基于蛋白质半胱氨酸的活性蛋白表达谱分析

蛋白质中的半胱氨酸对维持和调控细胞内的氧化还原平衡起着重要作用,同时它们也是多种蛋白质的功能活性位点,参与诸多生理过程。此外,蛋白质中的半胱氨酸上所发生的一系列翻译后修饰也扩展了蛋白质功能的多样性。随着化学蛋白质组学的发展,基于活性的蛋白表达谱(Activity-basedproteinprofiling,ABPP)分析技术在探究蛋白质半胱氨酸及其翻译后修饰方面取得了很多重要的研究成果。本文简要介绍了用于蛋白质半胱氨酸的活性蛋白表达谱分析方法,讨论了针对蛋白质半胱氨酸及其翻译后修饰的蛋白质组学领域的最新研究,并对该领域的未来发展趋势进行了展望。     

基于活性的蛋白质组分析

众多物种基因组解码工作的完成极大地丰富了我们对这些生命体系组成复杂度的认知, 然而下一个更为严峻的挑战是如何快速准确地解析这些基因编码蛋白的分子功能, 这也是当前蛋白质组学领域亟待解决的一个重要科学问题. 基于活性的蛋白质组分析是近些年来一项新兴的技术平台, 它致力于在复杂的生命体系中系统地鉴定某类具有特定功能的蛋白质分子. 在本篇短综述中, 我们将对该化学生物学技术的发展做一个简要的回顾, 重点介绍该技术在未知蛋白的功能解析、小分子抑制剂的筛选以及活性小分子靶标蛋白的鉴定等方面的工作, 最后将对该技术未来发展的走向及其拓展应用做前瞻性的讨论.     

基于分子信标探针和生物功能化纳米颗粒的蛋白质分析

随着人类基因组测序计划的完成,生命科学研究热点逐渐由基因组学向蛋白质组学转移.分析化学工作者利用分子信标探针和生物功能化纳米颗粒的固有优势,发展了一系列新原理、新方法和新技术并在蛋白质组学研究领域得到了广泛应用,极大地促进了蛋白质组学的发展和进步.本文主要综述了基于分子探针和生物功能化纳米颗粒开展的一系列实时、原位、灵敏、特异的蛋白质分析研究,包括:非特异性/特异性蛋白质的检测与分离、蛋白质/DNA相互作用研究、细胞表面蛋白质的识别,以及基于抗原-抗体反应的病原菌检测等,并进一步展望了基于分子信标探针和生物功能化纳米颗粒的蛋白质分析研究的发展前景与关键问题.     

Edman降解中异硫氰酸苯酯新修饰位点的发现和验证

Edman降解是最早建立的一种用于多肽和蛋白质氨基端测序的方法,该方法现在仍被广泛用于生物化学领域。随着高通量蛋白质组学技术的发展和应用,该方法中的异硫氰酸苯酯反应被用于修饰蛋白质氨基端,并用于检测蛋白质水解位点。但还没有异硫氰酸苯酯是否可以修饰其他氨基酸侧链并影响多肽序列分析的研究。为了探究其修饰其他氨基酸的可能性,本文利用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）和液相色谱-串联质谱（LC-MS/MS）研究了异硫氰酸苯酯对一个模型肽的化学修饰。质谱数据解析后发现在高浓度异硫氰酸苯酯的反应条件下,组氨酸上可以引入一个新的异硫氰酸苯酯修饰位点。这一修饰位点的发现预示着通过改变实验条件或分析方法,可以更准确地利用Edman降解和蛋白质组学技术分析多肽和蛋白质。     

牛视紫红质蛋白质中视黄醛的活性位点

视紫红质蛋白是一个跨膜蛋白, 视黄醛(RET)在该蛋白中的活性结合位点涉及到视觉过程机理, 与一些眼科疾病病理有关. 基于牛视紫红质蛋白1U19的蛋白质晶体结构数据, 采用密度泛函理论的B3LYP方法计算RET-Lys296残基与视黄醛分子周围半径为0.6 nm的空间范围30个氨基酸残基相互作用和结合能. 数值显示1U19蛋白中的残基Glu113、Glu181和Glu122是质子化的RET-Lys296残基的活性结合位点, 结合能分别为-333.38、-205.67和-194.56 kJ·mol-1. 这些氨基酸残基带有一个负电荷, 与质子化的RET-Lys296残基发生强烈的离子静电相互作用. 另外几个残基Ala292、Cys187、Phe293、Pro291以及Trp265等与质子化RET-Lys296残基也有相互吸引作用. 当RET-Lys296残基非质子化, 上述相互作用消失, 促使视黄醛分子与视蛋白分离. 研究发现残基Glu113和Glu181周围各自有一个结晶水分子通过双氢键形式起着稳定作用.     

“精准医疗”背景下的分子靶向药物研究——精准药物设计策略浅析

如何实现对肿瘤、病毒感染等严重危害人类健康的疾病的精准治疗是当前医学界的难题和研究热点。随着"精准医疗"计划的启动,当代药物设计也随之进入"精准"靶向药物分子设计时代。基于靶标结构的合理药物设计及特异性的药物递送系统是当代精准药物设计的重要方面。靶标-配体精准相互作用为基于靶标的合理药物设计奠定了理论基础;精准"制导"化学合成方法学的研究为药物合成提供了强有力的工具;灵敏、准确分子探针的研究为当代化学生物学及发现特异性的药物递送系统提供有效的辅助手段。本文从以上几方面,从药物化学的视角综述了"精准医疗"背景下的分子靶向诊疗药物研究。     

基于复小波能量谱的蛋白质活性位点识别新方法

蛋白质活性位点的识别对于理解蛋白质的功能及计算机辅助药物设计具有重要的意义. 基于复小波能量谱建立蛋白质活性位点识别新方法, 采用Morlet复小波对数字化的蛋白质序列进行一维连续小波变换. 结果表明, 通过时-频分析, 能量集中区域往往与蛋白质的活性位点具有密切联系, 并且同源蛋白质序列的复小波能量最大值通常分布于相同的频率处, 表明小波功率谱在预测蛋白质活性位点方面具有广阔的应用前景.     

活性天然产物靶标蛋白的鉴定

天然产物是创新药物的重要来源,确定活性天然产物的靶标蛋白和作用机制是新药开发的关键.在本篇综述中,对近二十年来在活性天然产物靶标鉴定领域出现的新技术和新方法进行简要的回顾和总结,通过实例重点介绍化学蛋白质组学和生物物理学方法在天然产物靶标蛋白鉴定方面的应用,讨论各种策略的优势和不足,并对其适用范围,应用前景和发展趋势作了前瞻性的展望.     

基于ICP-MS的蛋白质定量方法*

蛋白质组学已经成为生命科学研究中最为活跃的领域之一。研究蛋白质的生物功能,不但需要高通量的鉴定蛋白质,还需要定量分析动态变化的蛋白质,即定量蛋白质组学研究。蛋白质的定量研究有助于发现新的生物功能,并可以用于疾病的预警和药物靶点的发现。现有的定量蛋白质组学研究主要利用同位素标记结合生物质谱（电喷雾电离质谱ESI-MS,基质辅助激光解吸电离质谱MALDI-MS）技术而实现。近年来电感耦合等离子体质谱（ICP-MS）作为ESI-MS和MALDI-MS的补充,越来越多地应用于蛋白质的定量分析,特别是蛋白质的绝对定量分析。ICP-MS是检测生物分子中痕量元素的理想工具,具有灵敏度高、动态范围广,不易受基体的影响等优点。本文将讨论基于ICP-MS的分析方法,及其在蛋白质定量分析和免疫分析中的部分成功应用。     

多重反应监测(MRM):靶标蛋白质定量的新方法

多重反应监测(multiple reaction monitoring,MRM)是针对靶标分子的一种质谱分析技术.该技术采用三重四级杆质谱仪,检测靶标分子的母离子和子离子的质谱响应信号,从而获取较灵敏和高重现性的定性和定量信息,近年来在蛋白质组学领域得到了广泛应用.与全谱性的蛋白质组学分析不同,MRM注重有限目标的蛋白质定量测定,因此,它在蛋白质分析检测领域中的应用极有发展潜力.在临床检验中,酶联免疫吸附测定(enzyme linked immunosorbent assay,ELISA)是蛋白质定量分析的常规技术,但是ELISA在多重蛋白质生物标志物的测定方面具有一定限制.随着蛋白质组学的深入进行,MRM的定量分析优势可否应用于临床检测已提至日程,世界范围内多个研究团队一直致力于推动这一领域的发展,也取得了令人瞩目的成就.本文简单介绍了MRM技术的原理、优势及发展前景等,同时,对其在蛋白质组学研究及临床应用中的潜力进行了讨论.     

Activity-based probes: discovering new biology and new drug targets

The development and application of chemical technologies enabling direct analysis of enzyme activity in living systems has undergone explosive growth in recent years. Activity-based protein profiling (ABPP) is a key constituent of this broad field, and is among the most powerful and mature chemical proteomic technologies. This tutorial review introduces the essential features of ABPP and the design and application of activity-based probes (ABPs) from drug target elucidation and in vivo visualisation of enzyme activity to comprehensive profiling of the catalytic content of living systems, and the discovery of new biological pathways.     

Bioorthogonal Reactions in Activity-Based Protein Profiling

Activity-based protein profiling (ABPP) is a powerful technique to label and detect active enzyme species within cell lysates, cells, or whole animals. In the last two decades, a wide variety of applications and experimental read-out techniques have been pursued in order to increase our understanding of physiological and pathological processes, to identify novel drug targets, to evaluate selectivity of drugs, and to image probe targets in cells. Bioorthogonal chemistry has substantially contributed to the field of ABPP, as it allows the introduction of tags, which may be bulky or have unfavorable physicochemical properties, at a late stage in the experiment. In this review, we give an overview of the bioorthogonal reactions that have been implemented in ABPP, provide examples of applications of bioorthogonal chemistry in ABPP, and share some thoughts on future directions.     

Structure-based approaches to drug discovery against tuberculosis

Tuberculosis has become one of the deadliest global emergencies due to the widespread existence of multiple drug resistance strains of Mycobacterium tuberculosis and the increase of immuno-compromised populations in large parts of the world. Although the complete genome of M. tuberculosis became available in 1998, opening unprecedented opportunities for target-specific drug development, the progress since then has been slow, mainly due to a lack of a sufficiently strong interest by pharmaceutical and biotechnology industries. One of the most promising tools for future drug discovery lies in the elucidation of the molecular structures of potential drug targets from the M. tuberculosis proteome. During the last five years, the structures of about 200 unique targets have already been determined, which comprise about 5% of the entire M. tuberculosis proteome. As an example, we present the approach and some of the key achievements of the X-MTB consortium based in Germany. We summarize and discuss some recent highlights of potential drug targets of M. tuberculosis involved in lipid metabolism, protein phosphorylation/dephosphorylation and amino acid biosynthesis. The achievements of several structural genomics consortia that focus on targets from the M. tuberculosis proteome are now providing a solid framework to support coordinated international approaches for future structure-based drug discovery programs at the interface between industrial enterprises and academic research. One of the objectives will be to focus on target complexes, in addition to single targets that dominate the present repository of structures from the M. tuberculosis proteome.     

Applications of Activity‐Based Protein Profiling (ABPP) and Bioimaging in Drug Discovery

Activity‐based protein profiling (ABPP) and bioimaging have been developed in recent years as powerful technologies in drug discovery. Specifically, both approaches can be applied in critical steps of drug development, such as therapy target discovery, high‐throughput drug screening and target identification of bioactive molecules. We have been focused on the development of various strategies that enable simultaneous activity‐based protein profiling and bioimaging studies, thus facilitating an understanding of drug actions and potential toxicities. In this Minireview, we summarize these novel strategies and applications, with the aim of promoting these technologies in drug discovery.     

Potential drug targets in Mycobacterium tuberculosis through metabolic pathway analysis

The emergence of multidrug resistant varieties of Mycobacterium tuberculosis has led to a search for novel drug targets. We have performed an insilico comparative analysis of metabolic pathways of the host Homo sapiens and the pathogen M. tuberculosis. Enzymes from the biochemical pathways of M. tuberculosis from the KEGG metabolic pathway database were compared with proteins from the host H. sapiens, by performing a BLASTp search against the non-redundant database restricted to the H. sapiens subset. The e-value threshold cutoff was set to 0.005. Enzymes, which do not show similarity to any of the host proteins, below this threshold, were filtered out as potential drug targets. We have identified six pathways unique to the pathogen M. tuberculosis when compared to the host H. sapiens. Potential drug targets from these pathways could be useful for the discovery of broad spectrum drugs. Potential drug targets were also identified from pathways related to lipid metabolism, carbohydrate metabolism, amino acid metabolism, energy metabolism, vitamin and cofactor biosynthetic pathways and nucleotide metabolism. Of the 185 distinct targets identified from these pathways, many are in various stages of progress at the TB Structural Genomics Consortium. However, 67 of our targets are new and can be considered for rational drug design. As a case study, we have built a homology model of one of the potential drug targets MurD ligase using WHAT IF software. The model could be further explored for insilico docking studies with suitable inhibitors. The study was successful in listing out potential drug targets from the M. tuberculosis proteome involved in vital aspects of the pathogen's metabolism, persistence, virulence and cell wall biosynthesis. This systematic evaluation of metabolic pathways of host and pathogen through reliable and conventional bioinformatic methods can be extended to other pathogens of clinical interest.     

Quantitative analysis of highly homologous proteins: the challenge of assaying the “CYP-ome” by mass spectrometry

Cytochrome P450 enzymes comprise families of highly homologous proteins. These proteins play a pivotal role in oxidative drug metabolism and are important targets in drug discovery research. Proteomics today is a valuable tool for the analysis of proteins. In the past, qualitative analysis of the proteome was the main focus of research, but in the last few years interest in the mathematical modelling of protein networks has been growing and so has the demand on quantitative proteome analysis. As a thorough understanding of cytochrome P450 dependent metabolism is crucial for drug discovery, it is thus not astounding that cytochrome P450 enzymes are a target for quantitative proteomics research. In this article, we review the techniques available for quantitative proteome analysis and to what extent these techniques have been used for the quantification of cytochrome P450 enzymes and give a brief outlook of the techniques that have promising potential for the analysis of these proteins in the future.     

Recent advances in activity-based probes (ABPs) and affinity-based probes (AfBPs) for profiling of enzymes

Activity-based protein profiling (ABPP) is a technique that uses highly selective active-site targeted chemical probes to label and monitor the state of proteins. ABPP integrates the strengths of both chemical and biological disciplines. By utilizing chemically synthesized or modified bioactive molecules, ABPP is able to reveal complex physiological and pathological enzyme–substrate interactions at molecular and cellular levels. It is also able to provide critical information of the catalytic activity changes of enzymes, annotate new functions of enzymes, discover new substrates of enzymes, and allow real-time monitoring of the cellular location of enzymes. Based on the mechanism of probe-enzyme interaction, two types of probes that have been used in ABPP are activity-based probes (ABPs) and affinity-based probes (AfBPs). This review highlights the recent advances in the use of ABPs and AfBPs, and summarizes their design strategies (based on inhibitors and substrates) and detection approaches.

This review highlights the recent advances in the use of activity-based probes (ABPs) and affinity-based probes (AfBPs), and summarizes their design strategies (based on inhibitors and substrates) and detection approaches.     

Small-molecule microarrays as tools in ligand discovery

Small molecules that bind and modulate specific protein targets are increasingly used as tools to decipher protein function in a cellular context. Identifying specific small-molecule probes for each protein in the proteome will require miniaturized assays that permit screening of large collections of compounds against large numbers of proteins in a highly parallel fashion. Simple and general binding assays involving small-molecule microarrays can be used to identify probes for nearly any protein in the proteome. The assay may be used to identify ligands for proteins in the absence of knowledge about structure or function. In this tutorial review, we introduce small-molecule microarrays (SMMs) as tools for ligand discovery; discuss methods for manufacturing SMMs, including both non-covalent and covalent attachment strategies; and provide examples of ligand discovery involving SMMs.     

Protein posttranslational modifications: the chemistry of proteome diversifications

The diversity of distinct covalent forms of proteins (the proteome) greatly exceeds the number of proteins predicted by DNA coding capacities owing to directed posttranslational modifications. Enzymes dedicated to such protein modifications include 500 human protein kinases, 150 protein phosphatases, and 500 proteases. The major types of protein covalent modifications, such as phosphorylation, acetylation, glycosylation, methylation, and ubiquitylation, can be classified according to the type of amino acid side chain modified, the category of the modifying enzyme, and the extent of reversibility. Chemical events such as protein splicing, green fluorescent protein maturation, and proteasome autoactivations also represent posttranslational modifications. An understanding of the scope and pattern of the many posttranslational modifications in eukaryotic cells provides insight into the function and dynamics of proteome compositions.