中国在翻译后修饰的生物信息学研究领域的进展与前瞻

翻译后修饰在调控蛋白质构象变化、活性以及功能方面具有重要作用,并参与了几乎所有细胞通路和过程。蛋白质翻译后修饰的鉴定是阐明细胞内分子机理的基础。相对于劳动密集的、耗费时间的实验工作,利用各种生物信息学方法开展翻译后修饰预测,能够提供准确、简便和快速的研究方案,并产生有价值的信息为进一步实验研究提供参考。文章主要综述了中国生物信息学者在翻译后修饰生物信息学领域所取得的研究进展,包括修饰底物与位点预测的计算方法学设计与完善、在线或本地化工具的设计与维护、修饰相关数据库及数据资源的构建及基于修饰蛋白质组学数据的生物信息学分析。通过比较国内外的同类研究,发现优势和不足,并对未来的研究作出前瞻。     

“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.     

生物质谱在细胞信号转导研究中的应用

近几年快速发展起来的生物质谱技术 ,依靠 (酶解后肽段 )精确质量数测定和随机肽序列标签分析 ,实现了对蛋白质高通量的鉴定 ,并被成功地用于蛋白质相互作用和蛋白质磷酸化等翻译后修饰研究。与传统的研究手段相比 ,上述技术能够在一次实验中对多信号通路中所有磷酸化的蛋白质分子及其磷酸化位点进行鉴定 ,已成为蛋白质组学最新发展中令人关注的一个热点。简要综述质谱技术应用于上述工作中的 3种策略     

基于质谱的磷酸化蛋白质组学: 富集、检测、鉴定和定量

磷酸化是一种调控生命活动的重要翻译后修饰,调控生物的生长发育、信号转导、以及疾病的发生发展．从上世纪80年代开始,质谱应用于蛋白质磷酸化的检测中,极大地推动了磷酸化蛋白质组学的发展．质谱检测拥有高灵敏度、高通量的特点,更重要的是具有位点分辨率,因此基于质谱的磷酸化蛋白质组检测方法得到不断的发展和推广．常见的磷酸化蛋白质组研究,首先对磷酸化肽段进行富集,然后进行串联质谱分析,最后通过搜索引擎对修饰位点进行鉴定和定量．本文从这个三个基本方面,对磷酸化蛋白质组研究进行综述,并对未来研究发展方向进行讨论．     

非限制翻译后修饰鉴定方法的研究进展

蛋白质翻译后修饰在真核生物细胞内广泛存在,对蛋白质的结构和功能有着十分重要的影响.串联质谱技术的快速发展为翻译后修饰鉴定提供了高通量、高灵敏度和高分辨率的分析平台,但传统搜索引擎鉴定修饰的方法无法满足数据分析的需求,非限制翻译后修饰鉴定已成为目前蛋白质组修饰分析的重要手段之一.非限制翻译后修饰鉴定不需要在分析前指定修饰类型,可以直接从样品中找出大量已知或未知的修饰,对提高质谱图谱解析率以及揭示蛋白质的生物学功能具有十分重要的意义.本文首先介绍了非限制翻译后修饰鉴定的定义和发展历程,然后从序列匹配和谱图匹配两个方面详细综述了目前非限制翻译后修饰鉴定的主流算法,分析了非限制翻译后修饰鉴定的质量控制问题,最后结合非限制翻译后修饰鉴定的实际应用讨论了修饰鉴定算法的不足和发展方向.     

翻译后修饰蛋白质组学助力实现精准医疗

与基因组学相比,蛋白质组学能够即时反映病人在疾病状态与正常状态下的蛋白表达和修饰谱,从而进行精准诊断和治疗,因此在临床领域越来越具有应用前景。蛋白质组学早期聚焦于细胞不同发育时期或疾病状态下的蛋白质表达水平变化,然而,许多至关重要的生命进程不仅与蛋白质相对丰度有关,更重要的是被时空特异分布的、可逆的翻译后修饰调控。在蛋白质组学、抗体亲和色谱与质谱分析、生物信息学等跨学科知识基础上发展的PTMScan?技术,在定性、定量分析翻译后修饰蛋白质位点方面具有显著优势。介绍了PTMScan?翻译后修饰蛋白质组学技术,其能够快速、准确、高通量地分析肺癌、黑色素瘤等多种疾病中信号通路关键节点蛋白质磷酸化、乙酰化、甲基化、泛素化和类泛素化等修饰的变化,满足精准医疗与伴随诊断对于疾病生物标志物鉴定、药物靶点筛选、靶向用药指导、疾病预后指示的需求,真正实现精准医疗,将正确的药物在最恰当的时间治疗真正需要的患者。     

N-糖肽的规模化质谱解析方法进展

蛋白质糖基化修饰的鉴定是蛋白质翻译后修饰分析中最具挑战性的任务之一,近几年尤其受到关注.快速发展的质谱技术为规模化的蛋白质糖基化修饰研究提供了有效的手段.与其他基于质谱技术的翻译后修饰鉴定相比,糖基化鉴定的难点在于糖链是大分子而且存在微观不均一性,另外糖链本身可以在串联质谱中碎裂且与肽段的碎裂规律不同,导致蛋白质组学的质谱解析方法和软件难以完整地鉴定肽段序列和糖链结构.完整N-糖肽的鉴定是糖基化分析的热点内容之一,针对N-糖肽的鉴定,近年来,人们开发了多种多样的质谱解析方法,其中包括用N-糖酰胺酶切除糖链后鉴定N-糖基化位点的方法、基于电子转运裂解的糖肽肽段鉴定、基于高能碰撞裂解与电子转运裂解联用或碰撞诱导裂解与三级谱联用的完整N-糖肽鉴定等等.本文对这些质谱解析方法进行了整理和综述,简要指出了目前完整糖肽鉴定软件存在的一些不足,展望了未来的发展方向.     

质谱图聚类网络法在鉴定多肽翻译后修饰中的应用及研究进展

蛋白质组学多肽鉴定方法一直以基于质谱分析和数据库搜索的方法为主,随着质谱仪技术的发展,海量的质谱数据被获取,这为大规模蛋白质的鉴定提供了一个强大的数据仓库,使得以质谱数据为基础的蛋白质组学研究成为主流。传统的串联质谱图搜库方法鉴定多肽翻译后修饰时具有诸多局限,质谱网络方法可以在一定程度上弥补局限。文中系统综述了基于质谱聚类的质谱网络和质谱图库搜索方法的发展历程、理论研究和应用研究,讨论了质谱网络库方法在鉴定多肽翻译后修饰的优势,并进行了分析和展望。     

磷酸化蛋白质组学的新进展及其在肝脏生理和病理机制中的应用

蛋白质磷酸化是最常见的蛋白质翻译后修饰形式。由于蛋白质的磷酸化形式可以被磷酸酶和磷酸激酶进行可逆的调控,所以在众多的生命活动过程中蛋白质的磷酸化修饰起着重要的调控作用,因此对生物体内蛋白质磷酸化修饰的系统研究对于揭示生命科学的奥秘显得十分重要。近年来,随着质谱技术和生物信息学软件以及磷酸化肽段富集方法的发展,利用质谱对生物体内蛋白质磷酸化修饰研究的技术逐渐成熟。肝脏作为人体最重要的代谢和免疫器官,深入研究肝脏细胞内蛋白质磷酸化修饰形式对于理解其功能具有重要指导意义。目前,迅速发展的磷酸化蛋白质组学技术已经被广泛应用到肝脏功能的生物学研究中。这些研究加深了人们对肝脏的生理及病理状态的分子生物学机制的了解。本文综述了当前磷酸化蛋白质组学的研究进展和磷酸化蛋白质组学在肝脏中的研究。     

基于质谱的蛋白质修饰定位算法

蛋白质翻译后修饰在细胞中广泛存在,对生命活动起到重要的调节作用。串联质谱技术的快速发展为蛋白质及其修饰鉴定提供了高通量、高灵敏度和高分辨率的研究平台。由于蛋白质鉴定软件往往不能准确定位修饰发生的位点,因而需要专门的数据分析算法重新定位修饰位点。本文首先介绍蛋白质修饰定位问题的来源和挑战,继而分析已有修饰重定位算法的原理和特点,最后讨论修饰重定位算法的不足和发展方向。     

Modification-specific proteomics in plant biology

Post-translational modifications (PTMs) are involved in the regulation of a wide range of biological processes, and affect e.g. protein structure, activity and stability. Several hundred PTMs have been described in the literature, but relatively few have been studied using mass spectrometry and proteomics. In general, methods for PTM characterization are developed to study yeast and mammalian biology and later adopted to investigate plants. Our point of view is that it is advantageous to enrich for PTMs on the peptide level as part of a quantitative proteomics strategy to not only identify the PTM, but also to determine the functional relevance in the context of regulation, response to abiotic stress etc. Protein phosphorylation is the only PTM that has been studied extensively at the proteome wide level in plants using mass spectrometry based methods.     

Current strategies and findings in clinically relevant post-translational modification-specific proteomics

Mass spectrometry-based proteomics has considerably extended our knowledge about the occurrence and dynamics of protein post-translational modifications (PTMs). So far, quantitative proteomics has been mainly used to study PTM regulation in cell culture models, providing new insights into the role of aberrant PTM patterns in human disease. However, continuous technological and methodical developments have paved the way for an increasing number of PTM-specific proteomic studies using clinical samples, often limited in sample amount. Thus, quantitative proteomics holds a great potential to discover, validate and accurately quantify biomarkers in body fluids and primary tissues. A major effort will be to improve the complete integration of robust but sensitive proteomics technology to clinical environments. Here, we discuss PTMs that are relevant for clinical research, with a focus on phosphorylation, glycosylation and proteolytic cleavage; furthermore, we give an overview on the current developments and novel findings in mass spectrometry-based PTM research.     

Strategy for comprehensive identification of post-translational modifications in cellular proteins, including low abundant modifications: application to glyceraldehyde-3-phosphate dehydrogenase

Post-translational modifications (PTMs) play key roles in the regulation of biological functions of proteins. Although some progress has been made in identifying several PTMs using existing approaches involving a combination of affinity-based enrichment and mass spectrometric analysis, comprehensive identification of PTMs remains a challenging problem in proteomics because of the dynamic complexities of PTMs in vivo and their low abundance. We describe here a strategy for rapid, efficient, and comprehensive identification of PTMs occurring in biological processes in vivo. It involves a selectively excluded mass screening analysis (SEMSA) of unmodified peptides during liquid chromatography-electrospray ionization-quadrupole-time-of-flight tandem mass spectrometry (LC-ESI-q-TOF MS/MS) through replicated runs of a purified protein on two-dimensional gel. A precursor ion list of unmodified peptides with high mass intensities was obtained during the initial run followed by exclusion of these unmodified peptides in subsequent runs. The exclusion list can grow as long as replicate runs are iteratively performed. This enables the identifications of modified peptides with precursor ions of low intensities by MS/MS sequencing. Application of this approach in combination with the PTM search algorithm MODi to GAPDH protein in vivo modified by oxidative stress provides information on multiple protein modifications (19 types of modification on 42 sites) with >92% peptide coverage and the additional potential for finding novel modifications, such as transformation of Cys to Ser. On the basis of the information of precursor ion m/z, quantitative analysis of PTM was performed for identifying molecular changes in heterogeneous protein populations. Our results show that PTMs in mammalian systems in vivo are more complicated and heterogeneous than previously reported. We believe that this strategy has significant potential because it permits systematic characterization of multiple PTMs in functional proteomics.     

The Plant PTM Viewer,a central resource for exploring plant protein modifications

Post‐translational modifications (PTMs) of proteins are central in any kind of cellular signaling. Modern mass spectrometry technologies enable comprehensive identification and quantification of various PTMs. Given the increased numbers and types of mapped protein modifications, a database is necessary that simultaneously integrates and compares site‐specific information for different PTMs, especially in plants for which the available PTM data are poorly catalogued. Here, we present the Plant PTM Viewer (http://www.psb.ugent.be/PlantPTMViewer), an integrative PTM resource that comprises approximately 370 000 PTM sites for 19 types of protein modifications in plant proteins from five different species. The Plant PTM Viewer provides the user with a protein sequence overview in which the experimentally evidenced PTMs are highlighted together with an estimate of the confidence by which the modified peptides and, if possible, the actual modification sites were identified and with functional protein domains or active site residues. The PTM sequence search tool can query PTM combinations in specific protein sequences, whereas the PTM BLAST tool searches for modified protein sequences to detect conserved PTMs in homologous sequences. Taken together, these tools help to assume the role and potential interplay of PTMs in specific proteins or within a broader systems biology context. The Plant PTM Viewer is an open repository that allows the submission of mass spectrometry‐based PTM data to remain at pace with future PTM plant studies.     

Post-translational Modifications and Mass Spectrometry Detection

In this review, we provide a comprehensive bibliographic overview of the role of mass spectrometry and the recent technical developments in the detection of post-translational modifications (PTMs). We briefly describe the principles of mass spectrometry for detecting PTMs and the protein and peptide enrichment strategies for PTM analysis, including phosphorylation, acetylation and oxidation. This review presents a bibliographic overview of the scientific achievements and the recent technical development in the detection of PTMs is provided. In order to ascertain the state of the art in mass spectrometry and proteomics methodologies for the study of PTMs, we analyzed all the PTM data introduced in the Universal Protein Resource (UniProt) and the literature published in the last three years. The evolution of curated data in UniProt for proteins annotated as being post-translationally modified is also analyzed. Additionally, we have undertaken a careful analysis of the research articles published in the years 2010 to 2012 reporting the detection of PTMs in biological samples by mass spectrometry.     

The utility of proteases in proteomics,from sequence profiling to structure and function analysis

In mass spectrometry (MS)-based bottom-up proteomics, protease digestion plays an essential role in profiling both proteome sequences and post-translational modifications (PTMs). Trypsin is the gold standard in digesting intact proteins into small-size peptides, which are more suitable for high-performance liquid chromatography (HPLC) separation and tandem MS (MS/MS) characterization. However, protein sequences lacking Lys and Arg cannot be cleaved by trypsin and may be missed in conventional proteomic analysis. Proteases with cleavage sites complementary to trypsin are widely applied in proteomic analysis to greatly improve the coverage of proteome sequences and PTM sites. In this review, we survey the common and newly emerging proteases used in proteomics analysis mainly in the last 5 years, focusing on their unique cleavage features and specific proteomics applications such as missing protein characterization, new PTM discovery, and de novo sequencing. In addition, we summarize the applications of proteases in structural proteomics and protein function analysis in recent years. Finally, we discuss the future development directions of new proteases and applications in proteomics.     

Post-translational quantitation by SRM/MRM: applications in cardiology

Introduction: Post-translational modifications (PTMs) have an important role in the regulation of protein function, localization, and interaction with other molecules. PTMs apply a dynamic control of proteins in both physiological and pathological conditions. The study of disease-specific PTMs allows identifying potential biomarkers and developing effective drugs. Enrichment techniques combined with high-resolution mass spectrometry (MS)/MS analysis provide attractive results on PTM characterization. Selected reaction monitoring/multiple reaction monitoring (SRM/MRM) is a powerful targeted assay for the quantitation and validation of PTMs in complex biological samples.

Areas covered: The most frequent PTMs are described in terms of biological role and analytical methods commonly used to detect them. The applications of SRM/MRM for the absolute quantitation of PTMs are reported, and a specific section is focused on PTM detection in proteins that are involved in the cardiovascular system and heart diseases.

Expert commentary: PTM characterization in relation to disease pathology is still in progress, but targeted proteomics by LC-MS/MS has significantly upgraded our knowledge in the last few years. Advances in enrichment strategies and software tools will facilitate the interpretation of high PTM complexity. Promising studies confirm the great potential of SRM/MRM to study PTMs in the cardiovascular field, and PTMomics could be very useful in the clinical perspective.     

ModifiComb, a new proteomic tool for mapping substoichiometric post-translational modifications, finding novel types of modifications, and fingerprinting complex protein mixtures

A major challenge in proteomics is to fully identify and characterize the post-translational modification (PTM) patterns present at any given time in cells, tissues, and organisms. Here we present a fast and reliable method ("ModifiComb") for mapping hundreds types of PTMs at a time, including novel and unexpected PTMs. The high mass accuracy of Fourier transform mass spectrometry provides in many cases unique elemental composition of the PTM through the difference DeltaM between the molecular masses of the modified and unmodified peptides, whereas the retention time difference DeltaRT between their elution in reversed-phase liquid chromatography provides an additional dimension for PTM identification. Abundant sequence information obtained with complementary fragmentation techniques using ion-neutral collisions and electron capture often locates the modification to a single residue. The (DeltaM, DeltaRT) maps are representative of the proteome and its overall modification state and may be used for database-independent organism identification, comparative proteomic studies, and biomarker discovery. Examples of newly found modifications include +12.000 Da (+C atom) incorporation into proline residues of peptides from proline-rich proteins found in human saliva. This modification is hypothesized to increase the known activity of the peptide.     

Engineering Proteases for Mass Spectrometry‐Based Post Translational Modification Analyses

Protein post‐translational modifications (PTMs) are important modulators of virtually all cellular processes, and frequently correlate with not only the rate but also severity of diseases. There has been considerable interest to map all possible PTM sites to be used as drug targets. Current approaches for PTM analysis suffer from a number of challenges; one of which is the lack of a PTM specific cleaving reagent. A central technology for global quantitative PTM analysis, mass spectrometry (MS) based proteomics, is biased toward trypsin due to its high activity and specificity. This bias becomes a problem when a PTM is located at or near tryptic cleavage sites, in which case the PTM might block recognition by trypsin, resulting in missed cleavage and sequence coverage gaps. Reviewed here are recent advances in engineering new proteases for PTM analyses, and how these new proteases are beginning to address current challenges in the field.     

Proteomic analysis of phosphorylation, oxidation and nitrosylation in signal transduction

Signal transduction pathways control cell fate, survival and function. They are organized as intricate biochemical networks which enable biochemical protein activities, crosstalk and subcellular localization to be integrated and tuned to produce highly specific biological responses in a robust and reproducible manner. Post translational Modifications (PTMs) play major roles in regulating these processes through a wide variety of mechanisms that include changes in protein activities, interactions, and subcellular localizations. Determining and analyzing PTMs poses enormous challenges. Recent progress in mass spectrometry (MS) based proteomics have enhanced our capability to map and identify many PTMs. Here we review the current state of proteomic PTM analysis relevant for signal transduction research, focusing on two areas: phosphorylation, which is well established as a widespread key regulator of signal transduction; and oxidative modifications, which from being primarily viewed as protein damage now start to emerge as important regulatory mechanisms.