JVirGel: Calculation of virtual two-dimensional protein gels

We developed JVirGel, a collection of tools for the simulation and analysis of proteomics data. The software creates and visualizes virtual two-dimensional (2D) protein gels based on the migration behaviour of proteins in dependence of their theoretical molecular weights in combination with their calculated isoelectric points. The utilization of all proteins of an organism of interest deduced from genes of the corresponding genome project in combination with the elimination of obvious membrane proteins permits the creation of an optimized calculated proteome map. The electrophoretic separation behaviour of single proteins is accessible interactively in a Java(TM) applet (small application in a web browser) by selecting a pI/MW range and an electrophoretic timescale of interest. The calculated pattern of protein spots helps to identify unknown proteins and to localize known proteins during experimental proteomics approaches. Differences between the experimentally observed and the calculated migration behaviour of certain proteins provide first indications for potential protein modification events. When possible, the protein spots are directly linked via a mouse click to the public databases SWISS-PROT and PRODORIC. Additionally, we provide tools for the serial calculation and visualization of specific protein properties like pH dependent charge curves and hydrophobicity profiles. These values are helpful for the rational establishment of protein purification procedures. The proteomics tools are available on the World Wide Web at http://prodoric.tu-bs.de/proteomics.php.     

Shotgun identification of the structural proteome of shrimp white spot syndrome virus and iTRAQ differentiation of envelope and nucleocapsid subproteomes

White spot syndrome virus (WSSV) is a major pathogen that causes severe mortality and economic losses to shrimp cultivation worldwide. The genome of WSSV contains a 305-kb double-stranded circular DNA, which encodes 181 predicted ORFs. Previous gel-based proteomics studies on WSSV have identified 38 structural proteins. In this study, we applied shotgun proteomics using off-line coupling of an LC system with MALDI-TOF/TOF MS/MS as a complementary and comprehensive approach to investigate the WSSV proteome. This approach led to the identification of 45 viral proteins; 13 of them are reported for the first time. Seven viral proteins were found to have acetylated N termini. RT-PCR confirmed the mRNA expression of these 13 newly identified viral proteins. Furthermore iTRAQ (isobaric tags for relative and absolute quantification), a quantitative proteomics strategy, was used to distinguish envelope proteins and nucleocapsid proteins of WSSV. Based on iTRAQ ratios, we successfully identified 23 envelope proteins and six nucleocapsid proteins. Our results validated 15 structural proteins with previously known localization in the virion. Furthermore the localization of an additional 12 envelope proteins and two nucleocapsid proteins was determined. We demonstrated that iTRAQ is an effective approach for high throughput viral protein localization determination. Altogether WSSV is assembled by at least 58 structural proteins, including 13 proteins newly identified by shotgun proteomics and one identified by iTRAQ. The localization of 42 structural proteins was determined; 33 are envelope proteins, and nine are nucleocapsid proteins. A comprehensive identification of WSSV structural proteins and their localization should facilitate the studies of its assembly and mechanism of infection.     

Population proteomics: the concept, attributes, and potential for cancer biomarker research

This review outlines the concept of population proteomics and its implication in the discovery and validation of cancer-specific protein modulations. Population proteomics is an applied subdiscipline of proteomics engaging in the investigation of human proteins across and within populations to define and better understand protein diversity. Population proteomics focuses on interrogation of specific proteins from large number of individuals, utilizing top-down, targeted affinity mass spectrometry approaches to probe protein modifications. Deglycosylation, sequence truncations, side-chain residue modifications, and other modifications have been reported for myriad of proteins, yet little is know about their incidence rate in the general population. Such information can be gathered via population proteomics and would greatly aid the biomarker discovery efforts. Discovery of novel protein modifications is also expected from such large scale population proteomics, expanding the protein knowledge database. In regard to cancer protein biomarkers, their validation via population proteomics-based approaches is advantageous as mass spectrometry detection is used both in the discovery and validation process, which is essential for the detection of those structurally modified protein biomarkers.     

蛋白质组学在附睾研究中的应用

蛋白质组学是在后基因组时代出现的一个新的研究领域,它从整体水平对蛋白质的表达和功能模式进行分析.附睾是精子成熟和贮存的场所,因其基因表达呈现出高度的区域特异性,可能成为蛋白质组学,尤其是差异蛋白质组学研究的一个理想模型.本文主要对不同物种的附睾蛋白质组以及附睾不同区域的差异表达蛋白的研究进展予以综述,期望为附睾功能研究及开发新型男性避孕药带来新的思路和方法.     

Proteomics technologies and challenges

Proteomics is the study of proteins and their interactions in a cell. With the completion of the Human Genome Project, the emphasis is shifting to the protein compliment of the human organism. Because proteome reflects more accurately on the dynamic state of a cell, tissue, or organism, much is expected from proteomics to yield better disease markers for diagnosis and therapy monitoring. The advent of proteomics technologies for global detection and quantitation of proteins creates new opportunities and challenges for those seeking to gain greater understanding of diseases. High-throughput proteomics technologies combining with advanced bioinformatics are extensively used to identify molecular signatures of diseases based on protein pathways and signaling cascades. Mass spectrometry plays a vital role in proteomics and has become an indispensable tool for molecular and cellular biology. While the potential is great, many challenges and issues remain to be solved, such as mining low abundant proteins and integration of proteomics with genomics and metabolomics data. Nevertheless, proteomics is the foundation for constructing and extracting useful knowledge to biomedical research. In this review, a snapshot of contemporary issues in proteomics technologies is discussed.     

Current methodologies for proteomics of bacterial surface-exposed and cell envelope proteins

The study of surface-exposed proteins has received increasing attention following the advent of genomic sequencing, which in turn has enabled predictive tools and facilitated the technologies for their analysis by proteomics. The exterior topology of a bacterial pathogen is the interface between the cell and environment and thus is the initial mediator for infection, providing an important reservoir for components that may be used for novel vaccine development as well as the characterization of new drug targets. The study of such biological molecules has however, been under-represented in proteomics studies due to the difficulty involved in their analysis. Cell-envelope proteins in bacteria are typically difficult to characterize due to their low abundance, poor solubility, and the problematic isolation of pure surface fractions with only minimal contamination. Here, we describe different cell envelope preparations for proteomic characterization, focused principally on gel-free technologies. Fractionation techniques popularly used in proteomics are also explained with emphasis on surface and membrane-derived proteins/peptides. Conditional confirmation of localization is also explored with emphasis on different prediction algorithms as well as on analyses of surface peptide fractions by the use of different search programs and their implications for the unambiguous identification of surface-exposed and membrane-embedded proteins. Finally, different quantification techniques are discussed that are important for the validation of identifications and for highlighting novel proteins that may warrant further study by independent techniques.     

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.     

Characterization of the rat liver membrane proteome using peptide immobilized pH gradient isoelectric focusing

Membrane proteins are of particular interest in proteomics because of their potential therapeutic utility. Past proteomic approaches used to investigate membrane proteins have only been partially successful at providing a comprehensive analysis due to the inherently hydrophobic nature and low abundance for some of these proteins. Recently, these difficulties have been improved by analyzing membrane protein enriched samples using shotgun proteomics. In addition, the recent application of methanol-assisted trypsin digestion of membrane proteins has been shown to be a method to improve membrane protein identifications. In this study, a comparison of different concentrations of methanol was assessed for assisting membrane protein digestion with trypsin prior to analysis using a gel-based shotgun proteomics approach called peptide immobilized pH gradient isoelectric focusing (IPG-IEF). We demonstrate the use of peptide IEF on pH 3-10 IPG strips as the first dimension of two-dimensional shotgun proteomics for protein identifications from the membrane fraction of rat liver. Tryptic digestion of proteins was carried out in varying concentrations of methanol in 10 mM ammonium bicarbonate: 0% (v/v), 40% (v/v), and 60% (v/v). A total of 800 proteins were identified from 60% (v/v) methanol, which increased the protein identifications by 17% and 14% compared to 0% (v/v) methanol and 40% (v/v) methanol assisted digestion, respectively. In total, 1549 nonredundant proteins were identified from all three concentrations of methanol including 690 (42%) integral membrane proteins of which 626 of these proteins contained at least one transmembrane domain. Peptide IPG-IEF separation of peptides was successful as the peptides were separated into discrete pI regions with high resolution. The results from this study prove utility of 60% (v/v) methanol assisted digestion in conjunction with peptide IPG-IEF as an optimal shotgun proteomics technique for the separation and identification of previously unreported membrane proteins.     

膜转运蛋白的功能性超表达和纯化

膜转运蛋白结构和功能的研究是功能膜蛋白质组研究中的一个重要内容,而大量蛋白质的分离纯化是进行蛋白质的结构和功能研究的基础.目前,结构和功能膜蛋白质组学相关研究的瓶颈,在于不能有效地超量表达和纯化具有生物活性的膜转运蛋白.影响膜转运蛋白超量表达和纯化的关键因素,包括目标蛋白的拓扑学结构分析和去垢剂的选择.进行膜转运蛋白拓扑学结构的分析,对于构建用于活体表达的重组膜转运蛋白具有指导意义.去垢剂能够稳定去膜状态的膜蛋白,在膜转运蛋白的离体表达和亲和纯化以及包涵体的处理过程中具有重要的作用.本文就目前功能膜蛋白质组学研究中所涉及的有关膜转运蛋白功能性超表达和分离纯化策略及关键技术作一简述.     

Proteomic map of peripheral blood mononuclear cells

In the field of proteomics extensive efforts have been focused on the knowledge of proteins expressed by different cell types. In particular, enormous progress has been done in the characterization of blood cellular components. In this work, we have established a public 2-DE database for human peripheral blood mononuclear cells (PBMCs) proteins. Two hundred and forty-six spots corresponding to 174 different proteins have been identified on 2-DE gels from PBMCs isolated from six healthy individuals. All the identified proteins have been classified in thirteen categories on the basis of their differential functions or cellular localization and annotated at the http://physiology.unile.it/proteomics. The role of several proteins has been discussed in relation to their biological function. We intend to show the potentiality of PBMCs to investigate the proteomics changes possibly associated with a large number of pathologies such as autoimmune, neurodegenerative and cancer diseases.     

Terminal proteomics: N- and C-terminal analyses for high-fidelity identification of proteins using MS

In proteomics, MS plays an essential role in identifying and quantifying proteins. To characterize mature target proteins from living cells, candidate proteins are often analyzed with PMF and MS/MS ion search methods in combination with computational search routines based on bioinformatics. In contrast to shotgun proteomics, which is widely used to identify proteins, proteomics based on the analysis of N- and C-terminal amino acid sequences (terminal proteomics) should render higher fidelity results because of the high information content of terminal sequence and potentially high throughput of the method not requiring very high sequence coverage to be achieved by extensive sequencing. In line with this expectation, we review recent advances in methods for N- and C-terminal amino acid sequencing of proteins. This review focuses mainly on the methods of N- and C-terminal analyses based on MALDI-TOF MS for its easy accessibility, with several complementary approaches using LC/MS/MS. We also describe problems associated with MS and possible remedies, including chemical and enzymatic procedures to enhance the fidelity of these methods.     

Activity profiling of platelets by chemical proteomics

Chemical proteomics or activity based proteomics is a functional proteomics technology where molecular probes are used to target a selective group of functionally related proteins. Its emergence has enabled specific targeting of subproteomes, overcoming the limitations in dynamic range of traditional large‐scale proteomics experiments. Using a chemical proteomics strategy, we attempt to differentially profile the nucleotide‐binding proteome of active and resting platelets. We apply an affinity chromatography protocol using immobilized adenosine triphosphate, cyclic adenosine monophosphate, and cyclic guanosine monophosphate. The specificity of the immobilized nucleotides was demonstrated by competitive assays and by immunoblotting. LC coupled MS/MS was applied to identify the proteins recovered by our chemical proteomics strategy. When compared to a standard set of platelet lysate proteins, we confirmed that enrichment for nucleotide‐binding proteins was indeed taking place. Finally, by employing label‐free MS‐based comparative quantification, we found a small number of platelet proteins that show statistically significant difference between the active and resting nucleotide‐binding proteome.     

Ultrahigh pressure fast size exclusion chromatography for top‐down proteomics

Top‐down MS‐based proteomics has gained a solid growth over the past few years but still faces significant challenges in the LC separation of intact proteins. In top‐down proteomics, it is essential to separate the high mass proteins from the low mass species due to the exponential decay in S/N as a function of increasing molecular mass. SEC is a favored LC method for size‐based separation of proteins but suffers from notoriously low resolution and detrimental dilution. Herein, we reported the use of ultrahigh pressure (UHP) SEC for rapid and high‐resolution separation of intact proteins for top‐down proteomics. Fast separation of intact proteins (6–669 kDa) was achieved in < 7 min with high resolution and high efficiency. More importantly, we have shown that this UHP‐SEC provides high‐resolution separation of intact proteins using a MS‐friendly volatile solvent system, allowing the direct top‐down MS analysis of SEC‐eluted proteins without an additional desalting step. Taken together, we have demonstrated that UHP‐SEC is an attractive LC strategy for the size separation of proteins with great potential for top‐down proteomics.     

High-throughput proteomics using matrix-assisted laser desorption/ ionization mass spectrometry

It has become evident that the mystery of life will not be deciphered just by decoding its blueprint, the genetic code. In the life and biomedical sciences, research efforts are now shifting from pure gene analysis to the analysis of all biomolecules involved in the machinery of life. One area of these postgenomic research fields is proteomics. Although proteomics, which basically encompasses the analysis of proteins, is not a new concept, it is far from being a research field that can rely on routine and large-scale analyses. At the time the term proteomics was coined, a gold-rush mentality was created, promising vast and quick riches (i.e., solutions to the immensely complex questions of life and disease). Predictably, the reality has been quite different. The complexity of proteomes and the wide variations in the abundances and chemical properties of their constituents has rendered the use of systematic analytical approaches only partially successful, and biologically meaningful results have been slow to arrive. However, to learn more about how cells and, hence, life works, it is essential to understand the proteins and their complex interactions in their native environment. This is why proteomics will be an important part of the biomedical sciences for the foreseeable future. Therefore, any advances in providing the tools that make protein analysis a more routine and large-scale business, ideally using automated and rapid analytical procedures, are highly sought after. This review will provide some basics, thoughts and ideas on the exploitation of matrix-assisted laser desorption/ ionization in biological mass spectrometry – one of the most commonly used analytical tools in proteomics – for high-throughput analyses.     

Development and application of proteomics technologies in Saccharomyces cerevisiae

Proteomics research focuses on the identification and quantification of "all" proteins present in cells, organisms or tissue. Proteomics is technically complicated because it encompasses the characterization and functional analysis of all proteins that are expressed by a genome. Moreover, because the expression levels of proteins strongly depend on complex regulatory systems, the proteome is highly dynamic. This review focuses on the two major proteomics methodologies, one based on 2D gel electrophoresis and the other based on liquid chromatography coupled to mass spectrometry. The recent developments of these methodologies and their application to quantitative proteomics are described. The model system Saccharomyces cerevisiae is considered to be the optimal vehicle for proteomics and we review studies investigating yeast adaptation to changes in (nutritional) environment.     

Proteomics: a link between genomics,genetics and physiology

Thanks to spectacular advances in the techniques for identifying proteins separated by two-dimensional electrophoresis and in methods for large-scale analysis of proteome variations, proteomics is becoming an essential methodology in various fields of plant biology. In the study of pleiotropic effects of mutants and in the analysis of responses to hormones and to environmental changes, the identification of involved metabolic pathways can be deduced from the function of affected proteins. In molecular quantitative genetics, proteomics can be used to map translated genes and loci controlling their expression, which can be used to identify proteins accounting for the variation of complex phenotypic traits. Linking gene expression to cell metabolism on the one hand and to genetic maps on the other, proteomics is a central tool for functional genomics.     

Proteins at membrane surfaces-a review of approaches

Membrane proteins are critical for normal cellular differentiation and function, and alterations in these proteins often leads to cell dysfunction and disease. Membrane proteomics aims to identify the membrane protein constituents, their posttranslational modifications, protein-protein interactions, and dynamics. Efforts to identify membrane proteins and elucidate their dynamics have been plagued by the challenges presented by studying water insoluble proteins that are distributed among a range of membranes in a cell and often occur at a relatively low abundance. This brief review presents a summary of the literature related to membrane proteomics with an emphasis on efforts to develop effective protocols for the enrichment of membrane proteins, particularly those located in the plasma membrane.     

Proteomic profiling of human pleural effusion using two-dimensional nano liquid chromatography tandem mass spectrometry

Pleural effusion, an accumulation of pleural fluid, contains proteins originated from plasma filtrate and, especially when tissues are damaged, parenchyma interstitial spaces of lungs and/or other organs. This study details protein profiles in human pleural effusion from 43 lung adenocarcinoma patients by a two-dimensional nano-high performance liquid chromatography electrospray ionization tandem mass spectrometry (2D nano-HPLC-ESI-MS/MS) system. The experimental results revealed the identification of 1415 unique proteins from human pleural effusion. Among these 124 proteins identified with higher confidence levels, some proteins have not been reported in plasma and may represent proteins specifically present in pleural effusion. These proteins are valuable for mass identification of differentially expressed proteins involved in proteomics database and screening biomarker to further study in human lung adenocarcinoma. The significance of the use of proteomics analysis of human pleural fluid for the search of new lung cancer marker proteins, and for their simultaneous display and analysis in patients suffering from lung disorders has been examined.     

Label-free quantitation, an extension to 2DB

Determining the differential expression of proteins under different conditions is of major importance in proteomics. Since mass spectrometry-based proteomics is often used to quantify proteins, several labelling strategies have been developed. While these are generally more precise than label-free quantitation approaches, they imply specifically designed experiments which also require knowledge about peptides that are expected to be measured and need to be modified. We recently designed the 2DB database which aids storage, analysis, and publication of data from mass spectrometric experiments to identify proteins. This database can aid identifying peptides which can be used for quantitation. Here an extension to the database application, named MSMAG, is presented which allows for more detailed analysis of the distribution of peptides and their associated proteins over the fractions of an experiment. Furthermore, given several biological samples in the database, label-free quantitation can be performed. Thus, interesting proteins, which may warrant further investigation, can be identified en passant while performing high-throughput proteomics studies.