蛋白质组与生物质谱技术

蛋白质组是基因组研究的继续。蛋白质组的研究是为了识别及鉴定一个基因组、一个细胞或组织所表达的全部蛋白质以及它们的表达模式。以基质辅助激光解吸附飞行时间质谱和电喷雾质谱为代表的现代生物质谱技术,为蛋白质组的研究提供了必要的技术手段,被称为蛋白质组研究的三大关键性支撑技术之一。而质谱—质谱联用、质谱与其它技术联用以及高产出筛选技术的应用和发展,将使蛋白质组的研究在高准确度、高灵敏度以及大规模化水平上的发展成为可能。     

走在生命科学的前沿——ABI公司蛋白质组研究技术平台简介

本文介绍了美国应用生物系统公司蛋白质组研究技术平台的技术途径和关键技术及其应用,并提供了相关文献供读者查阅。     

蛋白质组和科学仪器现代化

基因组载有生物体的全部遗传信息 ,基因组的测序的完成是重要的里程碑之一。科学的进展已进入功能基因组时代。因为蛋白质是生物功能的行者 ,因此蛋白质组成为功能基因组的重要前沿。蛋白质组是指基因组表达的全部蛋白质。蛋白质组学是在细胞水平上研究各种蛋白质的性质 (比如表达水平 ,翻译后的修饰 ,蛋白质相互作用等 ) ,以便得到疾病过程 ,生理过程和生化过程中的整体的 ,整合的概况和在蛋白质水平上的调节网络。2 0 0 1年 4月在美国成立了ＨＵＰＯ(ＨｕｍａｎＰｒｏ ｔｅｏｍｉｃｓＯｒｇａｎｉｚａｔｉｏｎ) ,研究重点为人类疾病与健康…     

比较蛋白质组研究方法学进展

比较蛋白质组学针对不同空间、不同时间上动态变化着的蛋白质组的整体进行比较,分析不同蛋白质组之间在表达数量、表达水平和修饰状态上的差异,可以发现与病变相关的特异蛋白质,对疾病的诊断和治疗具有重要意义.本文对比较蛋白质组研究在方法学方面的最新进展加以系统综述,引用文献33篇.     

蛋白质组研究前沿技术

随着人类基因组计划的完成，蛋白质组研究已成为21世纪生命科学发展的先导，成为生命乃至自然科学最活跃的学科领域。2003年底，国际人类蛋白质组计划启动，“人类肝脏蛋白质组计划(HLPP)”和“人类血浆蛋白质组计划(HPPP)”两大项目首先开始执行，其中HLPP由中国科学家领导执行，将承担整个国际蛋白质组计划的20％以上的任务。     

蛋白质组研究中的分离方法

蛋白质组包括一个细胞或生命体中所有的蛋白质，对蛋白质组的研究是一种非常复杂、并具冒险性的行为。其中心除了基因表达的研究外，首先是对特定蛋白质的结构进行鳃析和辩认。通过联用技术，如液相色谱-质谱（LCMS／MS）联用或高效液相色谱-电喷雾离子源双质谱（HPLC／ESI／MS／MS）联用，可以将蛋白质从混合物中分离出来，并进行表征。[编者按]     

人唾液蛋白质组的纳升液相色谱-高分辨串联质谱鉴定及高丰度蛋白质分析

常规的定性分析质谱数据能否以及如何应用于蛋白质组学的相对定量分析是实际工作中经常遇到的问题。本研究采用一维纳升液相色谱-高分辨串联质谱（nano-HPLC-Triple TOF 5600）法鉴定人唾液蛋白质组,并分析其基本组成,以探讨质谱数据与唾液中高丰度蛋白质的相关性。本实验共鉴定了6044条特异性酶切肽段,归属于α-淀粉酶1、碳酸酐酶6、血清白蛋白、粘蛋白、半胱氨酸蛋白酶抑制剂和免疫球蛋白等各种已知的高丰度蛋白质在内的521个蛋白,这为一维纳升液相色谱分离条件下的唾液蛋白质组的基本组成分析提供了参考。结果表明,仅用蛋白质排序指标Unused值表征蛋白质的相对含量具有局限性,而蛋白质排序、检出肽段数目和肽段平均强度等综合指标与蛋白质相对含量具有正相关性,即排名较后的蛋白质均只有1条肽段被检出,且肽段强度较低。这说明,当蛋白质相对含量差别较大时,肽段的化学性质和电离效率差异对质谱信号的影响已不占主要地位,即可以认为当肽段检出数目、鉴定覆盖率和肽段强度均较低时,蛋白质的相对含量也较低。该结果可为利用Triple TOF 5600质谱仪的定性鉴定数据进行初步半定量判断提供参考,也可为唾液蛋白质组生物标志物研究提供借鉴。     

一种基于iSRM策略的蛋白质组质谱数据分析工具

由于重复性好、定量精度高,基于质谱的选择反应监测（SRM）技术在蛋白质定量分析中的应用越来越广泛。智能选择反应监测（iSRM）是一种新型的SRM实验策略。针对该策略产生的质谱数据,开发了一个肽段定量信息提取工具--iSQuant。该工具使用MATLAB脚本语言编写,简便易用。利用重复实验数据和标准实验数据对iSQuant的性能评估表明,iSQuant具有优越的可重复性能,定量结果和理论丰度线性度很高。     

美国应用生物系统公司与北京蛋白质组研究中心蛋白质组合作实验室成立

[本刊讯]2003年12月15日，北京蛋白质组研究中心在中关村生命科学园举行了入驻签字仪式，并宣告成立。该中心是在国家科技部和北京市科委的支持下，由军事医学科学院、中国科学院、中国医学科学院、清华大学、北京大学、江中集团以及北京新医药和生物技术发展促进中心等单位共同发起组建的国家级蛋白质组研究机构。集聚了以贺福初院士领衔的几十位海内外科学家，是促进中国蛋白质科学整体发展的科教研综合基地。同时，经国际人类蛋白质组组织（HUPO）认可，该中心已成为人类肝脏蛋白质组计划（HLPP）的执行总部，整体负责其组织管理、交流与合作、教育与培训、数据集散等工作;并经国家科技部和中国人类蛋白质组组织（CNHUPO）认可，该中心成为中国人类肝脏蛋白质组计划（CNHLPP）的牵头和主要承担单位。目前，中心已建立蛋白质表达谱、蛋白质修饰谱、蛋白质相互作用、蛋白质定位、抗体工程、生物信息学、功能蛋白质组、功能基因组、蛋白质组新技术和蛋白质工程等高通量的技术平台。     

Proteome analysis of apoptotic cells

Apoptosis, a genetically determined form of cell death, is a central and complex process involved in the development of multicellular organisms in the maintenance of cell homeostasis. During apoptosis, a large number of proteins involved in transducing signals are posttranslationally modified. Classical proteomics, the combination of protein separation by two-dimensional gel electrophoresis (2DGE) and protein identification by mass spectrometry (MS), enabled the discovery of more than 100 proteins altered during apoptosis. Functional data about protein degradation, modification, translocation, and synthesis were obtained. In addition to classical proteomics, some specifically designed proteome studies were carried out to analyze specific apoptotic components such as the mitochondrial releasing factors, death-inducing signaling complex (DISC), inhibitor of apoptosis (IAP) interacting proteins, and caspases. The identification of main regulators significantly influenced the elucidation of the concept underlying apoptosis signaling. Thus, the application of detailed protein analytical methods in the young field of apoptosis research was particularly fruitful.     

假单胞菌SJTD-1全蛋白质组学研究

近年来,石油泄漏对海洋和土壤的污染事件频发,解决这一污染问题非常重要。本实验从原油污染的土壤中分离得到一株新的铜绿假单胞菌株SJTD-1,使用SDS-PAGE-1DLC-MS和offline-2DLC-MS两种方法研究其全蛋白质组。利用这两种技术平台分别鉴定获得1453个和1623个可信蛋白,共鉴定到1912个可信蛋白。通过比较两种技术路线所鉴定到蛋白种类的差异,发现offline-2DLC-MS有利于SJTD-1菌株的膜蛋白、相对分子质量较大的蛋白和pI值不大于8的蛋白的鉴定。另外,对所有鉴定到的可信蛋白进行GO功能注释,亚细胞定位分析和代谢通路富集分析。通过对分离获得的SJTD-1菌株进行蛋白质组学分析,可为铜绿假单胞菌SJTD-1分解环烷烃以及SJTD-1在生物修复应用中的研究奠定基础。     

蛋白质组分析的开门技术--双向电泳

本文简述了蛋白质组学研究的主要技术之一———双向电泳的原理、方法、发展概况和相关设备的进展 ,并介绍了一些国际互联网上的双向电泳资源     

Proteome analysis of tissues by mass spectrometry

Tissues and biofluids are important sources of information used for the detection of diseases and decisions on patient therapies. There are several accepted methods for preservation of tissues, among which the most popular are fresh‐frozen and formalin‐fixed paraffin embedded methods. Depending on the preservation method and the amount of sample available, various specific protocols are available for tissue processing for subsequent proteomic analysis. Protocols are tailored to answer various biological questions, and as such vary in lysis and digestion conditions, as well as duration. The existence of diverse tissue‐sample protocols has led to confusion in how to choose the best protocol for a given tissue and made it difficult to compare results across sample types. Here, we summarize procedures used for tissue processing for subsequent bottom‐up proteomic analysis. Furthermore, we compare protocols for their variations in the composition of lysis buffers, digestion procedures, and purification steps. For example, reports have shown that lysis buffer composition plays an important role in the profile of extracted proteins: the most common are tris(hydroxymethyl)aminomethane, radioimmunoprecipitation assay, and ammonium bicarbonate buffers. Although, trypsin is the most commonly used enzyme for proteolysis, in some protocols it is supplemented with Lys‐C and/or chymotrypsin, which will often lead to an increase in proteome coverage. Data show that the selection of the lysis procedure might need to be tissue‐specific to produce distinct protocols for individual tissue types. Finally, selection of the procedures is also influenced by the amount of sample available, which range from biopsies or the size of a few dozen of mm² obtained with laser capture microdissection to much larger amounts that weight several milligrams.     

Proteome analysis in the study of lymphoma cells

This review provides an overview on recent studies in the field of proteome analysis of lymphoma cells, and highlights the potentials of such studies for a better knowledge of drug effects at the molecular level. After giving general information on the field of proteome analysis of lymphoma cells, some characteristics of the strategies used during this analysis are pointed out, such as cell extraction strategies and affinity captures. Therefore, the issue of proteome analysis of lymphoma cells content will be covered with respect to those protein extracts that can be prepared in saline solutions, such as cytoplasm proteins, or that are associated with the cell membranes. The question of which kinds of information have been retrieved from lymphoma-cell proteomics is discussed on the basis of several examples-lymphoma cell-mapping studies and constitution of protein databases, and comparative proteome analysis studies of the modifications that result from a drug treatment.     

Proteome analysis in the study of the bacterial heat-shock response

In recent years, it has become clear that, in addition to the regulation of the expression of specific genes, there are global regulatory systems that control the simultaneous expression of a large number of genes in response to a variety of environmental stresses. The first of these global control systems, and of substantial importance, is the heat-shock response. The heat-shock response is characterized by the induction of a large set of proteins (heat-shock proteins-HSPs) upon shifts to higher temperature and upon exposure to conditions in which proteins are denatured (i.e., alcohols, heavy metals). The heat-shock response is universal and many of the heat-shock proteins are highly conserved among species. In bacteria, the heat-shock response has been studied extensively in several Gram-positive bacteria (Bacillus subtilis) and in the Gram-negative bacteria (i.e., Escherichia coli, Agrobacterium tumefaciens). The first recognition of the molecular abundance of the bacterial heat-shock proteins took place with the introduction of high-resolution two-dimensional polyacrylamide gels (2D gels) to analyze complex mixtures of cellular proteins. Two-dimensional gels, followed by mass spectrometry, were used to define the heat-shock stimulons in several bacteria, and to study the regulatory elements that control the heat-shock response. Here, we review the heat-shock response and its regulation in bacteria. The review will emphasize the use of proteome analysis in the study of this response, and will point out those open questions that can be investigated with proteomics, including mass spectrometry techniques.     

热蛋白质组学分析鉴定TH588的靶标蛋白

通过对活性小分子靶标蛋白的鉴定,可以建立活性小分子与细胞表型之间的联系,阐明活性小分子的功能和作用机理,对小分子药物的研发具有重要意义.热蛋白组学分析(thermal proteome profi-ling,TPP)是基于蛋白-配体结合可以增加蛋白热稳定性这一原理发展的鉴定活性小分子靶标蛋白的新技术,相较于其他小分子靶...     

大弹涂鱼肝脏蛋白质双向电泳技术的建立及优化

通过对不同裂解液配方、等电聚焦程序和SDS—PAGE方式等的对比实验,建立和优化了大弹涂鱼肝脏蛋白质组双向电泳的相关技术体系。结果显示采用裂解液中添加Tris和TBP,聚焦时适当延长除盐时间,提高聚焦电压和功率（伏-小时）,能显著提高双向电泳图谱的分辨率,MSOG程序进行双向电泳（Multi—strips on One Gel MSOG）,提高了双向凝胶的匹配率及有效性,降低了人为修饰点的增加,匹配率高达90％。通过相关条件优化提高了大弹涂鱼肝脏蛋白双向电泳图谱的分辨率,为后续大弹涂鱼的毒理蛋白质组学研究提供技术保障。