Application of free-flow electrophoresis/2-dimentional gel electrophoresis for fractionation and characterization of native proteome of Pseudomonas putida KT2440

Free Flow Electrophoresis (FFE) is a liquid-based isoelectric focusing method. Unlike conventional in-gel fractionation of proteins, FFE can resolve proteins in their native forms and fractionation of subcellular compartments of the cell is also possible. To test the efficacy of the FFE method, the native cytosol proteome of a bacterium, Pseudomonas putida KT2440 was fractionated by FFE and the spectrum of protein elutes was characterized in association with 2-dimentional gel electrophoresis (2-DE). Major native proteins of P. putida KT2440 were eluted in the range of pH 4.8 approximately 6.0 in FFE, whereas the denatured proteome of P. putida KT2440 was widely distributed in the rage of pH 4 approximately 10 in the 2-DE analysis. In addition, one of the three FFE major fractions, which was eluted at pH 5.0, was further analyzed using 2-DE/MS-MS. Then, the pH range of identified proteins eluted in 2-DE/MS-MS was 4.72 approximately 5.89, indicating that observed pi values of native cytosolic proteomes in FFE were narrower than those of denatured cytosolic proteome. These results suggest that FFE fractionation and 2-DE/MS analysis may be useful tools for characterization of native proteomes of P. putida KT2440 and comparative analysis between denatured and native proteomes.     

The synaptic vesicle proteome: a comparative study in membrane protein identification

A proteomic analysis of the synaptic vesicle was undertaken to obtain a better understanding of vesicle regulation. Synaptic vesicles primarily consist of integral membrane proteins that are not well resolved on traditional isoelectric focusing/two-dimensional gel electrophoresis (IEF/2-DE) gels and are resistant to in-gel digestion with trypsin thereby reducing the number of peptides available for mass spectrometric analysis. To address these limitations, two complementary 2-DE methods were investigated in the proteome analysis: (a) IEF/sodium dodecyl sulfate-polyacrylamide gel electrophoresis (IEF/SDS-PAGE) for resolution of soluble proteins and, (b) Benzyl hexadecyl ammonium chloride/SDS-PAGE (16-BAC/SDS-PAGE) for resolution of integral membrane proteins. The IEF/SDS-PAGE method provided superior resolution of soluble proteins, but could only resolve membrane proteins with a single transmembrane domain. The 16-BAC/SDS-PAGE method improved separation, resolution and identification of integral membrane proteins with up to 12 transmembrane domains. Trypsin digestion of the integral membrane proteins was poor and fewer peptides were identified from these proteins. Analysis of both the peptide mass fingerprint and the tandem mass spectra using electrospray ionization quadrupole-time of flight mass spectrometry led to the positive identification of integral membrane proteins. Using both 2-DE separation methods, a total of 36 proteins were identified including seven integral membrane proteins, 17 vesicle regulatory proteins and four proteins whose function in vesicles is not yet known.     

Identification and characterization of the Sulfolobus solfataricus P2 proteome

Via combined separation approaches, a total of 1399 proteins were identified, representing 47% of the Sulfolobus solfataricus P2 theoretical proteome. This includes 1323 proteins from the soluble fraction, 44 from the insoluble fraction and 32 from the extra-cellular or secreted fraction. We used conventional 2-dimensional gel electrophoresis (2-DE) for the soluble fraction, and shotgun proteomics for all three cell fractions (soluble, insoluble, and secreted). Two gel-based fractionation methods were explored for shotgun proteomics, namely: (i) protein separation utilizing 1-dimensional gel electrophoresis (1-DE) followed by peptide fractionation by iso-electric focusing (IEF), and (ii) protein and peptide fractionation both employing IEF. Results indicate that a 1D-IEF fractionation workflow with three replicate mass spectrometric analyses gave the best overall result for soluble protein identification. A greater than 50% increment in protein identification was achieved with three injections using LC-ESI-MS/MS. Protein and peptide fractionation efficiency; together with the filtration criteria are also discussed.     

结核分枝杆菌菌体蛋白双向电泳技术的优化

目的：提取结核分枝杆菌菌体蛋白并建立一种利用双向电泳分离结核分枝杆菌蛋白质组的方法。方法：分离提取结核分枝杆菌菌体蛋白。样品采用不同pH梯度的鹏胶条进行第一向等电聚焦,12％SDS—PAGE凝胶进行二向电泳。银染后双向电泳图谱用Molecular Image Fx激光图像扫描仪扫描,PDQuest6．0软件完成配比分析。结果：优化了结核分枝杆菌菌体蛋白的提取方法,用裂解液8mol／L尿素结合2mol／L硫脲,140mmol／LDTT,0．5％biolyte,4％CHAPs,400mg／m1lOG处理,成功提取了蛋白,并通过结核分枝杆菌双向电泳技术体系的优化,建立了结核分枝杆菌菌体蛋白的分解图谱。pH4—7及pH7—10两胶面上共1387个点,占所检测到的蛋白总数的86％,绝大部分（1194个）蛋白位于pH4—7范围内。结论：为进一步开展结核分枝杆菌的比较蛋白质组学研究提供了方法学参考。     

The membrane proteome of Halobacterium salinarum

The identification of 114 integral membrane proteins from Halobacterium salinarum was achieved using liquid chromatography/tandem mass spectrometric (LC/MS/MS) techniques, representing 20% of the predicted alpha-helical transmembrane proteins of the genome. For this experiment, a membrane preparation with only minor contamination by soluble proteins was prepared. From this membrane preparation a number of peripheral membrane proteins were identified by the classical two dimensional gel electrophoresis (2-DE) approach, but identification of integral membrane proteins largely failed with only a very few being identified. By use of a fluorescently labeled membrane preparation, we document that this is caused by an irreversible precipitation of the membrane proteins upon isoelectric focusing (IEF). Attempts to overcome this problem by using alternative IEF methods and IEF strip solubilisation techniques were not successful, and we conclude that the classical 2-DE approach is not suited for the identification of integral membrane proteins. Computational analysis showed that the identification of integral membrane proteins is further complicated by the generation of tryptic peptides, which are unfavorable for matrix assisted laser desorption/ionization time of flight mass spectrometric peptide mass fingerprint analysis. Together with the result from the analysis of the cytosolic proteome (see preceding paper), we could identify 34% (943) of all gene products in H. salinarum which can be theoretically expressed. This is a cautious estimate as very stringent criteria were applied for identification. These results are available under www.halolex.mpg.de.     

箭毒木种子蛋白质样品制备及双向电泳改良方法

建立箭毒木（Antiaris toxicaria）种子总蛋白的提取方法,以及可以对其蛋白质组进行分析的双向电泳条件。通过各种条件的优化与组合,建立了以TCA-丙酮为基础的Tris—HCl提取法提取总蛋白,第1向电泳为固相pH梯度等电聚焦,第2向电泳为垂直平板SDS-PAGE的双向电泳体系。通过对样品制备、样品溶解、等电聚焦电泳、SDS-聚丙烯酰胺凝胶电泳以及染色方法等关键步骤进行分析,获得了满意的双向电泳图谱。在探索适合箭毒木种子蛋白质组学研究双向电泳方法中,比较了三氯乙酸-丙酮沉淀法、和Tris—HCl法,以及对双向电泳过程中的关键步骤的改良,认为Tris—HCl法为最适方法,所得图谱背景清晰,蛋白质信息量最大,为箭毒木属植物的差异蛋白质组学的后续研究打下了坚实的基础。     

Two-dimensional gel electrophoresis in bacterial proteomics

Two-dimensional gel electrophoresis (2-DE) is a gel-based technique widely used for analyzing the protein composition of biological samples. It is capable of resolving complex mixtures containing more than a thousand protein components into individual protein spots through the coupling of two orthogonal biophysical separation techniques: isoelectric focusing (first dimension) and polyacrylamide gel electrophoresis (second dimension). 2-DE is ideally suited for analyzing the entire expressed protein complement of a bacterial cell: its proteome. Its relative simplicity and good reproducibility have led to 2-DE being widely used for exploring proteomics within a wide range of environmental and medically-relevant bacteria. Here we give a broad overview of the basic principles and historical development of gel-based proteomics, and how this powerful approach can be applied for studying bacterial biology and physiology. We highlight specific 2-DE applications that can be used to analyze when, where and how much proteins are expressed. The links between proteomics, genomics and mass spectrometry are discussed. We explore how proteomics involving tandem mass spectrometry can be used to analyze (post-translational) protein modifications or to identify proteins of unknown origin by de novo peptide sequencing. The use of proteome fractionation techniques and non-gel-based proteomic approaches are also discussed. We highlight how the analysis of proteins secreted by bacterial cells (secretomes or exoproteomes) can be used to study infection processes or the immune response. This review is aimed at non-specialists who wish to gain a concise, comprehensive and contemporary overview of the nature and applications of bacterial proteomics.     

The role of mass spectrometry in proteome studies

Mass spectrometry (MS) is an important tool in modern protein chemistry. In proteome analyses the expression of hundreds or thousands of proteins can be monitored at the same time. First, complex protein mixtures are separated by two-dimensional gel electrophoresis (2-DE) and then individual proteins are identified by using MS followed by database searches. Recent developments in this field have made it possible to do automated, high-throughput protein identification that is needed in proteome analyses. MS can also be used to characterize post-translational modifications in proteins and to study protein complexes. This review will introduce the current MS methods used in proteome studies, and discuss their advantages and disadvantages. New instrumental MS developments are also presented that are useful in these analyses.     

Proteome profile of zebrafish kidney

The most imperative organ, kidney has been widely studied in zebrafish for its simplified structures and development. Understanding the proteomic component of kidney might lead to a better insight for understanding the structural and functional complexity of kidney. In this study we have analyzed the proteome profile of the zebrafish kidney based on gel based proteome mapping techniques involving single dimension gel electrophoresis nanoflow liquid chromatography mass spectrophotometer, single dimension gel electrophoresis microflow ESI liquid chromatography mass spectrophotometer and two dimensional gel electrophoresis matrix assisted laser desorption/ionization assay mass spectrophotometer analysis. A total of 385 proteins were identified consensually from the analysis as zebrafish kidney specific protein which includes 313, 55, and 87 proteins identified based on 1-DE FTMS/ITMSMS, 1-DE ESI-LCMS/MS and 2-DE MALDI MS/MS approaches respectively. The identified kidney proteome dataset was found to be representatives of diverse pI, mass, localization, process and functions. The kidney proteome dataset was found to be significantly associated with various metabolic, catabolic, cytoskeleton remodeling and rectal disease pathways. The engendered kidney protein catalog will serve as a template for understanding kidney functions and biomarker identification related to different kidney disorders.     

Efficient prefractionation of low-abundance proteins in human plasma and construction of a two-dimensional map

Human plasma is the most clinically valuable specimen, containing not only a dynamic concentration range of protein components, but also several groups of high-abundance proteins that seriously interfere with the detection of low-abundance potential biomarker proteins. To establish a high-throughput method for efficient depletion of high-abundance proteins and subsequent fractionation, prior to molecular analysis of proteins, we explored how coupled immunoaffinity columns, commercially available as multiple affinity removal columns (MARC) and free flow electrophoresis (FFE), could apply to the HUPO plasma proteome project. Here we report identification of proteins and construction of a human plasma 2-DE map devoid of six major abundance proteins (albumin, transferrin, IgG, IgA, haptoglobin, and antitrypsin) using MARC. The proteins were identified by PMF, matching with various internal 2-DE maps, resulting in a total of 144 nonredundant proteins that were identified from 398 spots. Tissue plasminogen activator, usually present at 10-60 ng/mL plasma, was also identified, indicative of a potentially low-abundance biomarker. Comparison of representative 2-D gel images of three ethnic groups (Caucasian, Asian-American, African-American) plasma exhibited minor differences in certain proteins between races and sample pretreatment. To establish a throughput fractionation of plasma samples by FFE, either MARC flow-through fractions or untreated samples of Korean serum were subjected to FFE. After separation of samples on FFE, an aliquot of each fraction was analyzed by 1-D gel, in which MARC separation was a prerequisite for FFE work. Thus, a working scheme of MARC --> FFE --> 1-D PAGE --> 2-D-nanoLC-MS/MS may be considered as a widely applicable standard platform technology for fractionation of complex samples like plasma.     

Analysis of the cytosolic proteome of Halobacterium salinarum and its implication for genome annotation

The halophilic archaeon Halobacterium salinarum (strain R1, DSM 671) contains 2784 protein-coding genes as derived from the genome sequence. The cytosolic proteome containing 2042 proteins was separated by two-dimensional gel electrophoresis (2-DE) and systematically analyzed by a semi-automatic procedure. A reference map was established taking into account the narrow isoelectric point (pI) distribution of halophilic proteins between 3.5 and 5.5. Proteins were separated on overlapping gels covering the essential areas of pI and molecular weight. Every silver-stained spot was analyzed resulting in 661 identified proteins out of about 1800 different protein spots using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) peptide mass fingerprinting (PMF). There were 94 proteins that were found in multiple spots, indicating post-translational modification. An additional 141 soluble proteins were identified on 2-D gels not corresponding to the reference map. Thus about 40% of the cytosolic proteome was identified. In addition to the 2784 protein-coding genes, the H. salinarum genome contains more than 6000 spurious open reading frames longer than 100 codons. Proteomic information permitted an improvement in genome annotation by validating and correcting gene assignments. The correlation between theoretical pI and gel position is exceedingly good and was used as a tool to improve start codon assignments. The fraction of identified chromosomal proteins was much higher than that of those encoded on the plasmids. In combination with analysis of the GC content this observation permitted an unambiguous identification of an episomal insert of 60 kbp ("AT-rich island") in the chromosome, as well as a 70 kbp region from the chromosome that has integrated into one of the megaplasmids and carries a series of essential genes. About 63% of the chromosomally encoded proteins larger than 25 kDa were identified, proving the efficacy of 2-DE MALDI-TOF MS PMF technology. The analysis of the integral membrane proteome by tandem mass spectrometric techniques added another 141 identified proteins not identified by the 2-DE approach (see following paper).     

Optimized peptide separation and identification for mass spectrometry based proteomics via free-flow electrophoresis

Multidimensional LC-MS based shotgun proteomics experiments at the peptide level have traditionally been carried out by ion exchange in the first dimension and reversed-phase liquid chromatography in the second. Recently, it has been shown that isoelectric focusing (IEF) is an interesting alternative approach to ion exchange separation of peptides in the first dimension. Here we present an improved protocol for peptide separation by continuous free-flow electrophoresis (FFE) as the first dimension in a two-dimensional peptide separation work flow. By the use of a flat pI gradient and a mannitol and urea based separation media we were able to perform high-throughput proteome analysis with improved interfacing between FFE and RPLC-MS/MS. The developed protocol was applied to a cytosolic fraction from Schneider S2 cells from Drosophila melanogaster, resulting in the identification of more than 10,000 unique peptides with high probability. To improve the accuracy of the peptide identification following FFE-IEF we incorporated the pI information as an additional parameter into a statistical model for discrimination between correct and incorrect peptide assignments to MS/MS spectra.     

Proteome analysis of Escherichia coli using high-performance liquid chromatography and Fourier transform ion cyclotron resonance mass spectrometry

The basic problem of complexity poses a significant challenge for proteomic studies. To date two-dimensional gel electrophoresis (2-DE) followed by enzymatic in-gel digestion of the peptides, and subsequent identification by mass spectrometry (MS) is the most commonly used method to analyze complex protein mixtures. However, 2-DE is a slow and labor-intensive technique, which is not able to resolve all proteins of a proteome. To overcome these limitations gel-free approaches are developed based on high performance liquid chromatography (HPLC) and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). The high resolution and excellent mass accuracy of FT-ICR MS provides a basis for simultaneous analysis of numerous compounds. In the present study, a small protein subfraction of an Escherichia coli cell lysate was prepared by size-exclusion chromatography and proteins were analyzed using C4 reversed phase (RP)-HPLC for pre-separation followed by C18 RP nanoHPLC/nanoESI FT-ICR MS for analysis of the peptide mixtures after tryptic digestion of the protein fractions. We identified 231 proteins and thus demonstrated that a combination of two RP separation steps - one on the protein and one on the peptide level - in combination with high-resolution FT-ICR MS has the potential to become a powerful method for global proteomics studies.     

Proteomic characterization of the Pseudomonas putida KT2440 global response to a monocyclic aromatic compound by iTRAQ analysis and 1DE-MudPIT

Pseudomonas putida KT2440 is a metabolically versatile soil bacterium. To examine the effects of an aromatic compound on the proteome of this bacterium, cytosolic proteins induced by the presence of benzoate and succinate were analyzed using two liquid chromatography (LC)-based proteomic approaches: an isobaric tag for relative and absolute quantitation (iTRAQ) for quantitative analysis and one-dimensional gel electrophoresis/multidimensional protein identification technology (1-DE MudPIT) for protein identification. In total, 1286 proteins were identified by 1-DE MudPIT; this represents around 23.3% of the total proteome. In contrast, 570 proteins were identified and quantified by iTRAQ analysis. Of these, 55 and 52 proteins were up- and down-regulated, respectively, in the presence of benzoate. The proteins up-regulated included benzoate degradation enzymes, chemotaxis-related proteins, and ABC transporters. Enzymes related to nitrogen metabolism and pyruvate metabolism were down-regulated. These data suggest that a combination of 1-DE MudPIT and iTRAQ is an appropriate method for comprehensive proteomic analysis of biodegradative bacteria.     

蛋白质组学研究中的对角线电泳

经典的蛋白质组学研究方法包括IEF/SDS-PAGE双向电泳和质谱技术的联用,但由于IEF的一些不足,限制了其应用范围。对角线电泳是蛋白质组学研究中的一项特殊分离技术,由于其原理与IEF/SDS-PAGE不同,正逐渐成为蛋白质组学中电泳分离技术的重要补充,特别是在膜蛋白和蛋白质相互关系的研究中将起到重要作用。本文综述了对角线双向电泳技术的特点、发展和在蛋白质组学研究中的最新进展,比较了双向电泳和对角线电泳的优缺点,展望了对角线电泳在蛋白质组学研究中的应用前景。     

Construction of a two-dimensional gel electrophoresis protein database for the Nicotiana tabacum cv. Bright Yellow-2 cell suspension culture

Using two-dimensional gel electrophoresis (2-DE) and electrospray-tandem mass spectrometry (ESI-MS/MS), we have started the proteome analysis of the cell line Nicotiana tabacum cv. Bright Yellow-2 (tobacco BY-2). The BY-2 cell suspension culture is widely used as a model system to study the growth and development of plant cells. We present a protocol describing the sample preparation and 2-DE, enabling us to separate and display more than 1000 proteins from this cell culture. A reference gel was generated, using immobilized pH gradient isoelectric focusing in a linear gradient from pH 3 to 10 and 12% Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Although the tobacco genome is not sequenced yet, a range of protein spots from this reference map was identified by means of a semi-automated liquid chromatography-ESI-quadrupole time of flight-tandem MS (LC-ESI-QTOF-MS-MS) setup and cross-species matching. These data were integrated in a database, which can be accessed at http://tby2-www.uia.ac.be/tby2/. On the on-line reference map, the identified protein spots are hyperlinked to individual protein entries. Each protein entry contains all identification information, as well as links to relevant entries in other on-line databases. Comprehensive search functions are implemented. Especially for an unsequenced but widespread model organism like tobacco BY-2, such a reference database is a convenient source for protein information that brings protein identification within reach without the need for extensive MS. This publicly accessible database provides a solid basis for tobacco BY-2 proteomics in the future.     

Qualitative and quantitative proteomics by two-dimensional gel electrophoresis, peptide mass fingerprint and a chemically-coded affinity tag (CCAT)

The chemically-coded affinity tag (CCAT) method combines standard electrophoresis protocols with MALDI-TOF-MS analysis to identify and quantify protein abundances in complex samples in one step. This method is designed to fit into the workflow of SDS-PAGE or two-dimensional electrophoresis (2-DE) only requiring basic proteome laboratory equipment. Prior to electrophoresis two protein samples are separately labelled with a heavy or a light version of the CCAT reagent via reduced cysteines in the proteins. Equal amounts are then combined and electrophoretically separated. Proteins can then be excised from the gel to obtain their peptide mass fingerprint by mass spectrometry. This fingerprint enabled not only identification, but also quantification by comparing relative peak intensities of CCAT-labelled peptides. In this article, we display how the CCAT method can be used to analyse two protein samples in one gel and that the peak intensities of labelled peptides reflect the abundance of a protein in it.     

Technical improvement to 2D-PAGE of rice organelle membrane proteins

Cytosolic and membrane-associated proteins prepared from rice cells were separated and compared by two different 2D-PAGE methods, isoelectric focusing (IEF)/SDS-PAGE and nonequilibrium pH gradient electrophoresis (NEPHGE)/SDS-PAGE. Although IEF/SDS-PAGE of the cytosolic proteins showed sufficient resolution, some mitochondrial and basic microsomal membrane-associated proteins were weakly or hardly detectable on the 2D gel. High-quality and -quantity separation of the organelle membrane-associated proteins was accomplished by NEPHGE/SDS-PAGE, the advantage of this method being more critical in tightly membrane-bound proteins that were unwashable with NaCl. These results indicate that NEPHGE/SDS-PAGE is a useful tool for the proteomic analysis of rice membrane-associated proteins.