蛋白质组学技术在植物类囊体膜蛋白研究中的应用

类囊体作为植物光合作用光反应的重要场所,在植物亚细胞蛋白质组学研究中倍受关注.介绍了植物蛋白质组学相关技术,包括双向凝胶电泳(2DE)、高效液相色谱(HPLC)、高效毛细管电泳(HPCE)、质谱(MS)和蛋白质组学数据库在植物类囊体膜蛋白研究中的应用.同时对类囊体膜蛋白质组学的研究趋势进行了探讨.     

大规模膜蛋白质组鉴定技术进展

生物膜是一类重要的亚细胞结构,含有大量的跨膜蛋白和非跨膜蛋白,负责细胞与外界的物质和信息交换以及行使细胞内的多种功能。鉴定并分析膜蛋白在生物膜上的表达是研究其功能必不可少的步骤。蛋白质组学技术可以在单次实验中一次性从全细胞裂解物中鉴定多达上万个蛋白质,从而为分析这些蛋白的表达提供了直接的证据。但由于膜蛋白具有较强的疏水性,从而导致对膜蛋白的蛋白质组学鉴定相对困难,这也使得对膜蛋白的结构与功能的研究进展相对比较缓慢。随着鸟枪法蛋白质组学(shot-gun proteomics)及滤膜辅助的样品制备(filter aided sample preparation)等为代表的现代蛋白质组学技术的快速发展,膜蛋白的鉴定水平及覆盖率也随之大幅提高。本文主要综述了过去20余年在膜蛋白质组学技术上的重要进展,并以光合作用模式蓝藻集胞藻(Synechocystis sp.PCC6803)的膜蛋白质组学研究为例,阐述了技术的进步如何提高膜蛋白鉴定的覆盖度,以期为其他物种膜蛋白质组全覆盖度的鉴定提供相应理论借鉴。     

大鼠背根神经节细胞膜蛋白的提取及双向凝胶电泳分离

为了开展大鼠背根神经节(DRG)细胞质膜蛋白质组学研究,取成年大鼠的背根神经节,用胰蛋白酶和胶原酶等消化处理后经密度梯度离心分离DRG细胞质膜;用裂解液裂解提取膜蛋白并通过双向凝胶电泳将膜蛋白分离.扫描凝胶图谱后进行图像分析,结果表明DRG细胞膜蛋白得到了有效的提取和分离.双向凝胶电泳图谱的建立为进一步进行DRG细胞质膜蛋白质组学的研究提供了重要的基础.     

植物液泡蛋白质组研究

植物液泡是植物生长、发育及逆境防御必要细胞器。细胞中有不同类型的液泡,执行不同的功能,如蛋白质降解、更新、解毒、代谢物储存和离子稳态等。随着质谱技术的快速发展,基于质谱的蛋白质组学分析将有助于鉴定液泡蛋白质组分及了解液泡膜上的一些特异载体和通道转运过程。本综述将从液泡功能,液泡蛋白质及液泡膜蛋白的提取、纯化,植物液泡蛋白质组及膜蛋白质组的研究,液泡磷酸化蛋白质组研究等方面进行阐述。     

盐胁迫对大麦叶片类囊体膜组成和功能的影响

盐胁迫下大麦叶片类囊体膜蛋白和叶绿素含量以及对绿素a／叶绿素b、磷脂／膜蛋白和膜脂结合半乳糖／膜蛋白的比值下降,膜脂中亚麻酸（18：3）摩尔百分数上升,不饱和指数上升,类囊体膜H －ATPase活性先升后降,希尔反应一直呈较高活性。类囊体膜组分和功能的变化可能与植物盐适应有关。     

膜蛋白结构研究方法新进展

膜蛋白是一类结构独特的蛋白质,执行很多基本的和重要的细胞生物学功能。了解膜蛋白在生物膜上的基本构象,对研究膜蛋白的精细拓扑结构、功能具有重要意义。但是膜蛋白的疏水特性使其需要与生物膜共同形成稳定的自然构象,至今在蛋白质组学的研究中对膜蛋白知之甚少。了解分子结构是了解生物大分子功能的一个重要途径。因此,本文对近来膜分子结构研究领域的进展作一简要概述。晶体学方法、单颗粒方法和原子力显微镜为膜蛋白的研究提供了大量的细节数据。固相核磁共振技术提供了跨膜α螺旋结构的方向约束数据和精确的分子间距离约束数据。直接位点标记的旋转电子顺磁共振可以得到更长的距离约束数据,但是目前的标记策略仍然具有局限性。位点特异的红外二色性分析可使得在脂双层中定向分析跨膜α螺旋束成为可能。     

光合膜蛋白晶体的结构与功能

文章对植物类囊体膜上最重要的4种光合膜蛋白的晶体结构和功能作了简要概述。     

脂质体重组和脂蛋白体在植物生物膜研究中的应用

脂质体是磷脂在一定条件下在水中形成的由脂质双分子层组成的内部为水相的闭合囊泡。在推动生物膜的研究进展中,它作为模式系统起着非常重要的作用,能用于研究膜蛋白的性质和功能;膜脂和膜蛋白的相互关系;膜的电化学性质等。近年来脂质体重组技术开始引入到植物学研究领域,用于对植物膜蛋白的研究。本文简要介绍了脂质体的制备和脂酶体重组的方法及其在植物生物膜研究中的应用。     

非模式植物蛋白质组学研究进展

蛋白质组学研究是对基因组学研究的重要补充,它是在蛋白质水平定量、动态、整体性研究生物体。该文简要介绍了蛋白质组学的含义,蛋白质组学及植物蛋白质组学产生的科学背景,蛋白质组学的研究内容。概述了非模式植物蛋白质组学的研究进展,主要包括非模式植物个体及群体蛋白质组学,组织和器官蛋白质组学,亚细胞蛋白质组学,响应环境变化的蛋白质组学以及非模式植物生物环境因子的蛋白质组学的研究情况,同时对植物蛋白质组学的发展前景进行了展望。     

细胞质膜蛋白质组学

随着基于质谱的大规模蛋白质鉴定技术的建立,蛋白质组学得到迅速发展。同时由于质膜在细胞生命活动中的重要作用,质膜蛋白质组学逐渐兴起,并发展成为蛋白质组学研究中的重要组成部分。但由于膜蛋白尤其是内在膜蛋白的强疏水性、低丰度,造成蛋白质提取、分离和鉴定相对困难,使质膜蛋白质组成为蛋白质组研究中的一个技术难点。     

The proteomics of plant cell membranes

Membrane proteins are involved in many different functions depending on their location in the cell. Characterization of the membrane proteome can bring new insights to the function of different plant membrane systems and the subcellular compartments where the proteins are found. Plant membrane proteomics can also provide valuable information about plant-specific biological processes. Despite recent advances in the separation and techniques for the analysis of plant membrane proteins, characterization of these proteins, especially the hydrophobic ones, is still challenging. In this review, plant membrane proteomics data, compiled from the literature on Arabidopsis thaliana, are described. In addition, initial attempts towards determining the physiological significance of some proteins identified from membrane proteomics in rice are also described.     

Plasma membrane proteome in Arabidopsis and rice

Plant cells contain many membrane systems that are specially adapted to perform particular functions. In plant cells, the processing of signals that are involved in responses to biotic and abiotic stressors occurs in the plasma membrane. Therefore, characterization of the plasma membrane proteome can provide new insights into the functions of various plant membrane systems. Plant plasma membrane proteomics can also provide valuable information for plant-specific biological investigations. Despite recent advances in preparative and analytical techniques for plant plasma membrane proteins, the characterization of these proteins, particularly the hydrophobic ones, remains challenging. In this review, plant plasma membrane proteomics data compiled from the literature on Arabidopsis thaliana are presented. Initial attempts to determine the physiological significance of some proteins identified from plasma membrane proteomics in rice and other plants are also described from the results of our research.     

Plant membrane proteomics

Plant membrane proteins are involved in many different functions according to their location in the cell. For instance, the chloroplast has two membrane systems, thylakoids and envelope, with specialized membrane proteins for photosynthesis and metabolite and ion transporters, respectively. Although recent advances in sample preparation and analytical techniques have been achieved for the study of membrane proteins, the characterization of these proteins, especially the hydrophobic ones, is still challenging. The present review highlights recent advances in methodologies for identification of plant membrane proteins from purified subcellular structures. The interest of combining several complementary extraction procedures to take into account specific features of membrane proteins is discussed in the light of recent proteomics data, notably for chloroplast envelope, mitochondrial membranes and plasma membrane from Arabidopsis. These examples also illustrate how, on one hand, proteomics can feed bioinformatics for a better definition of prediction tools and, on the other hand, although prediction tools are not 100% reliable, they can give valuable information for biological investigations. In particular, membrane proteomics brings new insights over plant membrane systems, on both the membrane compartment where proteins are working and their putative cellular function.     

Quantitative proteomics reveals dynamic changes in the plasma membrane during Arabidopsis immune signaling

The plant plasma membrane is a crucial mediator of the interaction between plants and microbes. Understanding how the plasma membrane proteome responds to diverse immune signaling events will lead to a greater understanding of plant immunity and uncover novel targets for crop improvement. Here we report the results from a large scale quantitative proteomics study of plasma membrane-enriched fractions upon activation of the Arabidopsis thaliana immune receptor RPS2. More than 2300 proteins were identified in total, with 1353 proteins reproducibly identified across multiple replications. Label-free spectral counting was employed to quantify the relative protein abundance between different treatment samples. Over 20% of up-regulated proteins have known roles in plant immune responses. Significantly changing proteins include those involved in calcium and lipid signaling, membrane transport, primary and secondary metabolism, protein phosphorylation, redox homeostasis, and vesicle trafficking. A subset of differentially regulated proteins was independently validated during bacterial infection. This study presents the largest quantitative proteomics data set of plant immunity to date and provides a framework for understanding global plasma membrane proteome dynamics during plant immune responses.     

Differential proteomics of plant development

In this mini-review, recent advances in plant developmental proteomics are summarized. The growing interest in plant proteomics continually produces large numbers of developmental studies on plant cell division, elongation, differentiation, and formation of various organs. The brief overview of changes in proteome profiles emphasizes the participation of stress-related proteins in all developmental processes, which substantially changes the view on functional classification of these proteins. Next, it is noteworthy that proteomics helped to recognize some metabolic and housekeeping proteins as important signaling inducers of developmental pathways. Further, cell division and elongation are dependent on proteins involved in membrane trafficking and cytoskeleton dynamics. These protein groups are less prevalently represented in studies concerning cell differentiation and organ formation, which do not target primarily cell division. The synthesis of new proteins, generally observed during developmental processes, is followed by active protein folding. In this respect, disulfide isomerase was found to be commonly up-regulated during several developmental processes. The future progress in plant proteomics requires new and/or complementary approaches including cell fractionation, specific chemical treatments, molecular cloning and subcellular localization of proteins combined with more sensitive methods for protein detection and identification.     

Vectorial proteomics

Vectorial proteomics is a methodology for the differential identification and characterization of proteins and their domains exposed to the opposite sides of biological membranes. Proteomics of membrane vesicles from defined isolated membranes automatically determine cellular localization of the identified proteins and reduce complexity of protein characterizations. The enzymatic shaving of naturally-oriented, or specifically-inverted sealed membrane vesicles, release the surface-exposed peptides from membrane proteins. These soluble peptides are amenable to various chromatographic separations and to sequencing by mass spectrometry, which provides information on the topology of membrane proteins and on their posttranslational modifications. The membrane shaving techniques have made a breakthrough in the identification of in vivo protein phosphorylation sites in membrane proteins form plant photosynthetic and plasma membranes, and from caveolae membrane vesicles of human fat cells. This approach has also allowed investigation of dynamics for in vivo protein phosphorylation in membranes from cells exposed to different conditions. Vectorial proteomics of membrane vesicles with retained peripheral proteins identify extrinsic proteins associated with distinct membrane surfaces, as well as a variety of posttranslational modifications in these proteins. The rapid integration of versatile vectorial proteomics techniques in the functional characterization of biological membranes is anticipated to bring significant insights in cell biology.     

The 8th International Conference of the Canadian Proteomics Initiative

The 8th International Conference of the Canadian Proteomics Initiative (CPI) was held at the Hilton Vancouver Metrotown Hotel in Burnaby, British Columbia from 3–5 May, 2008. With nearly 200 delegates, 32 speakers and more than 50 poster presentations, CPI 2008 covered a wide range of topics, including novel technologies, human and clinical proteomics, structural proteomics, bioinformatics, post-translational modifications, membrane and receptor proteomics, and plant and animal proteomics. This year’s conference was also highlighted by hands-on proteomics workshops at the University of Victoria Genome BC Proteomics Centre and the University of British Columbia, an inaugural meeting of the British Columbia Proteomics Network and focus meetings exploring opportunities for the formation of a nationwide association for Canadian proteomics and Genome British Columbia’s efforts towards large-scale global genomics and proteomics projects.     

Membrane trafficking in plants: new discoveries and approaches

The general organization and function of the endomembrane system is highly conserved in eukaryotic cells. In addition, increasing numbers of studies demonstrate that normal plant growth and development are dependent on specialized tissue and subcellular-specific components of the plant membrane trafficking machinery. New approaches, including chemical genomics and proteomics, will likely accelerate our understanding of the diverse functions of the plant endomembrane system.     

A workflow for peptide-based proteomics in a poorly sequenced plant: a case study on the plasma membrane proteome of banana

Membrane proteins are an interesting class of proteins because of their functional importance. Unfortunately their analysis is hampered by low abundance and poor solubility in aqueous media. Since shotgun methods are high-throughput and partly overcome these problems, they are preferred for membrane proteomics. However, their application in non-model plants demands special precautions to prevent false positive identification of proteins. In the current paper, a workflow for membrane proteomics in banana, a poorly sequenced plant, is proposed. The main steps of this workflow are (i) optimization of the peptide separation, (ii) performing de novo sequencing to allow a sequence homology search and (iii) visualization of identified peptide-protein associations using Cytoscape to remove redundancy and wrongly assigned peptides, based on species-specific information. By applying this workflow, integral plasma membrane proteins from banana leaves were successfully identified.     

Proteomic study of the Arabidopsis thaliana chloroplastic envelope membrane utilizing alternatives to traditional two-dimensional electrophoresis

With the completion of the sequencing of the Arabidopsis genome and with the significant increase in the amount of other plant genome and expressed sequence tags (ESTs) data, plant proteomics is rapidly becoming a very active field. We have pursued a high-throughput mass spectrometry-based proteomics approach to identify and characterize membrane proteins localized to the Arabidopsis thaliana chloroplastic envelope membrane. In this study, chloroplasts were prepared from plate- or soil-grown Arabidopsis plants using a novel isolation procedure, and "mixed" envelopes were subsequently isolated using sucrose step gradients. We applied two alternative methodologies, off-line multidimensional protein identification technology (Off-line MUDPIT) and one-dimensional (1D) gel electrophoresis followed by proteolytic digestion and liquid chromatography coupled with tandem mass spectrometry (Gel-C-MS/MS), to identify envelope membrane proteins. This proteomic study enabled us to identify 392 nonredundant proteins.