蛋白质组

蛋白质组研究是近来兴起的生命科学的前沿领域,是生命科学进入后基因组时代的标志之一。蛋白质组是指基因组表达的全部蛋白质,蛋白质组研究在细胞或组织水平上对蛋白质进行大规模的分离和分析。目前蛋白质组研究采用的主要技术是双向凝胶电泳和质谱方法。本文就蛋白质组研究和蛋白质组学的发展过程,研究意义,所涉及的研究范围及主要的研究策略,及最新的进展作一综述。     

基于生物质谱的蛋白质组学绝对定量方法研究进展

蛋白质组学定量方法包括相对定量和绝对定量两部分。相对定量蛋白质组学（也称比较蛋白质组学）是指对不同生理病理状态下细胞、组织或体液蛋白质表达量的相对变化进行比较分析;绝对定量蛋白质组学是测定细胞、组织或体液蛋白质组中每种蛋白质的绝对量或浓度。目前蛋白质组学的绝对定量方法主要有基于内标法的蛋白质组学绝对定量方法和基于质谱数据统计分析的非标记方法。蛋白质组学定量的信息可以帮助了解蛋白质在相互作用网络中所起的作用;通过对临床生物标记物绝对量的分析,可以帮助直观地判断疾病的发生和发展过程,这对于临床诊断和疾病治疗都具有现实的指导意义。     

RPLC—SDS—PAGE整体蛋白质分离与纳升毛细管液相色谱-串联质谱联用技术的研究及应用

采用RPLC-SDS-PAGE与纳升毛细管液相色谱-串联质谱联用技术对国际人类蛋白质组织(HUPO)提供的法国人肝脏蛋白质组表达谱的构建及其理化性质的统计分析,鉴定了2552条肽段,归结于402种蛋白质和240个蛋白质簇.蛋白质的pI分布范围为4.29～11.57,分子量范围为8,593～3,816,218Da,其中大于100kDa的蛋白质占到所鉴定蛋白质的10%,二者皆高于两维凝胶电泳的等电点和分子量范围(一般等电点3～10;分子量10～100kDa).另外,蛋白质组中不同蛋白质的疏水性分布显示大部分蛋白质为可溶性蛋白质,其中疏水性蛋白质占15%(GRAVY＞0的蛋白质).实验结果表明该技术路线兼顾了反相色谱分离度高和SAS-PAGE兼容性好的优点,可用于复杂组织或细胞中蛋白质组表达谱的构建或比较蛋白质组的研究,为蛋白质组学的方法学研究奠定了基础.     

我国蛋白质组学研究取得系列成果

由中国科学院院士贺福初领衔的北京蛋白质组研究中心／蛋白质组学国家重点实验室,在国际学术刊物《分子与细胞蛋白质组学》（Molecular ＆ Cellular Proteomics,MCP）2009年第三期上,同时发表了《亨廷顿疾病患者脑脊液的脑特异性蛋白含量下调》等3篇研究论文。3篇论文在该刊同期发表,创造了该刊单期同一单位发表论文数量之最。3篇研究论文分别从亨廷顿疾病（HD）发病机理、乙型肝炎病毒（HBV）相关疾病的诊断治疗方法、蛋白质组质谱数据筛查新模型等方面进行了深入研究。     

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。     

静电场轨道阱质谱的进展

静电场轨道阱是近年来新兴的一种质谱质量分析器,它利用离子在特定静电场中运动频率的不同对阱内离子进行质量分析,由于其具有较高的分辨率和质量准确度,被广泛应用于化学、生物学、医学等领域。本工作对静电场轨道阱质谱的形成过程和基本原理进行了详细介绍;对轨道阱质谱在蛋白质组学、代谢组学等方面的应用做了简要综述;对轨道阱质谱与常压电离技术联用的最新进展进行了评述,并指出电喷雾萃取电离、低温等离子体探针等常压质谱技术与轨道阱质谱的联用将在蛋白质分析、化学合成、化学反应机理研究等诸多领域发挥重要作用,旨在推动我国相关质谱仪器的国产化进程。     

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。     

非变性质谱相关技术的研究进展

蛋白质与其他分子的相互作用几乎在所有的生命活动中起着核心调控作用,这些相互作用力和形成的蛋白质复合物是现代生命科学的研究重点。由于传统的生物物理技术对蛋白质复合物和相互作用的研究存在样品纯度要求高的限制,因此迫切需要新技术的出现,为结构生物学和相互作用组学的研究提供补充。质谱技术可以从原理上对混合样品进行检测,降低对样品纯度的要求,其中非变性质谱展现出强大的连接与互补作用。本文从样品制备、离子源、质量分析器、质谱联用技术等4方面介绍非变性质谱相关技术及近年来的研究进展,并总结分析未来面临的挑战以及发展方向。     

SiLAD:一种蛋白质组学动态分析的新技术

随着蛋白质组学的产生和发展,对蛋白质组表达水平的差异和变化进行体内的和动态的分析已势所必然地成为蛋白质组学研究的发展趋势。但是传统差异蛋白质组学方法只能提供细胞或组织内各种蛋白累积总量的信息,而不是真正意义上的蛋白质差异表达分析。 SiLAD(~(35)S in vivo Labeling Analysis for Dynamic Proteomics)技术正是基于这种需求而在本实验室创立的。SiLAD技术由于其在差异检出的灵敏度和时间分辨率方面的明显优势,以及除提供蛋白质组表达动态以外,可以提供蛋白质代谢等相关动态变化的更多信息,所以可适用于对多种生物过程进行的动态蛋白质组学研究。本文对SiLAD技术的原理及特点进行简要综述。     

蛋白质组学的发展与科学仪器现代化

蛋白质组研究是当今生命科学发展的一个新的增长点。本文对蛋白组的历史及其重要性能做了简要论述,介绍了目前蛋白质组研究的两大主要技术,双向凝胶电泳和质谱技术,并对蛋白质组研究的应用领域和一些技术进展做了介绍。     

Applying proteomics technology to platelet research

Platelets are small enucleated cells that circulate in the blood, where they play a key role in hemostasis and contribute to the formation of vascular plugs. Pathologically, they are involved in thrombosis and heart disease. Because platelets do not have a nucleus, proteomics offers a powerful way to approach their biology. Proteomics technology is based on the huge analytical power offered by mass spectrometry in combination with several separation techniques, such as two-dimensional gel electrophoresis (2DGE) or multidimensional liquid chromatography. Proteomics provides information about the complete set of proteins present in platelets, the platelet proteome, including post-translational variants. Over the last years, several research groups have applied proteomics to platelet research. A detailed analysis of the general proteome and signaling cascades in human platelets will improve our knowledge of platelet function, and thereby aid the development of new therapeutic agents to treat thrombotic diseases. This review presents the major advances in mass spectrometry-based proteomics techniques and their application to platelet research, and analyzes in detail the most relevant contributions to the field.     

Mass spectrometry-based proteomics approaches applied in cataract research

Cataract, the opacification of the eye lens, is the leading cause of blindness worldwide--it accounts for approximately 42% of all cases. The lens fibers have the highest protein content within the body, more than 35% of their wet weight. Given the eye lens pure composition of highly abundant structural proteins crystallins (up to 90%), it seems to be an ideal proteomic entity to study and might be also hypothesized to model the other protein conformational diseases. Crystallins are extremely long-lived, and there is virtually no protein turnover. This provides great opportunities for post-translational modifications (PTM) to occur and to predispose lens to the cataract formation. Despite recent progress in proteomics, the human lens proteome remains largely unknown. Mass spectrometry hold great promise to determine which crystallin modifications lead to a cataract. Quantitative analysis of PTMs at the peptide level with proteomics is a powerful bioanalytical tool for lens-tissue samples, and provides more comprehensive results. New mass spectrometry-based approaches that are being applied to lens research will be highlighted. Finally, the future directions of proteomics cataract research will be outlined.     

A review of current applications of mass spectrometry for neuroproteomics in epilepsy

The brain is unquestionably the most fascinating organ, and the hippocampus is crucial in memory storage and retrieval and plays an important role in stress response. In temporal lobe epilepsy (TLE), the seizure origin typically involves the hippocampal formation. Despite tremendous progress, current knowledge falls short of being able to explain its function. An emerging approach toward an improved understanding of the complex molecular mechanisms that underlie functions of the brain and hippocampus is neuroproteomics. Mass spectrometry has been widely used to analyze biological samples, and has evolved into an indispensable tool for proteomics research. In this review, we present a general overview of the application of mass spectrometry in proteomics, summarize neuroproteomics and systems biology‐based discovery of protein biomarkers for epilepsy, discuss the methodology needed to explore the epileptic hippocampus proteome, and also focus on applications of ingenuity pathway analysis (IPA) in disease research. This neuroproteomics survey presents a framework for large‐scale protein research in epilepsy that can be applied for immediate epileptic biomarker discovery and the far‐reaching systems biology understanding of the protein regulatory networks. Ultimately, knowledge attained through neuroproteomics could lead to clinical diagnostics and therapeutics to lessen the burden of epilepsy on society. © 2009 Wiley Periodicals, Inc., Mass Spec Rev 29:197–246, 2010     

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.     

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。     

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.     

ICP‐MS‐Based strategies for protein quantification

In the post‐genomics era, proteomics has become a central branch in life sciences. An understanding of biological functions will not only rely on protein identification, but also on protein quantification in a living organism. Most of the existing methods for quantitative proteomics are based on isotope labeling combined with molecular mass spectrometry. Recently, a remarkable progress that utilizes inductively coupled plasma‐mass spectrometry (ICP‐MS) as an attractive complement to electrospray MS and MALDI MS for protein quantification, especially for absolute quantification, has been achieved. This review will selectively discuss the recent advances of ICP‐MS‐based technique, which will be expected to further mature and to become one of the key methods in quantitative proteomics. © 2009 Wiley Periodicals, Inc., Mass Spec Rev 29:326–348, 2010