化学生物学新前沿——化学蛋白质组学

随着包括人类在内的主要模式生物的基因组计划的完成,生命科学的研究重心转向蛋白质组的研究--在对应基因组的整体蛋白质水平上系统研究调控细胞生命活动的蛋白质.化学蛋白质组学是化学生物学在后基因组时代的最新发展:化学蛋白质组学利用化学小分子为工具和手段,以基于靶蛋白质功能的新战略探测体内蛋白质组,是新一代的功能蛋白质组学.本文综述了化学蛋白质组学的最新进展、有关技术及其在生物医学和药物研发等方面的应用,并对化学蛋白质组学的发展趋势和前景进行了讨论.     

Chip-based nano-LC-MS/MS identification of proteins in complex biological samples using a novel polymer microfluidic device

Although the proteome of each organism is unambiguously coded in its genome, the proteome shows the real biology in action in each particular organism. New powerful tools are being developed for biochemists and biologists to analyze complex biological samples for studying the complete protein supplement of the genome, i. e., the proteome. There are several methods available for proteome analysis including 2-DE and several forms of MS. In recent years, technologies such as microfluidics and array-based systems have appeared in the field of analysis, identification, and quantification of proteins. These novel approaches might help in solving current technical challenges in proteomics. This paper presents a practical application of the first commercially available microfluidic nano-ESI device coupled with nano-LC (i. e., HPLC-chip) for the analysis of samples of some biological protein mixtures.     

基于稳定同位素标记的蛋白质组学定量方法研究进展

定量蛋白质组学已经成为后基因时代的重要研究方向之一。目前该领域的研究主要采用无标记定量方法和稳定同位素标记定量法。其中,基于稳定同位素标记的蛋白质组定量方法发展非常迅速,已为生命科学研究提供了重要的技术支撑。本文分析了基于稳定同位素标记的蛋白质组学定量方法,包括相对定量方法和绝对定量方法,并对其发展进行了展望。     

Proteome-on-a-chip: mirage, or on the horizon?

Proteomics has emerged as the next great scientific challenge in the post-genome era. But even the most basic form of proteomics, proteome profiling, i.e., identifying all of the proteins expressed in a given sample, has proven to be a demanding task. The proteome presents unique analytical challenges, including significant molecular diversity, an extremely wide concentration range, and a tendency to adsorb to solid surfaces. Microfluidics has been touted as being a useful tool for developing new methods to solve complex analytical challenges, and, as such, seems a natural fit for application to proteome profiling. In this review, we summarize the recent progress in the field of microfluidics in four key areas related to this application: chemical processing, sample preconcentration and cleanup, chemical separations, and interfaces with mass spectrometry. We identify the bright spots and challenges for the marriage of microfluidics and proteomics, and speculate on the outlook for progress.     

Gel-based versus gel-free proteomics: a review

With the sequencing of the genome of over 150 organisms, the field of biology has been revolutionised. Instead of studying one gene or protein at the time, it is now possible to study the effect of physiological or pathological changes on the expression of all genes or proteins in the organism. Proteomics aims at the simultaneous analysis of all proteins expressed by a cell, tissue or organism in a specific physiological condition. Because proteins are the effector molecules in all organisms, it is evident that changes in the physiological condition of an organism will be reflected by changes in protein expression and/or processing. Since the formulation of the concept of proteomics in the mid 90's proteomics has relied heavily on 2 dimensional gel electrophoresis (2DGE) for the separation and visualization of proteins. 2DGE, however, has a number of inherent drawbacks. 2DGE is costly, fairly insensitive to low copy proteins and cannot be used for the entire proteome. Therefore, over the years, several gel-free proteomics techniques have been developed to either fill the gaps left by 2DGE or to entirely abolish the gel based techniques. This review summarizes the most important gel-free and gel-based proteomics techniques and compares their advantages and drawbacks.     

Systematic annotation and bioinformatics analyses of large-scale Oryza sativa proteome

Much has been now recognized on the rice (Oryza sativa L.) proteomics by using the powerful experimental tool two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). 2D-PAGE can be utilized for monitoring global changes of quantitative protein expression in specific tissues under various conditions. However, systematic annotations of the protein spots generated by 2D-PAGE are still limited for rice. In this study, a new approach for Oryza sativa proteome annotation based on the 2D-gel maps was developed. Based on the publicly available 2D-PAGE data of rice, 11,201 gel spots were annotated accounting for 87.2% of the total spots on the gel maps. Gel spot alignments were performed for the annotated gel maps belonging to 23 rice tissues or organelles. In summary, 253 alignments between 23 tissues or organelles were performed, and 26,207 co-expressed proteins were identified using our analytical strategy. Large-scale bi-cluster analysis of 23 tissues/organelles proteomes of rice was carried out to detect novel functional proteins. Function and pathway analysis identified a number of common gene products with great potential in regulating specific physiological and biochemical events within various rice tissues/organelles. It also suggested that the tissue- or organelle-specific proteins might be responsible for the functional divergence of these tissues or organelles. Taken together, this study provides us new strategies and informative resources for rice proteome research based on 2D-PAGE data.     

Proteomics in medical microbiology

The techniques of proteomics (high resolution two-dimensional electrophoresis and protein characterisation) are widely used for microbiological research to analyse global protein synthesis as an indicator of gene expression. The rapid progress in microbial proteomics has been achieved through the wide availability of whole genome sequences for a number of bacterial groups. Beyond providing a basic understanding of microbial gene expression, proteomics has also played a role in medical areas of microbiology. Progress has been made in the use of the techniques for investigating the epidemiology and taxonomy of human microbial pathogens, the identification of novel pathogenic mechanisms and the analysis of drug resistance. In each of these areas, proteomics has provided new insights that complement genomic-based investigations. This review describes the current progress in these research fields and highlights some of the technical challenges existing for the application of proteomics in medical microbiology. The latter concern the analysis of genetically heterogeneous bacterial populations and the integration of the proteomic and genomic data for these bacteria. The characterisation of the proteomes of bacterial pathogens growing in their natural hosts remains a future challenge.     

Recent advances in capillary separations for proteomics

The sequencing of several organisms' genomes, including the human's one, has opened the way for the so-called postgenomic era, which is now routinely coined as "proteomics". The most basic task in proteomics remains the detection and identification of proteins from a biological sample, and the most traditional way to achieve this goal consists of protein separations performed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Still, the 2-D PAGE-mass spectrometry (MS) approach remains lacking in proteome coverage (for proteins having extreme isoelectric points or molecular masses as well as for membrane proteins), dynamic range, sensitivity, and throughput. Consequently, considerable efforts have been devoted to the development of non-gel-based proteome separation technologies in an effort to alleviate the shortcomings in 2-D PAGE while reserving the ability to resolve complex protein and peptide mixtures prior to MS analysis. This review focuses on the most recent advances in capillary-based separation techniques, including capillary liquid chromatography, capillary electrophoresis, and capillary electrokinetic chromatography, and combinations of multiples of these mechanisms, along with the coupling of these techniques to MS. Developments in capillary separations capable of providing extremely high resolving power and selective analyte enrichment are particularly highlighted for their roles within the broader context of a state-of-the-art integrated proteome effort. Miniaturized and integrated multidimensional peptide/protein separations using microfluidics are further summarized for their potential applications in high-throughput protein profiling toward biomarker discovery and clinical diagnosis.     

结构蛋白质组学研究进展

蛋白质结构与其生物学功能直接相关，蛋白质功能的调控也主要依赖于其构象和相互作用的动态调节。对蛋白质结构和功能的研究一直是生命科学领域的研究热点，也是当前蛋白质组学研究的重要发展方向。该综述重点讨论了近年来基于质谱的结构蛋白质组学主要分析方法的原理、进展和应用，主要包括非变性质谱法、限制性蛋白质酶切法、化学交联法、氢氘交换法、共价化学标记法、热稳定性分析法等；最后对结构蛋白质组学的发展进行了总结与展望。     

Proteomics based on high-efficiency capillary separations

Identifying and quantifying in a high throughput manner the proteins expressed by cells, tissues or an organism provides the basis for understanding the functions of its constituents at a "systems" level. As a result, proteome analysis has increasingly become the focus of significant interest and research over the past decade. This is especially true following the recent stunning achievements in genomics analyses. However, unlike the static genome, the complexities and dynamism of the proteome present significant analytical challenges and demand highly efficient separations and detection technologies. A number of recent technological advancements have been in direct response to these challenges. Currently, strategically mated combinations of sophisticated separations techniques and advanced mass spectrometric detection represent the best approach to addressing the intricacies of the proteome. Liquid-phase separations, often within capillaries, are increasingly recognized as the best separations technique for this approach. In combination on-line with mass spectrometry, liquid-phase separations provide the improved analytical sensitivity, sample throughput, and quantitation capabilities necessitated by the multifaceted problems within proteomics analyses. This review focuses primarily on current high-efficiency capillary separations techniques, including both capillary liquid chromatography and capillary electrophoresis, applied to the analysis of complex proteomic samples. We emphasize developments at our laboratory and illustrate technical advances that attempt to review the role of separations within the broader context of a state-of-the-art integrated proteomics effort.     

Nascent Proteomics: Chemical Tools for Monitoring Newly Synthesized Proteins

Cellular proteins are dynamically regulated in response to environmental stimuli. Conventional proteomics compares the entire proteome in different cellular states to identify differentially expressed proteins, which suffers from limited sensitivity for analyzing acute and subtle changes. To address this challenge, nascent proteomics has been developed, which selectively analyzes the newly synthesized proteins, thus offering a more sensitive and timely insight into the dynamic changes of the proteome. In this Minireview, we discuss recent advancements in nascent proteomics, with an emphasis on methodological developments. Also, we delve into the current challenges and provide an outlook on the future prospects of this exciting field.     

Strategies for shotgun identification of post-translational modifications by mass spectrometry

The global identification of post-translationally modified proteins is a difficult challenge that is currently being addressed by many researchers in the field of mass spectrometry (MS)-based proteomics. The ability to identify thousands of proteins by shotgun-based strategies has made the mere idea of a global analysis of a particular protein modification seem reasonable. There has been much progress in the development of methods that make use of shotgun-based protein identification in the analysis of a wide variety of protein modifications, some of which will be discussed here.     

Profiling the specific reactivity of the proteome with non-directed activity-based probes

BACKGROUND: The field of proteomics aims to characterize dynamics in protein function on a global level. However, several classes of proteins, in particular low abundance proteins, remain difficult to characterize using standard proteomics technologies. Recently, chemical strategies have emerged that profile classes of proteins based on activity rather than quantity, thereby greatly facilitating the analysis of low abundance constituents of the proteome. RESULTS: In order to expand the classes of proteins susceptible to analysis by activity-based methods, we have synthesized a library of biotinylated sulfonate esters and applied its members to complex proteomes under conditions that distinguish patterns of specific protein reactivity. Individual sulfonates exhibited unique profiles of proteome reactivity that in extreme cases appeared nearly orthogonal to one another. A robustly labeled protein was identified as a class I aldehyde dehydrogenase and shown to be irreversibly inhibited by members of the sulfonate library. CONCLUSIONS: Through screening the proteome with a non-directed library of chemical probes, diverse patterns of protein reactivity were uncovered. These probes labeled protein targets based on properties other than abundance, circumventing one of the major challenges facing contemporary proteomics research. Considering further that the probes were found to inhibit a target enzyme's catalytic activity, the methods described herein should facilitate the identification of compounds possessing both selective proteome reactivities and novel bioactivities.     

比较蛋白质组学研究中的稳定同位素标记技术

比较蛋白质组学是指在蛋白质组学水平上研究正常和病理情况下细胞或组织中蛋白质表达变化,以期发现具有重要功能的生物标识物,为疾病的早期诊断提供依据。近年来它正成为蛋白质组学研究的热点和发展趋势。比较蛋白质组学的研究方法和策略有多种,本文就最近几年来稳定同位素标记技术(体内代谢标记技术和体外化学标记技术)在比较蛋白质组学研究中的进展进行综述。     

Gel and polymer-solution mediated separation of biopolymers by capillary electrophoresis

In the age of genomics and proteomics, high-resolution separation techniques are routinely utilized in an integrated and automated fashion to solve formidable separation problems and provide the means for large-scale analysis of biological samples with excellent resolution. By automating the current manual procedures, capillary gel (CGE) and polymer-solution mediated electrophoresis greatly enhance the productivity of biopolymer analysis while also reducing both analysis time and the human intervention necessary from sample loading to data processing. The advent of this novel and high-performance bioseparation technique has made it possible to sequence the human genome and revealed global changes in the genome and proteome level, bringing about a revolutionary transition in our views of living systems on the molecular basis. CGE and polymer-solution mediated electrophoresis and related microseparation methods (e.g., electrophoresis microchips) are quickly becoming important separation and characterization tools in analytical biochemistry and molecular biology. This review gives an overview of the key application areas of DNA, protein, and complex carbohydrate analysis, and summarizes the latest developments on CGE column technology, including capillary coatings and sieving polymer matrices. Micropreparative aspects and related microseparation techniques are also discussed.     

Quantitative proteomics reveals the kinetics of trypsin-catalyzed protein digestion

Trypsin is the popular protease to digest proteins into peptides in shotgun proteomics, but few studies have attempted to systematically investigate the kinetics of trypsin-catalyzed protein digestion in proteome samples. In this study, we applied quantitative proteomics via triplex stable isotope dimethyl labeling to investigate the kinetics of trypsin-catalyzed cleavage. It was found that trypsin cleaves the C-terminal to lysine (K) and arginine (R) residues with higher rates for R. And the cleavage sites surrounded by neutral residues could be quickly cut, while those with neighboring charged residues (D/E/K/R) or proline residue (P) could be slowly cut. In a proteome sample, a huge number of proteins with different physical chemical properties coexists. If any type of protein could be preferably digested, then limited digestion could be applied to reduce the sample complexity. However, we found that protein abundance and other physicochemical properties, such as molecular weight (Mw), grand average of hydropathicity (GRAVY), aliphatic index, and isoelectric point (pI) have no notable correlation with digestion priority of proteins. Graphical Abstract

Sequence logos of four cleavage site types with different kinetics (very fast, fast, slow, and very slow sites)     

Analytical platforms for activity-based protein profiling--exploiting the versatility of chemistry for functional proteomics

The field of proteomics aims to develop and apply technologies for the characterization of protein function on a global scale. Toward this end, synthetic chemistry has played a major role by providing new reagents to profile segments of the proteome based on activity rather than abundance. Small molecule probes for activity-based protein profiling have been created for more than a dozen enzyme classes and used to discover several enzyme activities elevated in disease states. These innovations have inspired complementary advancements in analytical chemistry, where new platforms have been introduced to augment the information content achievable in chemical proteomics experiments. Here, we will review these analytical platforms and discuss how they have exploited the versatility of chemical probes to gain unprecedented insights into the function of proteins in biological samples of high complexity.     

Proteomics and Its Application in Biomarker Discovery and Drug Development

Proteomics is a research field aiming to characterize molecular and cellular dynamics in protein expression and function on a global level. The introduction of proteomics has been greatly broadening our view and accelerating our path in various medical researches. The most significant advantage of proteomics is its ability to examine a whole proteome or sub-proteome in a single experiment so that the protein alterations corresponding to a pathological or biochemical condition at a given time c…