Matrix-assisted laser desorption/ionization imaging mass spectrometry: a promising technique for reproductive research

Matrix-assisted laser desorption/ionization (MALDI) tissue imaging mass spectrometry is particularly promising among the numerous applications of mass spectrometry. It is used for probing and analyzing the spatial arrangement of a wide range of molecules, including proteins, peptides, lipids, drugs, and metabolites, directly in thin slices of tissue. In the field of proteomics, the technology avoids tedious and time-consuming extraction and fractionation steps classically required for sample analysis. MALDI imaging mass spectrometry is increasingly recognized as a powerful method for clinical proteomics, particularly in cancer research. The technology has particular potential for the discovery of new tissue biomarker candidates, classification of tumors, early diagnosis or prognosis, elucidating pathogenesis pathways, and therapy monitoring. Over recent years, MALDI imaging mass spectrometry has been used for molecular profiling and imaging directly in male and female reproductive tissues. This review will consider some of the recent publications in the field, addressing a range of issues covering embryo development, gene expression product profiling during gametogenesis, and seeking and identifying biomarkers of reproductive cancers. The wealth of advances in mass spectrometry imaging will inevitably attract biologists and clinicians as the advantages and power of this technology become more widely known. This review will also discuss bottlenecks and the many technical issues that remain to be resolved before laboratories in the field can adopt the technology. We foresee that MALDI imaging mass spectrometry will have a major impact in reproductive research by opening new avenues to the understanding of various molecular mechanisms and the diagnosis of reproductive pathologies.     

基于质谱技术筛选差异表达蛋白的统计学策略研究进展

随着质谱技术的快速发展,蛋白质组学已成为继基因组学、转录组学之后的又一研究热点,寻找可靠的差异表达蛋白对于生物标记物的发现至关重要.因此,如何准确、灵敏地筛选出差异蛋白已成为基于质谱的定量蛋白质组学的主要研究内容之一.目前,针对该问题的研究方法众多,但这些方法策略的适用范围不尽相同.总体来说,基于质谱技术筛选差异蛋白的统计学策略可以分为3类:基于经典统计学派的策略、基于贝叶斯学派的统计检验策略和其他策略,这3类方法有各自的应用范围、特点及不足.此外,筛选过程还将产生部分假阳性结果,可以采用其他方法对差异表达蛋白的质量进行控制,以提高统计检验结果的可靠性.     

蛋白质组学在结核分枝杆菌研究中的应用

蛋白质组学是在基因组学基础上发展起来的新兴学科, 其基本技术包括样品制备、蛋白质分离和蛋白质鉴定分析, 其中的核心技术是双向凝胶电泳技术(2-Dimensional Electrophoresis, 2-DE)和质谱技术(Mass Spectrometry, MS)。近年来, 蛋白质组学技术已应用于结核分枝杆菌的研究领域。应用蛋白质组学技术分离、鉴定、检测结核分枝杆菌致病株的全菌蛋白及分泌蛋白, 分析其蛋白组成, 可深入解析结核分枝杆菌的致病机理和耐药机制。通过对结核分枝杆菌致病株抗原的分析, 为研制预防结核病的新型疫苗拓展了空间。通过对结核分枝杆菌临床分离株的蛋白组成分析还发现了一些有意义的结核病早期诊断标志物。蛋白质组学技术还应用于寻找新的药物靶标, 在研制和筛选新的抗结核药物等方面展示了一些有价值的研究成果, 为更好地开展结核病的预防、早期诊断及治疗打下了基础。     

Mycobacterium tuberculosis functional network analysis by global subcellular protein profiling

Trends in increased tuberculosis infection and a fatality rate of approximately 23% have necessitated the search for alternative biomarkers using newly developed postgenomic approaches. Here we provide a systematic analysis of Mycobacterium tuberculosis (Mtb) by directly profiling its gene products. This analysis combines high-throughput proteomics and computational approaches to elucidate the globally expressed complements of the three subcellular compartments (the cell wall, membrane, and cytosol) of Mtb. We report the identifications of 1044 proteins and their corresponding localizations in these compartments. Genome-based computational and metabolic pathways analyses were performed and integrated with proteomics data to reconstruct response networks. From the reconstructed response networks for fatty acid degradation and lipid biosynthesis pathways in Mtb, we identified proteins whose involvements in these pathways were not previously suspected. Furthermore, the subcellular localizations of these expressed proteins provide interesting insights into the compartmentalization of these pathways, which appear to traverse from cell wall to cytoplasm. Results of this large-scale subcellular proteome profile of Mtb have confirmed and validated the computational network hypothesis that functionally related proteins work together in larger organizational structures.     

Dendritic cell trafficking and antigen presentation in the human immune response to Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) is an extraordinarily successful human pathogen, one of the major causes of death by infectious disease worldwide. A key issue for the study of tuberculosis is to understand why individuals infected with Mtb experience different clinical outcomes. To better understand the dynamics of Mtb infection and immunity, we coupled nonhuman primate experiments with a mathematical model we previously developed that qualitatively and quantitatively captures important processes of cellular priming and activation. These processes occur between the lung and the nearest draining lymph node where the key cells mediating this process are the dendritic cells (DC). The nonhuman primate experiments consist of bacteria and cell numbers from tissues of 17 adult cynomolgus macaques (Macaca fascicularis) that were infected with Mtb strain Erdman ( approximately 25 CFU/animal via bronchoscope). The main result of this work is that delays in either DC migration to the draining lymph node or T cell trafficking to the site of infection can alter the outcome of Mtb infection, defining progression to primary disease or latent infection and reactivated tuberculosis. Our results also support the idea that the development of a new generation of treatment against Mtb should optimally elicit a fast DC turnover at the site of infection, as well as strong activation of DCs for maximal Ag presentation and production of key cytokines. This will induce the most protective T cell response.     

基于蛋白质组芯片的结核分枝杆菌系统生物学研究进展

近些年全球结核病疫情愈发严重,耐药性结核病使其雪上加霜。一个重要原因是结核病新药的匮乏以及结核分枝杆菌相关基础研究的不足。因此迫切需要开发新的技术以促进结核病系统生物学基础研究,并在此基础上研究新机制,发现新靶标,开发新药物。结核分枝杆菌功能蛋白质组芯片的出现旨在促进结核病相关研究工作。考虑到结核分枝杆菌高毒力、复制周期长和需要在生物安全三级实验室中开展研究等特点和难点,该工具为结核病相关研究人员提供了一个强有力的武器。目前这一技术手段的应用已经使我们对结核分枝杆菌-宿主相互作用、小分子-蛋白结合以及抗生素耐药性机制等关键生物过程有了更深入的了解。为了更好地帮助同行了解这一有效的工具,本文综述了结核分枝杆菌功能蛋白组芯片的几种主要应用,期望同行专家能更好地将其应用于结核病相关的基础研究中。     

Proteomics without polyacrylamide: qualitative and quantitative uses of tandem mass spectrometry in proteome analysis

Proteomics can be thought of as an attempt to understand the information encoded in genomic sequences from the perspective of proteins; i.e. the structure, function and regulation of biological processes at the protein level. In practice it stands in stark contrast to the hypothesis-driven serial approach practiced in the last century that was so successful for protein chemists and is built on the basic understanding of protein physicochemical properties developed during that era. Proteomics attempts to study biological processes comprehensively or globally by systematic parallel analysis of proteins expressed in a cell. While there are many analytical techniques in use and under development in proteomics, mass spectrometry is currently one of the field's most important discovery-based tools. This article will review some of the current approaches for qualitative and quantitative uses of tandem mass spectrometry in the field of proteomics specifically avoiding a discussion of the use of gel electrophoresis prior to mass spectrometry. Electronic Publication     

Central carbon metabolism in Mycobacterium tuberculosis: an unexpected frontier

Recent advances in liquid chromatography and mass spectrometry have enabled the highly parallel, quantitative measurement of metabolites within a cell and the ability to trace their biochemical fates. In Mycobacterium tuberculosis (Mtb), these advances have highlighted major gaps in our understanding of central carbon metabolism (CCM) that have prompted fresh interpretations of the composition and structure of its metabolic pathways and the phenotypes of Mtb strains in which CCM genes have been deleted. High-throughput screens have demonstrated that small chemical compounds can selectively inhibit some enzymes of Mtb's CCM while sparing homologs in the host. Mtb's CCM has thus emerged as a frontier for both fundamental and translational research.     

蛋白质组学研究中的分离分析技术及其研究进展

在后基因组时代,蛋白质组学成为新的研究热点。蛋白质组学的研究目标是为复杂蛋白质样品建立一个高通量、大规模、自动化的分离分析技术平台,从而实现准确、快速地筛选功能蛋白质。蛋白质的分离分析在蛋白组学研究中起着非常重要的作用。本文主要综述在蛋白质组学研究中二维凝胶电泳、毛细管电泳及其与质谱联用、多维液相分离技术及其与质谱联用和蛋白质芯片等高效分离分析技术的应用研究进展。     

PeptideAtlas: a resource for target selection for emerging targeted proteomics workflows

A crucial part of a successful systems biology experiment is an assay that provides reliable, quantitative measurements for each of the components in the system being studied. For proteomics to be a key part of such studies, it must deliver accurate quantification of all the components in the system for each tested perturbation without any gaps in the data. This will require a new approach to proteomics that is based on emerging targeted quantitative mass spectrometry techniques. The PeptideAtlas Project comprises a growing, publicly accessible database of peptides identified in many tandem mass spectrometry proteomics studies and software tools that allow the building of PeptideAtlas, as well as its use by the research community. Here, we describe the PeptideAtlas Project, its contents and components, and show how together they provide a unique platform to select and validate mass spectrometry targets, thereby allowing the next revolution in proteomics.     

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。     

Developments in mass spectrometry for the analysis of complex protein mixtures.

State-of-the-art proteomics workflows involve multiple interdependent steps: sample preparation, protein-peptide separation, mass spectrometry and data analysis. While improvements in any of these steps can increase the depth and breadth of analysis, advances in mass spectrometry have catalysed many of the most important developments. We discuss common classes of mass analysers and how these analysers are put together to produce some of the most popular mass spectrometry platforms. The capabilities of these platforms determine how they can be used in a variety of common proteomic strategies and, in turn, what types of biological questions can be addressed. Moving forward, powerful new hybrid mass spectrometers and application of emerging types of tandem mass spectrometry promise that our ability to analyse complex mixtures of proteins will continue to advance.     

2014蛋白质组学专刊序言

蛋白质组学研究是后基因组学时代最重要的功能基因组学研究之一,与医学生物学、化学、物理学、信息学以及现代技术等关系十分密切。为了检阅近年来国内外蛋白质组学某些重要研究进展,探索其可能的应用范围,讨论其存在的问题,展望其发展前景,特组织出版"蛋白质组学专刊"。本期专刊包括综述和研究论文两部分,内容主要涉及不同物种(包括人类、哺乳类动物、原核生物、放线菌等)蛋白质组学研究、蛋白质组学重要方法学与技术研究(包括串联质谱分析、尿蛋白膜保存法、定量蛋白质组学分折、meta分析等)和蛋白质组功能与应用研究(包括蜘蛛毒素蛋白质组、磷酸化蛋白质组、卵母细胞和早期胚胎蛋白质组、肝脏纤维化蛋白质组、分枝杆菌耐药的蛋白质组等)。     

Advances in shotgun proteomics and the analysis of membrane proteomes

The emergence of shotgun proteomics has facilitated the numerous biological discoveries made by proteomic studies. However, comprehensive proteomic analysis remains challenging and shotgun proteomics is a continually changing field. This review details the recent developments in shotgun proteomics and describes emerging technologies that will influence shotgun proteomics going forward. In addition, proteomic studies of integral membrane proteins remain challenging due to the hydrophobic nature in integral membrane proteins and their general low abundance levels. However, there have been many strategies developed for enriching, isolating and separating membrane proteins for proteomic analysis that have moved this field forward. In summary, while shotgun proteomics is a widely used and mature technology, the continued pace of improvements in mass spectrometry and proteomic technology and methods indicate that future studies will have an even greater impact on biological discovery.     

Mass spectrometry of RNA: linking the genome to the proteome.

Ribonucleic acids (RNAs) are continuing to attract increased attention as they are found to play pivotal roles in biological systems. Just as genomics and proteomics have been enabled by the development of effective analytical techniques and instrumentation, the large-scale analysis of non-protein coding (nc)RNAs will benefit as new analytical methodologies, such as mass spectrometry (MS), are developed for their analysis. Mass spectrometry offers a number of advantages for RNA analysis arising from its ability to provide mass and sequence information starting with limited amounts of sample. This review will highlight recent developments in the field of MS that enable the characterization of RNA modification status, RNA tertiary structures, and ncRNA expression levels. These developments will also be placed in perspective of how MS of RNAs can help elucidate the link between the genome and proteome.     

The mmaA2 gene of Mycobacterium tuberculosis encodes the distal cyclopropane synthase of the alpha-mycolic acid

Infection with Mycobacterium tuberculosis (Mtb) remains a severe global health problem that has prompted an aggressive search for new antibiotic targets and vaccine strategies for this persistent pathogen. Recently, a wide variety of genetic determinants of Mtb pathogenicity have been identified, including several genes involved in the biogenesis of the complex Mtb cell envelope. Among these are the mycolic acid cyclopropane synthases, a family of proteins that modify the major cell envelope lipids of Mtb with a diversity of cyclopropane rings. Despite substantial sequence identity, these proteins catalyze highly specific cyclopropane modifications, including proximal modification of the alpha-mycolate (pcaA) and trans-cyclopropane modification (cmaA2). Here we report the mycolic acid modification function of a third cyclopropane synthase, mmaA2, through the creation and analysis of an M. tuberculosis mmaA2 null mutant. Unexpectedly, mmaA2 is essential for the distal cyclopropane modification of the alpha-mycolate, a function previously attributed to cmaA1. alpha-Mycolates of a cmaA1 null mutant were unaffected, demonstrating that cmaA1 is not required for alpha-mycolate modification. Although fully cyclopropanated methoxymycolates are produced in the mmaA2 mutant, cis-cyclopropanation is impaired, leading to accumulation of unsaturated methoxymycolate derivatives. This study establishes mmaA2 as a distal cyclopropane synthase of the alpha-mycolates of M. tuberculosis and the first cyclopropane synthase to modify both alpha- and oxygenated mycolates. These results expand our knowledge of the biosynthesis of the Mtb cell envelope and will allow further elucidation of the relationship between Mtb pathogenesis and the fine structure of mycolic acids.     

Mycobacterium thermoresistibile as a source of thermostable orthologs of Mycobacterium tuberculosis proteins

The genus Mycobacterium comprises major human pathogens such as the causative agent of tuberculosis, Mycobacterium tuberculosis (Mtb), and many environmental species. Tuberculosis claims ~1.5 million lives every year, and drug resistant strains of Mtb are rapidly emerging. To aid the development of new tuberculosis drugs, major efforts are currently under way to determine crystal structures of Mtb drug targets and proteins involved in pathogenicity. However, a major obstacle to obtaining crystal structures is the generation of well-diffracting crystals. Proteins from thermophiles can have better crystallization and diffraction properties than proteins from mesophiles, but their sequences and structures are often divergent. Here, we establish a thermophilic mycobacterial model organism, Mycobacterium thermoresistibile (Mth), for the study of Mtb proteins. Mth tolerates higher temperatures than Mtb or other environmental mycobacteria such as M. smegmatis. Mth proteins are on average more soluble than Mtb proteins, and comparison of the crystal structures of two pairs of orthologous proteins reveals nearly identical folds, indicating that Mth structures provide good surrogates for Mtb structures. This study introduces a thermophile as a source of protein for the study of a closely related human pathogen and marks a new approach to solving challenging mycobacterial protein structures.     

Multivariate approaches in plant science

The objective of proteomics is to get an overview of the proteins expressed at a given point in time in a given tissue and to identify the connection to the biochemical status of that tissue. Therefore sample throughput and analysis time are important issues in proteomics. The concept of proteomics is to encircle the identity of proteins of interest. However, the overall relation between proteins must also be explained. Classical proteomics consist of separation and characterization, based on two-dimensional electrophoresis, trypsin digestion, mass spectrometry and database searching. Characterization includes labor intensive work in order to manage, handle and analyze data. The field of classical proteomics should therefore be extended to also include handling of large datasets in an objective way. The separation obtained by two-dimensional electrophoresis and mass spectrometry gives rise to huge amount of data. We present a multivariate approach to the handling of data in proteomics with the advantage that protein patterns can be spotted at an early stage and consequently the proteins selected for sequencing can be selected intelligently. These methods can also be applied to other data generating protein analysis methods like mass spectrometry and near infrared spectroscopy and examples of application to these techniques are also presented. Multivariate data analysis can unravel complicated data structures and may thereby relieve the characterization phase in classical proteomics. Traditionally statistical methods are not suitable for analysis of the huge amounts of data, where the number of variables exceed the number of objects. Multivariate data analysis, on the other hand, may uncover the hidden structures present in these data. This study takes its starting point in the field of classical proteomics and shows how multivariate data analysis can lead to faster ways of finding interesting proteins. Multivariate analysis has shown interesting results as a supplement to classical proteomics and added a new dimension to the field of proteomics.     

Defining the mandate of proteomics in the post-genomics era: workshop report

Research in proteomics is the next step after genomics in understanding life processes at the molecular level. In the largest sense proteomics encompasses knowledge of the structure, function and expression of all proteins in the biochemical or biological contexts of all organisms. Since that is an impossible goal to achieve, at least in our lifetimes, it is appropriate to set more realistic, achievable goals for the field. Up to now, primarily for reasons of feasibility, scientists have tended to concentrate on accumulating information about the nature of proteins and their absolute and relative levels of expression in cells (the primary tools for this have been 2D gel electrophoresis and mass spectrometry). Although these data have been useful and will continue to be so, the information inherent in the broader definition of proteomics must also be obtained if the true promise of the growing field is to be realized. Acquiring this knowledge is the challenge for researchers in proteomics and the means to support these endeavors need to be provided. An attempt has been made to present the major issues confronting the field of proteomics and two clear messages come through in this report. The first is that the mandate of proteomics is and should be much broader than is frequently recognized. The second is that proteomics is much more complicated than sequencing genomes. This will require new technologies but it is highly likely that many of these will be developed. Looking back 10 to 20 years from now, the question is: Will we have done the job wisely or wastefully? This report summarizes the presentations made at a symposium at the National Academy of Sciences on February 25, 2002.