯化和亚细胞组分的分离,降低了样品的复杂性,增大了相应蛋白质组分的富集,有利于由此分离获得的蛋白质的序列分析及功能鉴定。叶绿体蛋白质组为植物亚细胞蛋白质组学研究中相对全面的一部分,利用亚细胞分离结合双向电泳技术系统地鉴定叶绿体中蛋白质组分是获取叶绿体蛋白质信息、确定其功能的重要技术手段。本文就近年来植物叶绿体蛋白质组涵盖的叶绿体内、外被膜、叶绿体基质、类囊体膜和类囊体腔蛋白的研究进行综述,以全面认识叶绿体蛋白的组成、特点及其在叶绿体生理生化代谢网络中的作用。     

亚细胞蛋白质组学

蛋白质组研究应用了分辨蛋白分离技术、质谱法、生物信息学工具来鉴定生物体蛋白质性质。但由于高等真核细胞的复杂性 ,对其全部蛋白质组进行一步鉴定难于达到。为了分析细胞的全蛋白质组 ,目前提议以亚细胞蛋白质组作为切入点 ,即建立细胞的大分子结构 ,如亚细胞区室、细胞器、大分子结构和多蛋白复合物相应的蛋白质组 ,其优点是减少全细胞分析的复杂性 ,研究结果更具体。本文将介绍在亚细胞蛋白质组方面的已开展研究的概况。1 .细胞膜蛋白质组学在ＳＤＳ ＰＡＧＥ分析中 ,由于膜蛋白提取量少和 /或分辨率差而难以显示 ,这首先涉及膜蛋白自…     

长双歧杆菌BBMN68 可溶性蛋白质图谱的建立

为了建立长双歧杆菌BBMN68蛋白质图谱,采用双向电泳的方法建立了2-D参考图谱,通过MALDI-TOF/MS质谱鉴定和数据库搜索,鉴定到206个蛋白质(占长双歧杆菌BBMN68基因预测总蛋白的11.4%)。通过2-D胶分析,共有800±15(对数期)和800±20(稳定期)个蛋白质,其中282个蛋白点成功鉴定,代表206个不同的蛋白质。另外,分析了实验鉴定蛋白质的等电点和分子量,蛋白功能,密码子偏好性,蛋白质疏水性以及蛋白质细胞定位的分析。研究结果为长双歧杆菌的比较蛋白质组学研究提供了参考图谱和蛋白质基础信息数据。     

叶绿体的蛋白质组学研究进展

蛋白质组学是以基因组编码的所有蛋白为研究对象,高通量地从细胞及整体水平上研究蛋白质的组成及其功能的新兴学科。在后基因组时代的今天,蛋白质组学的研究正逐渐深入到生命科学的各个领域,21世纪蛋白质组学将成为生命科学中最热门的学科。蛋白质组分析已成为鉴定植物功能的有力工具之一,叶绿体作为比较重要的细胞器,在植物蛋白质组学中已有较多的研究,,随着双向电泳技术的改进和质谱法的出现,并与不断增多的拟南芥、水稻、玉米等植物的序列数据相结合,叶绿体蛋白质组可以被快速鉴定。本文主要介绍了植物蛋白质组学、叶绿体及其蛋白质组学研究技术和研究进展,并对蛋白质组学的研究趋势进行了展望。     

肝癌亚细胞结构的蛋白质组分比较分析

运用亚细胞蛋白质组学的研究策略,分离纯化亚细胞结构,可以提高低丰度蛋白质在双向电泳中检出的数量。通过对比分析肝癌细胞与正常肝细胞线粒体、细胞核蛋白质组的差异表达情况,为肝癌发病机理的研究提供更多、更有价值的信息。以体外培养的人体肝癌细胞QGY-7703与正常肝细胞LO2为研究模型,通过超离心的方法分离细胞的线粒体和细胞核。双向电泳分离线粒体和细胞核的蛋白质,图像分析筛选差异表达蛋白斑点,MALDI-TOF-MS鉴定蛋白质。从线粒体、细胞核的蛋白质电泳图谱中筛选出54个候选差异表达的蛋白质斑点,质谱鉴定出22种差异表达蛋白质,其中17种在肝癌细胞中表达上调,5种在肝癌细胞中表达下调。筛选出的差异表达蛋白质涉及到细胞的能量代谢、蛋白质合成、细胞骨架与核骨架的改变、mRNA的加工成熟及凋亡调控等许多方面,表明癌变细胞的组织结构和代谢状态都发生了很大的变化。     

表阿霉素对肝癌亚细胞蛋白质组影响的研究

表阿霉素是临床应用的抗癌药物之一,它干扰DNA的复制和转录,并能诱导癌细胞凋亡。表阿霉素对癌细胞蛋白质表达谱的影响,涉及到表阿霉素药理作用的机制,对此进行研究,有助于抗癌药物作用机制的理解。运用亚细胞蛋白质组学的研究策略,对比分析表阿霉素干预前后,肝癌细胞线粒体、细胞核蛋白质组的表达差异。通过超离心分离纯化细胞器,双向电泳分离蛋白质,图像分析筛选差异表达蛋白,MALDI-TOF-MS分析鉴定蛋白质。从线粒体、细胞核两个亚细胞组分中一个鉴定了15种差异表达蛋白,其中5种在表阿霉素干预后的肝癌细胞中表达上调,10种干预后表达下调。这些差异表达蛋白质涉及到细胞的能量代谢、蛋白质合成、细胞骨架的改变、mRNA的加工成熟、细胞应急状态的形成及凋亡调控等许多方面。     

叶绿体蛋白质组学研究进展

蛋白质组分析是鉴定蛋白质种类和功能的有力工具之一。叶绿体作为光合作用的重要细胞器,叶绿体蛋白质组学成为了研究的热点,涉及的领域包括叶绿体的总蛋白质组学、亚细胞蛋白质组学、差异蛋白质组学和蛋白质的功能等。现主要介绍蛋白质组学的常用技术以及叶绿体蛋白质组学的最新研究进展。     

活细胞内亚细胞结构蛋白质组学研究新技术——几种邻近标记策略的应用及比较

真核细胞内多种无膜及有膜细胞器为各种生物学过程的发生提供场所.被膜细胞器通过它们之间的膜接触位点所进行的信息交流和物质交换是维持生命活动所必需的.绘制活细胞中细胞器或膜接触位点等处的蛋白质组图谱,将有助于解析这些部位的生物学功能及作用机制,并为研究细胞器相互作用提供基础.但由于无膜细胞器或膜接触位点很难分离纯化,传统的生化方法难以系统解析其中的蛋白质组.最近报道的几种基于酶类的蛋白质邻近标记技术,则为系统分析上述空间受限的蛋白质组这一难题提供了有效的解决方案.通过将能催化产生活性自由基(最常见的是生物素及其衍生物的自由基)的酶连接到目标蛋白上,可对其邻近的蛋白质组进行共价标记,从而使后者的分离和鉴定成为可能,并可以运用于活细胞中的动态标记.我们在此综述了几种最新的邻近标记策略的原理及应用,并对它们的优势与局限性进行了比较,以期为细胞器互作的蛋白质组学研究提供参考.     

桶形芋螺毒液蛋白质组分析及二硫键异构酶的鉴定

蛋白质二硫键异构酶(PDI)是内质网新生肽链折叠中一个重要的折叠酶。在热带药用海洋生物芋螺的毒液中,富含PDI酶,该酶对于毒液中芋螺毒素神经肽的体内氧化折叠至关重要。研究上样量、水化方式与时间、除盐步骤、聚焦电压和时间、平衡时间、凝胶电泳方法和染色方法等方面,优化并建立了较为理想的芋螺毒液蛋白样品双向电泳的实验条件与方法;通过双向电泳和MALDI-TOF-MS质谱分析技术,从海南产桶形芋螺(Conus betulinus Linnaeus)毒液蛋白中成功分离鉴定出PDI等9种蛋白;通过双向电泳和PDQuest软件分析了并比较了三种不同大小桶形芋螺毒液总蛋白的差异性,并质谱鉴定出5个明显的差异蛋白点。研究建立了桶形芋螺毒液蛋白双向电泳分析及质谱鉴定的技术方法,为后续大量分离纯化出天然的芋螺PDI酶以及利用蛋白质组学技术方法深入研究芋螺毒液特征提供了重要的基础。     

激光捕获显微切割技术结合^18O标记定量蛋白质组技术在胃癌标志物筛查中的应用研究

建立一种更加精确地分离鉴定胃癌特异肿瘤标志物的定量蛋白质组学技术.首先采用激光捕获显微切割技术(LCM)纯化胃腺癌细胞及胃黏膜良性上皮细胞,将裂解的样本总蛋白经过1D SDS-PAGE预分离,然后采用17O/16O分别标记两种样本酶切后的多肽混合物.结合纳升级液相色谱(Nano-HPLC-MS/MS)定量地鉴定胃癌细胞和胃黏膜良性上皮细胞的差异表达蛋白.共筛选出78个差异表达蛋白,其中42个蛋白质在胃癌组织中表达上调,36个蛋白质下调.Western blot技术验证了其中几个差异蛋白(moesin,periostin,annexin A2,annexin A4)的表达,与蛋白质组学研究的结果一致.LOM技术结合18O稳定同位素标记的定量蛋白质组学技术,为研究胃癌发生机制、筛选胃癌的分子标志物提供了新的思路,亦为诸如胃癌等复杂体系蛋白质的分离鉴定提供了新的技术选择.     

Signal-anchor sequences are an essential factor for the Golgi-plasma membrane localization of type II membrane proteins

Despite studies of the mechanism underlying the intracellular localization of membrane proteins, the specific mechanisms by which each membrane protein localizes to the endoplasmic reticulum, Golgi apparatus, and plasma membrane in the secretory pathway are unclear. In this study, a discriminant analysis of endoplasmic reticulum, Golgi apparatus and plasma membrane-localized type II membrane proteins was performed using a position-specific scoring matrix derived from the amino acid propensity of the sequences around signal-anchors. The possibility that the sequence around the signal-anchor is a factor for identifying each localization group was evaluated. The discrimination accuracy between the Golgi apparatus and plasma membrane-localized type II membrane proteins was as high as 90%, indicating that, in addition to other factors, the sequence around signal-anchor is an essential component of the selection mechanism for the Golgi and plasma membrane localization. These results may improve the use of membrane proteins for drug delivery and therapeutic applications.     

IntraGolgi distribution of the Conserved Oligomeric Golgi (COG) complex

The Conserved Oligomeric Golgi (COG) complex is an eight-subunit (Cog1-8) peripheral Golgi protein involved in membrane trafficking and glycoconjugate synthesis. COG appears to participate in retrograde vesicular transport and is required to maintain normal Golgi structure and function. COG mutations interfere with normal transport, distribution, and/or stability of Golgi proteins associated with glycoconjugate synthesis and trafficking, and lead to failure of spermatogenesis in Drosophila melanogaster, misdirected migration of gonadal distal tip cells in Caenorhabditis elegans, and type II congenital disorders of glycosylation in humans. The mechanism by which COG influences Golgi structure and function is unclear. Immunogold electron microscopy was used to visualize the intraGolgi distribution of a functional, hemagglutinin epitope-labeled COG subunit, Cog1-HA, that complements the Cog1-deficiency in Cog1-null Chinese hamster ovary cells. COG was found to be localized primarily on or in close proximity to the tips and rims of the Golgi's cisternae and their associated vesicles and on vesicles and vesiculo-tubular structures seen on both the cis and trans-Golgi Network faces of the cisternal stacks, in some cases on COPI containing vesicles. These findings support the proposal that COG is directly involved in controlling vesicular retrograde transport of Golgi resident proteins throughout the Golgi apparatus.     

Proteomic analysis of chlorosome-depleted membranes of the green sulfur bacterium Chlorobium tepidum

Green sulfur bacteria are obligate anaerobic phototrophs, which in addition to outer and plasma membranes contain chlorosomes. The analysis of the membrane proteome of Chlorobium tepidum from chlorosome-depleted membranes is described in this study. The membranes were purified by sucrose density centrifugation and characterized by 1-DE and 2-DE coupled with MS, absorption spectroscopy, and electron microscopy. 1-DE and 2-DE were employed to analyze the membrane proteins and to characterize the capabilities of the methods. Solubilization of the membrane proteins prior to 2-DE was improved by using a series of zwitterionic detergents. Based on the resolved spots after 2-DE, the combination of amidosulfobetaine 14 with Triton X-100 is more efficient than the combination of CHAPS, N-decyl-N,N-dimethyl-3-ammonio-1-propane sulfonate, and Triton X-100. From the application of 1-DE and 2-DE, 167 and 202 unique proteins were identified, respectively, using PMF by MALDI-TOF MS. Both methods resulted in the detection of 291 different proteins of which only 88 were predicted membrane proteins, indicating the limitation of membrane protein detection after separation with electrophoresis methods. In addition, 53 of these proteins were identified as outer membrane proteins.     

Genome-wide identification of the subcellular localization of the Escherichia coli B proteome using experimental and computational methods

Escherichia coli K-12 and B strains have most widely been employed for scientific studies as well as industrial applications. Recently, the complete genome sequences of two representative descendants of E. coli B strains, REL606 and BL21(DE3), have been determined. Here, we report the subproteome reference maps of E. coli B REL606 by analyzing cytoplasmic, periplasmic, inner and outer membrane, and extracellular proteomes based on the genome information using experimental and computational approaches. Among the total of 3487 spots, 651 proteins including 410 non-redundant proteins were identified and characterized by 2-DE and LC-MS/MS; they include 440 cytoplasmic, 45 periplasmic, 50 inner membrane, 61 outer membrane, and 55 extracellular proteins. In addition, subcellular localizations of all 4205 ORFs of E. coli B were predicted by combined computational prediction methods. The subcellular localizations of 1812 (43.09%) proteins of currently unknown function were newly assigned. The results of computational prediction were also compared with the experimental results, showing that overall precision and recall were 92.16 and 92.16%, respectively. This work represents the most comprehensive analyses of the subproteomes of E. coli B, and will be useful as a reference for proteome profiling studies under various conditions. The complete proteome data are available online (http://ecolib.kaist.ac.kr).     

结核分枝杆菌菌体蛋白双向电泳技术的优化

目的：提取结核分枝杆菌菌体蛋白并建立一种利用双向电泳分离结核分枝杆菌蛋白质组的方法。方法：分离提取结核分枝杆菌菌体蛋白。样品采用不同pH梯度的鹏胶条进行第一向等电聚焦,12％SDS—PAGE凝胶进行二向电泳。银染后双向电泳图谱用Molecular Image Fx激光图像扫描仪扫描,PDQuest6．0软件完成配比分析。结果：优化了结核分枝杆菌菌体蛋白的提取方法,用裂解液8mol／L尿素结合2mol／L硫脲,140mmol／LDTT,0．5％biolyte,4％CHAPs,400mg／m1lOG处理,成功提取了蛋白,并通过结核分枝杆菌双向电泳技术体系的优化,建立了结核分枝杆菌菌体蛋白的分解图谱。pH4—7及pH7—10两胶面上共1387个点,占所检测到的蛋白总数的86％,绝大部分（1194个）蛋白位于pH4—7范围内。结论：为进一步开展结核分枝杆菌的比较蛋白质组学研究提供了方法学参考。     

Characterizing the dynamic nature of the Yersinia pestis periplasmic proteome in response to nutrient exhaustion and temperature change

The periplasmic proteome of Yersinia pestis strain KIM6+ was characterized using differential 2-DE display of proteins isolated from several subcellular fractions. Circa 160 proteins were designated as periplasmic, including 62 (putative) solute-binding proteins of ATP-binding cassette (ABC) transporters (SBPs) and 46 (putative) metabolic enzymes. More than 30 SBPs were significantly increased in abundance during stationary phase cell growth, compared to the exponential phase. The data suggest that nutrient exhaustion in the stationary phase triggers cellular responses resulting in the induced expression of numerous ABC transporters, which are responsible for the import of solutes/nutrients. Limited availability of inorganic phosphate (P(i)) also caused dramatic proteomic changes. Nine proteins were functionally linked to the mobilization and import of three small molecules (P(i), phosphonate and glycerol-3-phosphate) and accounted for nearly half of the total protein mass in the periplasm of P(i)-starved cells. When cells were grown at 26 degrees C versus 37 degrees C, corresponding to ambient temperatures in the flea vector and mammalian hosts, respectively, several periplasmic proteins with no known roles in the Y. pestis life cycle were strongly altered in abundance. This included a putative nitrate/sulfonate/bicarbonate-specific SBP (Y1004), encoded by the virulence-associated plasmid pMT1 and increased in abundance at 37 degrees C.     

双向电泳法在家蚕蛋白质分离中的应用

家蚕Bombyxmori(L.)既是重要的经济昆虫,又是鳞翅目昆虫研究的典型模式生物。开展家蚕蛋白质组研究,将有助于阐明家蚕绢丝蛋白的分泌机理,也是研究鳞翅目昆虫及其他生物生命本质的需要。双向电泳是蛋白质分离的关键技术。为探讨适宜家蚕蛋白质组研究的双向电泳条件,以家蚕丝腺、丝腺内容物、蚕卵和血液为材料,在不同条件下进行双向电泳,并对分离的蛋白点进行质谱分析。结果表明:通过改进的蛋白质裂解液辅以超声破碎制备的蛋白质,双向电泳后能够得到较好的2-DE图,也能满足进行MALDI-TOFMS分析的需要。因此本研究方法适用于家蚕不同组织中蛋白质的提取和双向电泳。