Characterization and quantitation of membrane proteomes using multidimensional MS-based proteomic technologies

A major goal of proteomics is to develop methods that enable the systematic characterization of every protein within the cell or particular subcellular proteome using a single analytical platform. Although the equivalent has already been achieved in genomics, reaching this goal in proteomics represents a much greater challenge due to the wide dynamic range of protein expression, numerous post-translational modifications and remarkable physicochemical heterogeneity of proteins. A major analytical challenge has involved developing more effective means for proteome-scale investigations of membrane proteins, whose solubility differs drastically from that of cytoplasmic proteins. Fortunately, rapid progress has increased the ability to characterize this critically important class of proteins on a scale analogous to that of aqueous soluble proteins.     

真核细胞质膜蛋白质组研究进展

细胞膜（质膜）蛋白质是细胞的“门铃”与“门户”,是许多药物的作用靶标。细胞质膜蛋白质组的研究正成为蛋白质组研究的热点,这方面的研究有利于具有重要功能的低丰度蛋白质的发掘,为药物研发和疾病的诊断提供靶体与标记蛋白质。然而,质膜蛋白质组的研究在强疏水性跨膜蛋白质和低丰度膜蛋白质的分离和鉴定上遇到了方法学的挑战。本文对质膜及其微区的纯化、质膜蛋白质组的分离与鉴定、生物信息学,以及亚细胞定位研究的近期进展作扼要介绍。     

Strategies for quantitation of phosphoproteomic data

Recent developments in phosphoproteomic sample-preparation techniques and sensitive mass spectrometry instrumentation have led to large-scale identifications of phosphoproteins and phosphorylation sites from highly complex samples. This has facilitated the implementation of different quantitation strategies in order to study the biological role of protein phosphorylation during disease progression, differentiation or during external stimulation of a cellular system. In this article, a brief summary of the most popular strategies for phosphoproteomic studies is given; however, the main focus will be on different quantitation strategies. Methods for metabolic labeling, chemical modification and label-free quantitation and their applicability or inapplicability in phosphoproteomic studies are discussed.     

Peptide fractionation in proteomics approaches

Peptide fractionation is extremely important in proteomics approaches. Full proteome characterization is desired from complex organisms, and with growing interest in post-translational modifications an extended protein sequence coverage is required. Peptide fractionation techniques have the great challenge of feeding current mass spectrometers in a way in which these issues are met. Peptide fractionation can be divided into three simple components: the column characteristics; the mobile phase; and peptide properties (charge, polarity, hydrophobicity and size). The current challenges are in the combination of these three components to allow comprehensive proteomics studies to be improved.     

基于生物质谱的蛋白质C末端研究进展

蛋白质的C末端在蛋白质进行各项生命活动过程中都起着极其重要的作用。它不仅标志着DNA转录翻译成蛋白质过程的初步完成,更是参与和调控了蛋白质的各种生理功能。研究蛋白质的C末端不仅有利于完整蛋白质的鉴定,对于在分子水平理解蛋白质的信号传导和生化功能是十分必要的。文中结合我们的研究工作,综述了近年来基于生物质谱的蛋白质C末端研究的相关进展,包括了C末端的识别、鉴定以及蛋白质C末端肽段富集的新方法和新技术。     

膜整合蛋白质的“鸟枪法”蛋白质组学分析策略

疾病状态下生物膜表面蛋白质分子标记的表达量和修饰状态会发生改变。但由于其低丰度和不易溶解等特性,制约了膜蛋白质组学的研究,同时也制约了相关药物靶标的设计。近年来,为克服这些困难,学者们提出了"鸟枪法"的膜蛋白质组学研究策略。基于此,本文论述了"鸟枪法"的蛋白质组学分析的基本过程及其后续的部分改进工作。随着新的策略不断被采用,更多膜蛋白质的拓扑学特征和功能的相关研究一定会走上新的台阶。     

Molecular constituents of the postsynaptic density fraction revealed by proteomic analysis using multidimensional liquid chromatography-tandem mass spectrometry

Protein constituents of the postsynaptic density (PSD) fraction were analysed using an integrated liquid chromatography (LC)-based protein identification system, which was constructed by coupling microscale two-dimensional liquid chromatography (2DLC) with electrospray ionization (ESI) tandem mass spectrometry (MS/MS) and an automated data analysis system. The PSD fraction prepared from rat forebrain was solubilized in 6 m guanidium hydrochloride, and the proteins were digested with trypsin after S-carbamoylmethylation under reducing conditions. The tryptic peptide mixture was then analysed with the 2DLC-MS/MS system in a data-dependent mode, and the resultant spectral data were automatically processed to search a genome sequence database for protein identification. In triplicate analyses, the system allowed assignments of 5264 peptides, which could finally be attributed to 492 proteins. The PSD contained various proteins involved in signalling transduction, including receptors, ion channel proteins, protein kinases and phosphatases, G-protein and related proteins, scaffold proteins, and adaptor proteins. Structural proteins, including membrane proteins involved in cell adhesion and cell-cell interaction, proteins involved in endocytosis, motor proteins, and cytoskeletal proteins were also abundant. These results provide basic data on a major protein set associated with the PSD and a basis for future functional studies of this important neural machinery.     

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.     

基于质谱技术的蛋白质组定量方法

对不同状态下的蛋白质在表达和修饰水平上进行精确定量,对于探索蛋白质的生物功能、发现疾病的生物标志物都具有重要意义,也是当前蛋白质组学的一个重要研究前沿。近年来,各种蛋白质组定量的新技术和新方法不断涌现,但仍面临着巨大挑战。本文就基于质谱技术的多种蛋白质组定量方法的基本原理、近几年的研究进展和应用进行评述。     

Characterizing phosphoproteins and phosphoproteomes using mass spectrometry.

The reversible phosphorylation of proteins plays a major role in many vital cellular processes by modulating protein function and transmitting signals within cellular pathways and networks. Because phosphorylation is dynamic and the sites of modification cannot be predicted by an organism's genome, proteomic measurements are required to identify sites of and changes in the phosphorylation state of proteins. The low stoichiometry of phosphorylation sites that accompany the multifarious nature of protein phosphorylation in biological systems continues to challenge the dynamic range of present mass spectrometry (MS) technologies and proteomic measurements, despite the preponderance of research and analytical methods devoted to this area. This review addresses some of the strategies and limitations involving the use of MS to map and quantify changes in protein phosphorylation sites for samples that range from a single protein to an entire proteome, and presents several compelling reasons as to why comprehensive phosphorylation site analysis has proven to be so elusive without a hypothesis-driven experimental approach to elicit more meaningful and confident results.     

Advanced proteomic technologies for cancer biomarker discovery

Proteomic technologies have experienced major improvements in recent years. Such advances have facilitated the discovery of potential tumor markers with improved sensitivities and specificities for the diagnosis, prognosis and treatment monitoring of cancer patients. This review will focus on four state-of-the-art proteomic technologies, namely 2D difference gel electrophoresis, MALDI imaging mass spectrometry, electron transfer dissociation mass spectrometry and reverse-phase protein array. The major advancements these techniques have brought about and examples of their applications in cancer biomarker discovery will be presented in this review, so that readers can appreciate the immense progress in proteomic technologies from 1997 to 2008. Finally, a summary will be presented that discusses current hurdles faced by proteomic researchers, such as the wide dynamic range of protein abundance, standardization of protocols and validation of cancer biomarkers, and a 5-year view of potential solutions to such problems will be provided.     

Sample preparation for the analysis of membrane proteomes by mass spectrometry

The low abundance and highly hydrophobic nature of most membrane proteins make their analysis more difficult than that for common soluble proteins. Successful membrane protein identification is largely dependent on the sample preparation including the enrichment and dissolution of the membrane proteins. A series of conventional and newly developed methods has been applied to the enrichment of low-abundance membrane proteins at membrane and/or protein levels and to the dissolution of hydrophobic membrane proteins. However, all the existing methods have inherent advantages and limitations. Up to now, there has been no unique method that can universally be employed to solve all the problems and more efforts are needed in improving sample preparation for the analysis of membrane proteomes.     

Large‐scale identification of membrane proteins with properties favorable for crystallization

Membrane protein crystallography is notoriously difficult due to challenges in protein expression and issues of degradation and structural stability. We have developed a novel method for large‐scale screening of native sources for integral membrane proteins that have intrinsic biochemical properties favorable for crystallization. Highly expressed membrane proteins that are thermally stable and nonaggregating in detergent solutions were identified by mass spectrometry from Escherichia coli, Saccharomyces cerevisiae, and Sus scrofa cerebrum. Many of the membrane proteins identified had been crystallized previously, supporting the promise of the approach. Most identified proteins have known functions and include high‐value targets such as transporters and ATPases. To validate the method, we recombinantly expressed and purified the yeast protein, Yop1, which is responsible for endoplasmic reticulum curvature. We demonstrate that Yop1 can be purified with the detergent dodecylmaltoside without aggregating.     

Combining bioinformatics and MS-based proteomics: clinical implications

Clinical proteomics research aims at i) discovery of protein biomarkers for screening, diagnosis and prognosis of disease, ii) discovery of protein therapeutic targets for improvement of disease prevention, treatment and follow-up, and iii) development of mass spectrometry (MS)-based assays that could be implemented in clinical chemistry, microbiology or hematology laboratories. MS has been increasingly applied in clinical proteomics studies for the identification and quantification of proteins. Bioinformatics plays a key role in the exploitation of MS data in several aspects such as the generation and curation of protein sequence databases, the development of appropriate software for MS data treatment and integration with other omics data and the establishment of adequate standard files for data sharing. In this article, we discuss the main MS approaches and bioinformatics solutions that are currently applied to accomplish the objectives of clinical proteomic research.     

Proteomic analysis of heart mitochondria from <Emphasis Type="Italic">Bos taurus:</Emphasis> I. Application of proteomic methods to identification of transmembrane domains of proteins of the internal mitochondrial membrane

This study is part of a large-scale investigation of the proteome of mitochondria from the heart muscle of Bos taurus. We developed a special approach to simplification of the protein mixture by separation of mitochondrial fractions with stable protein compositions. At the first stage of this approach, we isolated and purified internal mitochondrial membranes. The protein composition of this fraction was analyzed by the following proteomic methods: enzymatic or/and chemical cleavage of the proteins, chromatographic fractionation of the complex mixture of the resulting peptides, mass-spectrometric identification of these peptides, and a search for proteins in databases of amino acid sequences. We reliably identified 147 unique proteins with the use of the SwissProt database. The subcellular location and functions of these proteins were analyzed. Approaches to studies of transmembrane domains of integral membrane proteins of the internal mitochondrial membrane were proposed on the basis of proteomic methods of analysis. Considerable coincidence of the experimental data with the results of determination of the 3D structures of the proteins by X-ray analysis was shown.     

Characterization of mature rat oligodendrocytes: a proteomic approach

Oligodendrocytes are glial cells responsible for the synthesis and maintenance of myelin in the central nervous system (CNS). Oligodendrocytes are vulnerable to damage occurring in a variety of neurological diseases. Understanding oligodendrocyte biology is crucial for the dissemination of de- and remyelination mechanisms. The goal of the present study is the construction of a protein database of mature rat oligodendrocytes. Post-mitotic oligodendrocytes were isolated from mature Wistar rats and subjected to immunocytochemistry. Proteins were extracted and analyzed by means of two-dimensional gel electrophoresis and two-dimensional liquid chromatography, both coupled to mass spectrometry. The combination of the gel-based and gel-free approach resulted in confident identification of a total of 200 proteins. A minority of proteins were identified in both proteomic strategies. The identified proteins represent a variety of functional groups, including novel oligodendrocyte proteins. The results of this study emphasize the power of the applied proteomic strategy to study known or to reveal new proteins and to investigate their regulation in oligodendrocytes in different disease models.     

A proteomic study reveals the diversified distribution of plasma membrane-associated proteins in rat hepatocytes

To investigate the heterogeneous protein composition of highly polarized hepatocyte plasma membrane (PM), three PM-associated subfractions were obtained from freshly isolated rat hepatocytes using density gradient centrifugation. The origins of the three subfractions were determined by morphological analysis and western blotting. The proteins were subjected to either one-dimensional (1-D) SDS-PAGE or two-dimensional (2-D) benzyldimethyl-n-hexadecylammonium chloride (BAC)/SDS-PAGE before nano-Liquid Chromatography-Electrospray Ionization--tandem mass spectrometry analysis (LC-ESI-MS/MS). A total of 613 non-redundant proteins were identified, among which 371 (60.5%) proteins were classified as PM or membrane-associated proteins according to GO annotations and the literatures and 32.4% had transmembrane domains. PM proteins from microsomal portion possessed the highest percentage of transmembrane domain, about 46.5% of them containing at least one transmembrane domain. In addition to proteins known to be located at polarized liver PM regions, such as asialoglycoprotein receptor 2, desmoplakin and bile salt export pump, several proteins which had the potential to become novel subfraction-specific proteins were also identified, such as annexin a6, pannexin and radixin. Our analysis also evaluated the application of 1-D SDS-PAGE and 2-D 16-BAC/SDS-PAGE on the separation of integral membrane proteins.     

Cancer proteomics: many technologies,one goal

A major goal of the National Cancer Institute is to alleviate patient pain, suffering and death associated with cancer by the year 2015. This goal does not insinuate a cure for cancer, but rather the development of diagnostics and therapeutics that will eventually decrease cancer morbidity and mortality. A part of meeting this goal is to leverage the enormous data-gathering capabilities of proteomic technologies to discover disease-specific biomarkers in serum, plasma, urine, tissues and other biologic samples. The rapid advance in available technologies that have been spurred by the -omics era, has enabled biologic samples to be surveyed for biomarkers in ways never before possible. However, it is not yet clear which specific technologies will be the most successful. Therefore, proteomic laboratories within the National Cancer Institute are taking a multipronged approach to identify disease-specific biomarkers. This review discusses some of these approaches in their context of meeting the National Cancer Institute’s 2015 goal.     

Mycobacterial proteomics: analysis of expressed proteomes and post-translational modifications to identify candidate virulence factors

The Mycobacterium tuberculosis bacillus has a number of unique features that make it a particularly effective human pathogen. Although genomic analysis has added to our current understanding of the molecular basis by which M. tuberculosis damages its host, proteomics may be better suited to describe the dynamic interactions between mycobacterial and host systems that underpin this disease. The M. tuberculosis proteome has been investigated using proteomics for over a decade, with increasingly sophisticated mass spectrometry technology and sensitive methods for comparative proteomic profiling. Deeper coverage of the M. tuberculosis proteome has led to the identification of hundreds of putative virulence determinants, as well as an unsurpassed coverage of post-translational modifications. Proteomics is therefore uniquely poised to contribute to our understanding of this pathogen, which may ultimately lead to better management of the disease.     

A proteomic and phosphoproteomic analysis of Oryza sativa plasma membrane and vacuolar membrane

Proteomic and phosphoproteomic analyses of rice shoot and root tonoplast-enriched and plasma membrane-enriched membrane fractions were carried out to look at tissue-specific expression, and to identify putative regulatory sites of membrane transport proteins. Around 90 unique membrane proteins were identified, which included primary and secondary transporters, ion channels and aquaporins. Primary H(+) pumps from the AHA family showed little isoform specificity in their tissue expression pattern, whereas specific isoforms of the Ca(2+) pump ECA/ACA family were expressed in root and shoot tissues. Several ABC transporters were detected, particularly from the MDR and PDR subfamilies, which often showed expression in either roots or shoots. Ammonium transporters were expressed in root, but not shoot, tissue. Large numbers of sugar transporters were expressed, particularly in green tissue. The occurrence of phosphorylation sites in rice transporters such as AMT1;1 and PIP2;6 agrees with those previously described in other species, pointing to conserved regulatory mechanisms. New phosphosites were found in many transporters, including H(+) pumps and H(+):cation antiporters, often at residues that are well conserved across gene families. Comparison of root and shoot tissue showed that phosphorylation of AMT1;1 and several further transporters may be tissue dependent.