蛋白质芯片技术

以前对蛋白质的研究集中在一次研究一种蛋白质 ,通常费时费力 ;而蛋白质芯片技术是研究蛋白质组的新技术 ,是高通量、微型化和自动化的蛋白质分析技术。它可以用来研究蛋白质的亚细胞定位和蛋白质与蛋白质之间的相互作用 ,以及对蛋白质的功能进行生物化学分析 ,将对蛋白质组研究及医学生物学的发展有很大的推动作用。较系统地介绍了蛋白质芯片的概念、制作及检测方法 ;同时也讨论了蛋白质芯片的两种功能形式、存在问题和应用前景。     

蛋白质芯片构建技术进展

蛋白质芯片是一项实现蛋白质高通量分析和检测的新型技术,在生命科学的各个领域具有重要的应用前景。但是由于蛋白质本身的敏感性质和芯片的独特结构,蛋白质芯片技术的发展面临许多难点,这些正是蛋白质芯片研究者需要共同讨论的问题。本文从芯片构造、探针的固定方法和检测方法三个方面对芯片的构造进行分析和讨论,并对其相关技术的进展进行综述。     

蛋白质点阵/芯片技术的新进展

蛋白质点阵/芯片技术是分子生物学技术的重要进展,在功能蛋白质组研究方面具有广阔的潜在应用价值.目前发展起来的印迹蛋白微阵列、分子扫描技术和传感器生物芯片质谱,将应用于药靶检测、疾病诊断、蛋白质结构鉴定和/或蛋白质之间的相互作用分析等方面,具有分析速度快、效率高、样品消耗少等特点,将成为生命科学与医学领域新的研究工具.     

蛋白质芯片技术研究进展

蛋白质芯片技术是近年发展起来的一项新技术,这项技术是将蛋白质的分析微缩到小型芯片上进行,利用荧光或酶显色进行探测,最后用特 计算机软件加以分析。该技术在基因表达、抗原抗体检测、药物开发、疾病诊断等研究方面显示出快速、高效、高通量处理信息的能力。     

蛋白质芯片

随着人类基因组计划 (ＨＧＰ)顺利实施 ,以生命活动的执行者———蛋白质为研究对象的蛋白质组学越来越显得重要[1] ,并构想和发展了快捷、高效、并行、高通量的蛋白质组检测新技术———蛋白质芯片技术。1.蛋白质芯片的基本构成蛋白质芯片是高通量、微型化和自动化的蛋白质分析技术。目前蛋白质芯片主要分两种[2 ] :一种类似于ＤＮＡ芯片 ,即在固相支持物表面高密度排列的探针蛋白点阵 ,可特异地捕获样品中的靶蛋白 ,然后通过检测器对靶蛋白进行定性或定量分析 (如图1)。另一种就是微型化的凝胶电泳板。在电场作用下 ,样品中的蛋白质通过芯…     

蛋白质芯片技术研究进展

蛋白质芯片是继基因芯片后的又一种用于生命科学研究的技术平台。目前这一技术已经被广泛应用到生命科学研究的多个领域,如蛋白质组学研究,新药的开发以及疾病的临床诊断等。就蛋白质芯片的基本原理、研究进展及应用状况做一介绍。     

蛋白质芯片在微生物学领域的应用进展

蛋白质芯片是蛋白质组学研究的一种新技术。具有高通量、微型化、连续化、自动化、快速和准确等特点。已成功地应用于蛋白质的鉴定、量化和基础功能及蛋白质组学的研究。在微生物的监测、检测、分离、鉴定分类和微生物资源开发等方面拥有巨大的应用前景。就蛋白质芯片技术及其在微生物领域的应用进展作了概述。     

蛋白质芯片及其应用

人类基因组计划的实施和大量珍贵的功能基因组数据的获得,使得蛋白质的研究显得越发重要。蛋白质芯片技术的出现为我们提供了一种较传统的凝胶电泳技术更为方便和快速的研究方法。     

SELDI蛋白质芯片检测技术

从基因组学到蛋白质组学,到当前有关小RNA对蛋白质合成调控的研究无一例外地说明蛋白质是直接发挥对生命活动调控的物质。同基因研究相比较,由于蛋白质分子种类繁多,有复杂的修饰成份和空间结构,使得蛋白质研究比较困难。新近发展起来的蛋白质芯片技术为蛋白质的检测和研究提供了新的技术平台,比如荧光标记技术,蛋白质指纹图谱．飞行时间．质谱联用技术（SELDI蛋白质芯片）,表面等离子基元共振生物传感器技术（SPR芯片）以及初步应用的光学蛋白质芯片技术,其中,后三种是新兴的无需标记进行蛋白质检测的技术。就SELDI蛋白质芯片及其新近研究作一综述。     

生物传感芯片质谱及其在蛋白质组研究中的应用

目前蛋白质组研究技术在已有的研究基础上取得了长足进展 ,包括固相 ｐＨ梯度双向凝胶电泳、生物质谱技术、蛋白质双向电泳图谱的数字化和各种分析技术、蛋白质间相互作用分析的方法如酵母双杂交系统、噬菌体展示、表面等离子共振等。但均各有其利弊。目前质谱是进行蛋白质组成分鉴定的支撑技术 ,但是它在蛋白质间相互作用分析及结构与功能的关系分析方面显得无能为力 ,而生物分子相互作用分析技术 (Ｂｉｏｍｏｌｅｃｕｌａｒｉｎｔｅｒａｃｔｉｏｎａｎａｌｙｓｉｓ ,ＢＩＡ)是进行蛋白质相互作用分析的较好技术 ,将两者结合起来在蛋白质组…     

The European Proteomics Association (EuPA) is in the field. Report of the formal inauguration of the European Proteome Association (Munich, Germany, August 29, 2005)

After one year of preparation the European Proteomics Association (EuPA) was formally inaugurated on August 29, 2005, on the occasion of the 4(th) HUPO World Congress in Munich, Germany. Delegates from 16 European countries elected Dr. Friedrich Lottspeich, President of the German Proteome Society, as the first EuPA President. The EuPA Board also comprises Professor Mathias Uhlen as Vice President, along with Professor Michael Dunn (Ireland), Professor Concha Gil (Spain), Dr. Jean Charles Sanchez (Switzerland) and Professor Pier Giorgio Righetti (Italy) as Coordinators for the presently defined focus activities of the EuPA. The general objectives of the EuPA are to promote proteomic activities throughout Europe, emphasising the benefits and contribution of proteomics to biological researchers, industry, the general public and politicians.     

Is protein overlap in two‐dimensional gels a serious practical problem?

In a recently published article, Campostrini et al. [Proteomics 2005, 5, 2385-2395] raised questions regarding the utility of 2-D gels in proteomics research. We believe that the authors have overlooked several key issues including the dynamic range of protein expression and the sensitivity of the analytical methods used to explore a proteome. We argue that 2-D gels have and will continue to provide meaningful quantitative data when applied to proteomic analysis and that the practical significance of spot overlap has been overstated.     

Proteome-based identification of signal peptides for improved secretion of recombinant cyclomaltodextrin glucanotransferase in Escherichia coli

Secretion of recombinant proteins in heterologous host has drawn attention for its simpler purification and downstream processes. Searching for secretion aid molecules to improve protein secretion can be done through synthetic biology, screening of genome data and proteome-based approach. In the present study, the extracellular proteome on starch-containing medium of Bacillus lehensis G1 was analyzed to identify naturally secreted proteins with signal peptide. A total of 87 protein spots were identified by mass spectrometry, which were categorized mostly in the metabolism of carbohydrates and related molecules (20%). Over-expression and secretion studies were performed for all the 14 selected signal peptides fused to a reporter protein, cyclomaltodextrin glucanotransferase (CGTase). All clones were found to allow CGTase to be excreted into the medium, as observed and measured from the iodine plate assay and enzyme activity assay. Compared to native signal peptide (G1) of CGTase, signal peptide of GlcNAc-binding protein A (GAP) significantly improved CGTase activities by 735% and 205% in extracellular and periplasmic compartment, respectively, with an increase of only ∼1.7 fold the amount of β-galactosidase (cell lysis) in the medium. GAP has the highest secretion rate of 45.6 U/ml/h among all clones, where physicochemical characteristics of signal peptide play significant role.     

Analysis of the Shewanella oneidensis proteome by two-dimensional gel electrophoresis under nondenaturing conditions

Proteomes are dynamic, i.e., the protein components of living cells change in response to various stimuli. Protein changes can involve shifts in the abundance of protein components, in the interactions of protein components, and in the activity of protein components. Two-dimensional gel electrophoresis (2-DE) coupled with peptide mass spectrometry is useful for the analysis of relative protein abundance, but the denaturing conditions of classical 2-DE do not allow analysis of protein interactions or protein function. We have developed a nondenaturing 2-DE method that allows analysis of protein interactions and protein functions, as demonstrated in our analysis of the cytosol and crude membrane fractions of the facultative anaerobe Shewanella oneidensis MR-1. Our experiments demonstrate that enzymatic activity is retained under the sample and protein separation methods described, as shown by positive malate dehydrogenase activity results. We have also found protein interactions within both the soluble and membrane fractions. The method described will be useful for the characterization of the functional proteomes of microbial systems.     

Proteome dynamics in complex organisms: using stable isotopes to monitor individual protein turnover rates

The complete definition of changes in a proteome requires information about dynamics and specifically the rate at which the individual proteins are turned over intracellularly. Whilst this can be achieved in single-cell culture using stable isotope precursors, it is more challenging to develop methods for intact animals. In this study, we show how dietary administration of stable isotope-labelled amino acids can obtain information on the relative rates of synthesis and degradation of individual proteins in a proteome. The pattern of stable isotope-labelling in tryptic peptides can be deconstructed to yield a highly reliable measure of the isotope abundance of the precursor pool, a parameter that is often difficult to acquire. We demonstrate this approach using chickens fed a semisynthetic diet containing [(2)H(8)]valine at a calculated relative isotope abundance (RIA) of 0.5. When the labelling pattern of gel-resolved muscle proteins was analyzed, the intracellular precursor isotope abundance was 0.35, consistent with dilution of the amino acid precursor pool with unlabelled amino acids derived from degradation of pre-existing proteins. However, the RIA was stable over an extended labelling window, and permitted calculation of the rates of synthesis and degradation of individual proteins isolated by gel electrophoresis. For the first time, it is feasible to contemplate the analysis of turnover of individual proteins in intact animals.     

Upregulation of dihydropyrimidinase-related protein 2, spectrin α II chain, heat shock cognate protein 70 pseudogene 1 and tropomodulin 2 after focal cerebral ischemia in rats—A proteomics approach

In recent years, there are an increasing number of proteomics studies that investigated the alterations in the protein expression relevant to human diseases but none for stroke. We, therefore, attempted such a study in a paradigm of focal cerebral ischemia in rat. Rats were subjected to cerebral ischemia by unilateral occlusion of the middle cerebral artery. Global protein analysis was performed after 24 h on the lesioned and sham-control cerebral cortex using two-dimensional gel electrophoresis. Protein spots with more than a 3-fold change in intensity were identified by mass spectrometry. Middle cerebral artery occlusion (MCAO) caused infarct volume of 18–22% predominantly in the cortex of the lesioned hemisphere. Two-dimensional gel electrophoresis resolved about 1500 protein spots of which only 12 were significantly upregulated by 3–46-fold. Three spots were identified to be dihydropyrimidinase-related protein 2 (DRP-2, also known as collapsin response mediator protein 2 (CRMP-2) or turned on after division, 64 kD protein (TOAD-64)). The spots varied in pI values only and this may reflect different phosphorylation status of the same protein. Two spots were identified as spectrin α II chain (rat fragment, also known as α-fodrin or non-erythroid α chain, SPNA-2); and one spot each for heat shock cognate protein 70 pseudogene 1 (HSC70-ps1, also known as heat shock protein 8 pseudogene 1), and tropomodulin 2 (Tmod2). The upregulation of protein expression was corroborated by observed upregulation of mRNA expression. The remaining five spots were not identified satisfactorily. As DRP-2, spectrin, and Tmod2 are involved in axonal and neurite growth as well as synaptic plasticity and maturation, the presently observed upregulation of the expression of these proteins may indicate active neuroregeneration and repair at 24 h after the induction of cerebral ischemia.