蛋白质组学研究相关技术及进展

蛋白质组学以蛋白质组为研究对象,应用相关研究技术,从整体水平上来认识蛋白的存在及活动方式。随着人类基因组计划的完成,蛋白质组学的研究也得到了快速发展,与蛋白质组学研究相关的一些技术也日益得到完善和提高。简要综述了近年来蛋白质组学研究中最为重要的样品制备、蛋白质分离、蛋白质鉴定等技术及研究进展。     

蛋白质芯片在蛋白质组学研究中的作用

蛋白质芯片是以高度并行性、高通量、微型化和自动化为特点的蛋白质组检测技术。本文综述了蛋白质芯片在蛋白质组学研究中的多种作用,包括普通蛋白质芯片在微量蛋白质分离、蛋白质与蛋白质之间以及蛋白质与其他小分子间相互作用和蛋白质定量检测方面的作用,普通蛋白质芯片通过与质谱技术、生物传感器技术的结合而拓展其应用范围,以及蛋白质组芯片、活性的蛋白质芯片在蛋白质组学研究中应用的进展。     

蛋白质NMR样品制备技术

在结构基因组学和结构生物学的研究中,核磁共振技术被广泛地用于测定蛋白质在溶液中的空间结构及其动力学,研究蛋白质与配体的相互作用。但是,核磁共振技术的应用在很大程度上受到样品制备技术的制约。该文就蛋白质NMR样品的制备技术进行综述,主要介绍几种常用的重组蛋白质表达体系、蛋白质同位素标记技术以及缓冲液的选择等方面的进展。     

数据挖掘在蛋白质组学研究中的应用

介绍蛋白质组学研究的背景;阐述数据挖掘的原理、方法,重点讲述数据挖掘技术在蛋白质组学研究中取得的新的进展。最后,对数据挖掘目前存在的问题作分析,并对它的发展的前景作展望。     

蛋白质组学技术在感染研究中的应用

蛋白质组学的目标在于阐明特定生物体、组织、细胞或亚细胞结构中全部蛋白质的表达模式和功能模式,其技术平台由高通量的蛋白质分离技术、鉴定技术和生物信息学组成。在许多研究领域,蛋白质组学技术为阐明疾病过程和生命现象的分子机制提供了全面、网络和动态的蛋白质组信息。感染是重要的基本致病因素之一,蛋白质组学的研究策略和技术方法有利于快速分离鉴定病原体蛋白质组、宿主免疫细胞蛋白质组、感染相关蛋白、疫苗候：选抗原蛋白、生物标志物和药物靶标,从而明显加快病原体、宿主反应、感染发病机制以及感染预防、诊断和治疗等相关研究的进程。     

Phos-tag技术在磷酸化蛋白质组学中的应用

Phos-tag是新研制出的一种对磷酸基团具有特殊亲和力的化合物。由于其对磷酸化蛋白质具有高特异性、高亲和力等特点使其迅速在磷酸化蛋白质的检测、分离和纯化等方面得到广泛的应用。本文综述了Phos-tag的化学性质、原理及其近年来在磷酸化蛋白质组学中的应用,并与传统的磷酸化蛋白质组学研究技术做了比较,对未来磷酸化蛋白质组学的研究技术作了展望。     

蛋白质组学技术在网络药理学中的应用

蛋白质组学发展至今已日趋成熟,在生物医药相关领域研究中的应用显著增加,与之相关的样品制备技术、蛋白定量方法及先进的质谱仪器也得到了快速发展。网络药理学是近年来提出的新药发现新策略,是药理学的新兴分支学科,它从整体的角度探索药物与疾病的关联性,发现药物靶标,指导新药研发。将蛋白质组学技术应用于网络药理学研究,能使研究人员系统地预测和解释药物的作用,加速药物靶点的确认,从而设计多靶点药物或药物组合。综述了蛋白质组学技术的新近研究进展,并简单概述了其在网络药理学中的应用。     

胆管癌胆汁蛋白质样品制备和双向电泳条件的优化

采用合适的裂解液和沉淀方法,提取胆管癌胆汁中的蛋白质,获得分辨率高、重复性好的蛋白质双向电泳图谱.通过不同裂解液和蛋白质沉淀方法提取胆汁蛋白的效果比较,设计了不同的样品制备方法,并且对双向电泳(2-DE)的条件进行优化.结果显示,试验确定了适合胆管癌胆汁的裂解液配方(LSⅣ),丙酮沉淀的蛋白分布完整,沉淀效率相对较高.高伏时、长时间的等电聚焦可以获得高分辨率、重复性好的蛋白质双向电泳图谱.因此,本方法可以应用到胆管癌胆汁蛋白的提取,也可以对其他体液蛋白质样品的制备和双向电泳提供借鉴.     

现代质谱技术在蛋白质组学中的应用及其最新进展

简述了蛋白质组学的概念、内容和意义,重点综述了现代质谱技术在蛋白质组学中的应用,主要包括蛋白质和肽段的鉴定和定量、蛋白质翻译后修饰的鉴定和蛋白质间相互作用的检测等。随着新的高质量精确度、分辨率、灵敏度和通量质谱仪的出现,现代质谱技术在蛋白质组学中的应用将越来越广泛,并给蛋白质组学研究带来新的机遇。     

酵母双杂交技术及其在蛋白质组研究中的应用

蛋白质组学是后基因组时代出现的一个新兴的研究领域,它的主要任务是识别鉴定细胞,组织或机体的全部蛋白质,并分析蛋白质的功能及其模式。因此,揭示蛋白质组中蛋白质间的相互作用关系也是蛋白质组学的重要内容之一。酵母双杂交技术是用来检测蛋白质间是否相互作用的一个非常有效的手段,该技术在酵母蛋白质组研究中的初步成功应用,表明它有望在人类蛋白质且研究中发挥重要作用。     

基于超滤辅助样品制备方法的优化简

目的：基于超滤辅助样品制备（FASP）方法的出现使得使用去污剂（如SDS）的蛋白质提取方法与溶液内酶切方法得以兼容,因此提高了难溶性蛋白的鉴定数量。然而,超滤膜的非特异性吸附作用依然会造成蛋白的损失。我们拟针对该方法存在的问题对其进行改进。方法：对FASP方法进行了蛋白酶切条件、洗脱液选择、洗脱次数的改进;为测试优化方法的有效性和适用范围,选择标准蛋白BSA、鼠肝和鼠脑等3种样品进行考察。结果：相比报道的FASP方法,采用改进后的FASP方法使BSA的回收率提高了20％;经高精度质谱检测,对鼠肝、鼠脑的蛋白质鉴定结果分别比采用未优化的FASP多鉴定到2086和3592条特异肽段。结论：通过对FASP方法的优化,蛋白鉴定数量得到较大提高,该方法为蛋白质组深度覆盖研究提供了可靠的技术手段。     

Concepts and strategies of soybean seed proteomics using the shotgun proteomics approach

ABSTRACT

Introduction: The last decade has yielded significant developments in the field of proteomics, especially in mass spectrometry (MS) and data analysis tools. In particular, a shift from gel-based to MS-based proteomics has been observed, thereby providing a platform with which to construct proteome atlases for all life forms. Nevertheless, the analysis of plant proteomes, especially those of samples that contain high-abundance proteins (HAPs), such as soybean seeds, remains challenging.

Areas covered: Here, we review recent progress in soybean seed proteomics and highlight advances in HAPs depletion methods and peptide pre-fractionation, identification, and quantification methods. We also suggest a pipeline for future proteomic analysis, in order to increase the dynamic coverage of the soybean seed proteome.

Expert opinion: Because HAPs limit the dynamic resolution of the soybean seed proteome, the depletion of HAPs is a prerequisite of high-throughput proteome analysis, and owing to the use of two-dimensional gel electrophoresis-based proteomic approaches, few soybean seed proteins have been identified or characterized. Recent advances in proteomic technologies, which have significantly increased the proteome coverage of other plants, could be used to overcome the current complexity and limitation of soybean seed proteomics.     

Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale quantitative analysis of formalin fixed and paraffin embedded (FFPE) tissue. The procedure comprises four distinct steps. The first one is the preparation of sections from the FFPE material and microdissection of cells of interest. In the second step the isolated cells are lysed and processed using ''filter aided sample preparation'' (FASP) technique. In this step, proteins are depleted from reagents used for the sample lysis and are digested in two-steps using endoproteinase LysC and trypsin. After each digestion, the peptides are collected in separate fractions and their content is determined using a highly sensitive fluorescence measurement. Finally, the peptides are fractionated on ''pipette-tip'' microcolumns. The LysC-peptides are separated into 4 fractions whereas the tryptic peptides are separated into 2 fractions. In this way prepared samples allow analysis of proteomes from minute amounts of material to a depth of 10,000 proteins. Thus, the described workflow is a powerful technique for studying diseases in a system-wide-fashion as well as for identification of potential biomarkers and drug targets.     

Comparative evaluation of pectolytic and proteolytic enzyme production by free and immobilized cells of some strains of the phytopathogenic Erwinia chrysanthemi

Whole cells of the phytopathogenic Erwinia chrysanthemi strains were immobilized in k-carrageenan and grown in high-calcium Xanthomonas campestris medium containing sodium polypectate as carbon source. All the strains used survived immobilization into k-carrageenan beads. Immobilized E. chrysanthemi strains displayed higher pectolytic and proteolytic enzyme activities than free cells in liquid suspension. Carrageenan immobilization techniques could provide a system to mimic the conditions of E. chrysanthemi cells in the infected plant tissue. This could prompt a thorough study of the factors governing the biosynthesis of virulence factors by this bacterium. Journal of Industrial Microbiology & Biotechnology (2001) 27, 215–219. Received 04 April 2001/ Accepted in revised form 12 June 2001     

葡萄蛋白质组学研究进展

人类基因组计划的完成标志着生命科学已进入后基因组时代,蛋白质组学的研究被提升到了前所未有的高度,蛋白质组学旨在阐明基因组所表达的真正执行生命活动的全部蛋白质的表达规律和生物功能。伴随葡萄基因组测序工作的完成,有关葡萄蛋白质组学的研究迅速发展。对近年来蛋白质组学在葡萄上的研究进行了综述,内容主要包括:葡萄蛋白质样品的提取制备,葡萄果实发育和品质形成过程中蛋白质组的变化,葡萄果皮、细胞壁、质膜等特定组织材料的蛋白质组研究,及蛋白质组学在葡萄逆境胁迫、体细胞胚的发生等方面的研究,并对葡萄蛋白质组学的发展趋势进行了展望。     

Purification et propriétés d'une protéase neutre de Tricholoma columbetta

An endopeptidase was partially purified from the fruiting bodies of Tricholoma columbetta. The enzyme, MW ca 14 500, pH optimum 7.0, hydrolyses the natural substrates, casein and fibrin, but is inactive with the usual synthetic substrates. It is relatively insensitive to most inhibitors of proteases except for high concentrations of KCN, whereas high concentrations of EDTA greatly increase its caseinolytic activity.     

Enhancement of the acrosome reaction of hamster spermatozoa by the proteolytic enzymes,kallikrein, trypsin,and chymotrypsin

The involvement of a kallikrein−kinin system in the motility of mammalian spermatozoa has been suggested by several investigators. We found that incorporation of kallikrein (0.1–1.0) unit/ml) in the sperm incubation medium did not enhance the motility of hamster spermatozoa that were already active. However, this enzyme significantly increased the incidence of the acrosome reaction. Trypsin (1.8–18 units/ml) and chymotrypsin (0.34–3.4 units/ml) also increased the incidence of the acrosome reaction, and accelerated its onset. Kinins (bradykinin and kallidin) added to the medium in a wide concentration range (1 ng/ml to 1 mg/ml) had no marked effects on either the motility or the acrosome reaction. A kallikrein−kinin system is apparently not of primary importance at least for the acrosome reaction. The enhancement of the acrosome reaction by exogenous proteinases may be due in part to accelerated removal or alteration of the sperm surface coat (glycoprotein) by the enzyme peior to the acrosome reaction. Exogenous proteinases may also act synergistically with endogenous (acrosomal) proteinases (and other enzymes) in altering membrane proteins and dispersing the acrosome matrix during the course of teh acrosome reaction.     

An aminopeptidase from Agave americana,chemical properties of the enzyme

Agave aminopeptidase, a new enzyme obtained from the plant Agave americana displayed activity towards a variety of substrates. A free alphaamino group on these substrates was essential, but the enzyme did not need any metal ions for optimal activity. Aliphatic, aromatic and basic amino acids situated at the amino terminal end of substrates could be hydrolysed by the enzyme. The enzyme had no endopeptidase or other proteolytic activity. Values of the apparent Michaelis constants for different amino acid substrates, all in the range from 0.1 to 0.6 × 10^?3 M, suggested a relative wide specificity. The pK-values of the two dissociating groups on the enzyme taking part in the catalytic process were pH 6.3 to 6.8 and pH 7.5 to 7.8. These and other studies suggested that histidine plays an active role in the catalytic process. The enzyme was inhibited competitively by free amino acids and this, together with other results, implied a compulsory order of product release.