化学交联质谱技术的研究进展

化学交联质谱技术是解析蛋白质结构和研究蛋白质相互作用的重要工具。近5年以来,该技术在方法和应用上都取得了很大的进步。方法上,一方面可断裂交联剂与新型分离富集方法展现了较好的应用前景,另一方面更加高效的交联肽段搜索引擎和质量控制方法为交联质谱数据分析提供了有力的工具。应用上,一方面与冷冻电镜技术结合解析了大量蛋白质的结构,另一方面从研究蛋白质复合物的相互作用发展到研究全蛋白质组水平的相互作用网络。化学交联质谱技术在方法和应用上的蓬勃发展,体现了这一技术的重要作用。本文对化学交联质谱技术的各个环节进行了详细的综述,包括交联剂选择、交联反应、酶切、交联肽段富集、液质联用、交联肽段鉴定、质量控制和生物学应用,重点介绍了最近5年的研究进展。最后,讨论了化学交联质谱技术面临的挑战及未来的发展方向。     

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.     

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…     

生物质谱技术在蛋白质组学研究中的应用

随着技术的进步,蛋白质组学的研究重心由最初旨在鉴定细胞或组织内基因组所表达的全部蛋白质转移到从整个蛋白质组水平上阐述包括蛋白翻译后修饰、生物大分子相互作用等反映蛋白质功能的层次。多种质谱离子化技术的突破使质谱技术成为蛋白质组学研究必不可少的手段。质谱技术联合蛋白质组学多角度、深层次探索生命系统分子本质成为现阶段生命科学研究领域的主旋律之一。本文简要综述了肽和蛋白质等生物大分子质谱分析的原理、方式和应用,并对其发展前景做出展望。     

质谱MRM技术在蛋白质组学研究中的应用

质谱多反应监测(multiple reaction monitoring,MRM)技术是一种基于已知信息或假定信息有针对性地获取数据,进行质谱信号采集的技术,具有灵敏、准确和特异等优点.在基于蛋白组学的生物标志物研究、蛋白质翻译后修饰.定量蛋白质组和蛋白质相互作用等研究领域的应用逐渐受到重视.该文概述了该技术在蛋白质组学研究中的应用特点及其最新应用进展.     

双杂交和质谱技术在蛋白质-蛋白质相互作用研究中的应用

基因的功能是由蛋白质来执行的,而蛋白质要通过与其他生物分子相互作用来完成其各种生物功能。因此,如果能够快速做出蛋白质在不同时间、空间和不同环境中的相互作用图谱,就会帮助我们了解这些蛋白质的功能,进而了解许多生命活动的机制。目前,用于大规模研究蛋白质间相互作用的方法主要有酵母双杂交系统及其衍生系统、亲和纯化与质谱分析联用技术,前者用于研究蛋白分子间的两两相互作用,后者用于研究蛋白质复合物间的相互作用。本文主要阐述了酵母双杂交、细菌双杂交、哺乳动物细胞双杂交、亲和纯化与质谱联用技术在大规模蛋白质相互作用研究中的应用。     

蛋白质相互作用研究的新技术与新方法

目前,蛋白质相互作用已成为蛋白质组学研究的热点. 新方法的建立及对已有技术的改进标志着蛋白质相互作用研究的不断发展和完善．在技术改进方面,本文介绍了弥补酵母双杂交的蛋白定位受限等缺陷的细菌双杂交系统;根据目标蛋白特性设计和修饰TAP标签来满足复合体研究要求的串联亲和纯化技术,以及在双分子荧光互补基础上发展的动态检测多个蛋白质间瞬时、弱相互作用的多分子荧光互补技术．还综述了近两年建立的新方法：与免疫共沉淀相比,寡沉淀技术直接研究具有活性的蛋白质复合体;减量式定量免疫沉淀方法排除了蛋白质复合体中非特异性相互作用的干扰;原位操作的多表位－配基绘图法避免了样品间差异的影响,以及利用多点吸附和交联加固研究弱蛋白质相互作用的固相蛋白质组学方法.     

“自顶向下(top-down)”的蛋白质组学——蛋白质变体的规模化鉴定

高分辨率质谱技术的快速发展使得"自顶向下"的蛋白质组学(top-down proteomics)研究逐渐成熟起来.在完整蛋白质水平上研究蛋白质组可以提供更精准、更丰富的生物学信息,特别是对于蛋白质上发生了多种关联性的翻译后修饰的情况.另外,由于基因突变、RNA可变剪接和大量蛋白质翻译后修饰的存在,同一个基因往往最终会产生多个"蛋白质变体"(proteoform),而要准确地鉴定这些蛋白质变体,也离不开"自顶向下"的蛋白质组学.在蛋白质水平上的分离技术、质谱技术与生物信息学技术是完整蛋白质鉴定最关键的三项技术.高效的分离技术是实现规模化蛋白质变体鉴定的前提,有效的质谱碎裂是提供可靠鉴定的核心,而快速准确的质谱鉴定算法则是数据分析效率的保障.本文对这三项技术进行了详细总结,重点集中在生物信息学相关技术上,包括对完整蛋白质的质谱数据预处理、数据库搜索鉴定以及翻译后修饰定位等几个计算问题的讨论.     

质谱MRM技术在蛋白质组学研究中的应用

质谱多反应监测（multiple reaction monitoring,MRM）技术是一种基于已知信息或假定信息有针对性地获取数据,进行质谱信号采集的技术,具有灵敏、准确和特异等优点。在基于蛋白组学的生物标志物研究、蛋白质翻译后修饰、定量蛋白质组和蛋白质相互作用等研究领域的应用逐渐受到重视。该文概述了该技术在蛋白质组学研究中的应用特点及其最新应用进展。     

基于信息技术的蛋白质识别研究

随着质谱技术和NMR等仪器测定技术的飞速发展,利用大规模质谱技术可以得到海量蛋白质的质谱数据。同时,利用NMR技术也可获得蛋白质的精确三维结构数据。这些数据以及面向这些数据的分析方法使得蛋白质组研究迅速发展,其基础理论和技术方法,都在不断地进步和完善。利用信息工程技术辅助生物学家开展面向质谱技术的高温量蛋白识别,面向NMR的蛋白质三维结构识别,以及功能发现研究,已经成为蛋白质组中的生命科学与信息科学交叉研究的热点和挑战问题。本文侧重从信息技术的角度对上述问题进行综述,并提出我们解决问题的思路和方法。     

Errata

Abstract

Mass spectrometry (MS)-based proteomics is an unrivaled tool for studying complex biological systems and diseases in the post-genomic era. In recent years, MS has emerged as a powerful structural biological tool to characterize protein conformation and conformational dynamics. The advantages of MS in structural studies are most evident for membrane proteins such as GPCRs (G protein-coupled receptors), where other well-established structural methods such as X-ray crystallography and NMR remain challenging. For proteins with available high-resolution structures, MS-based structural strategies can provide valuable, previously inaccessible information on protein conformational changes and dynamics, protein motion/flexibility, ligand–protein binding, and protein–protein interfaces. In the past several years, we have developed and adapted a number of MS-based structural approaches, such as CDSiL-MS (Conformational changes and Dynamics using Stable-isotope Labeling and MS), CXMS (Crosslinking/MS) and HDXMS (Hydrogen-Deuterium Exchange MS), to study protein structures and conformational dynamics in human β2-adrenegic receptor (β2AR) signaling. In this mini-review, we will highlight several examples demonstrating the power of MS in structural analysis to better elucidate the structural basis of GPCR signaling, particularly through the β-arrestin-mediated GPCR signaling pathway.     

核磁共振波谱法在蛋白质三维结构解析中的应用

蛋白质特定的三维结构与其生物功能密切相关,因此,研究蛋白质的三维结构有助于揭示其生物功能机制。将核磁共振（NMR）波谱法应用于研究溶液状态下蛋白质的三维结构,能够更加准确地揭示蛋白质结构与生物功能之间的关系。本文综述了NMR解析蛋白质三维结构的理论和技术方法,以及NMR结合其他生物物理手段,并辅以分子建模计算法研究蛋白质三维结构的研究进展和最新方法,为精准解析蛋白质的三维结构提供思路及策略。     

An integrated workflow for charting the human interaction proteome: insights into the PP2A system

Protein complexes represent major functional units for the execution of biological processes. Systematic affinity purification coupled with mass spectrometry (AP‐MS) yielded a wealth of information on the compendium of protein complexes expressed in Saccharomyces cerevisiae. However, global AP‐MS analysis of human protein complexes is hampered by the low throughput, sensitivity and data robustness of existing procedures, which limit its application for systems biology research. Here, we address these limitations by a novel integrated method, which we applied and benchmarked for the human protein phosphatase 2A system. We identified a total of 197 protein interactions with high reproducibility, showing the coexistence of distinct classes of phosphatase complexes that are linked to proteins implicated in mitosis, cell signalling, DNA damage control and more. These results show that the presented analytical process will substantially advance throughput and reproducibility in future systematic AP‐MS studies on human protein complexes.     

Proteomic Profiling and Neurodegeneration in Alzheimer's Disease

Quantitative proteome analysis of Alzheimer's disease (AD) brains was performed using 2-D gels to identify disease specific changes in protein expression. The task of characterizing the proteome and its components is now practically achievable because of the development and integration of four important tools: protein, EST, and complete genome sequence databases, mass spectrometry, matching software for protein sequences and protein separation technology. Mass spectrometry (MS) instrumentation has undergone a tremendous change over the past decade, culminating in the development of highly sensitive, robust instruments that can reliably analyze biomolecules, particularly proteins and peptides; we identified 35 proteins from over 100 protein spots on a 2-D gel. Using this current technology, protein-expression profiling, which is actually a specialized form of mining, is an important principal application of proteomics. The information obtained has tremendous potential as a means of determining the pathogenesis, and detecting disease markers and potential targets for drug therapy in AD.     

Oncoproteomics: current trends and future perspectives

Oncoproteomics is the application of proteomics technologies in oncology. Functional proteomics is a promising technique for the rational identification of biomarkers and novel therapeutic targets for cancers. Recent progress in proteomics has opened new avenues for tumor-associated biomarker discovery. With the advent of new and improved proteomics technologies, such as the development of quantitative proteomic methods, high-resolution, -speed and -sensitivity mass spectrometry and protein arrays, as well as advanced bioinformatics for data handling and interpretation, it is now possible to discover biomarkers that can reliably and accurately predict outcomes during cancer management and treatment. However, there are several difficulties in the study of proteins/peptides that are not inherent in the study of nucleic acids. New challenges arise in large-scale proteomic profiling when dealing with complex biological mixtures. Nevertheless, oncoproteomics offers great promise for unveiling the complex molecular events of tumorigenesis, as well as those that control clinically important tumor behaviors, such as metastasis, invasion and resistance to therapy. In this review, the development and advancement of oncoproteomics technologies for cancer research in recent years are expounded.     

世纪之交的蛋白质序列测定技术

对蛋白质序列测定技术的发展过程作了简要回顾,介绍了近年在该方法学领域的主要进展,其中包括Ｎ－端顺序测定的微量化、毛细管ＨＰＬＣ与毛细管电泳的应用,新的Ｃ－端化学降解测序方法,以及快原子轰击（ＦＡＢ）、电喷雾（ＥＳＩ）和基质辅助激光解吸电离－飞行时间（ＭＡＬＤＩ－ＴＯＦ）质谱在蛋白质序列测定中的应用,还对世纪之交该领域的发展趋势作了扼要的展望。     

Small scale affinity purification and high sensitivity reversed phase nanoLC-MS N-glycan characterization of mAbs and fusion proteins

N-glycosylation is a complex post-translational modification with potential effects on the efficacy and safety of therapeutic proteins and known influence on the effector function of biopharmaceutical monoclonal antibodies (mAbs). Comprehensive characterization of N-glycosylation is therefore important in biopharmaceutical development. In early development, e.g. during pool or clone selection, however, only minute protein amounts of multiple samples are available for analytics. High sensitivity and high throughput methods are thus needed. An approach based on 96-well plate sample preparation and nanoLC-MS of 2- anthranilic acid or 2-aminobenzoic acid (AA) labeled N-glycans for the characterization of biopharmaceuticals in early development is reported here. With this approach, 192 samples can be processed simultaneously from complex matrices (e.g., cell culture supernatant) to purified 2-AA glycans, which are then analyzed by reversed phase nanoLC-MS. Attomolar sensitivity has been achieved by use of nanoelectrospray ionization, resulting in detailed glycan maps of mAbs and fusion proteins that are exemplarily shown in this work. Reproducibility, robustness and linearity of the approach are demonstrated, making use in a routine manner during pool or clone selection possible. Other potential fields of application, such as glycan biomarker discovery from serum samples, are also presented.     

Small scale affinity purification and high sensitivity reversed phase nanoLC-MS N-glycan characterization of mAbs and fusion proteins

N-glycosylation is a complex post-translational modification with potential effects on the efficacy and safety of therapeutic proteins and known influence on the effector function of biopharmaceutical monoclonal antibodies (mAbs). Comprehensive characterization of N-glycosylation is therefore important in biopharmaceutical development. In early development, e.g. during pool or clone selection, however, only minute protein amounts of multiple samples are available for analytics. High sensitivity and high throughput methods are thus needed. An approach based on 96-well plate sample preparation and nanoLC-MS of 2- anthranilic acid or 2-aminobenzoic acid (AA) labeled N-glycans for the characterization of biopharmaceuticals in early development is reported here. With this approach, 192 samples can be processed simultaneously from complex matrices (e.g., cell culture supernatant) to purified 2-AA glycans, which are then analyzed by reversed phase nanoLC-MS. Attomolar sensitivity has been achieved by use of nanoelectrospray ionization, resulting in detailed glycan maps of mAbs and fusion proteins that are exemplarily shown in this work. Reproducibility, robustness and linearity of the approach are demonstrated, making use in a routine manner during pool or clone selection possible. Other potential fields of application, such as glycan biomarker discovery from serum samples, are also presented.     

False discovery rate estimation for cross-linked peptides identified by mass spectrometry

The mass spectrometric identification of chemically cross-linked peptides (CXMS) specifies spatial restraints of protein complexes; these values complement data obtained from common structure-determination techniques. Generic methods for determining false discovery rates of cross-linked peptide assignments are currently lacking, thus making data sets from CXMS studies inherently incomparable. Here we describe an automated target-decoy strategy and the software tool xProphet, which solve this problem for large multicomponent protein complexes.