A comprehensive two-dimensional map of cytosolic proteins of Bacillus subtilis

Proteomics relying on two-dimensional (2-D) gel electrophoresis of proteins followed by spot identification with mass spectrometry is an excellent experimental tool for physiological studies opening a new perspective for understanding overall cell physiology. This is the intriguing outcome of a method introduced by Klose and O'Farrell independently 25 years ago. Physiological proteomics requires a 2-D reference map on which most of the main proteins were identified. In this paper, we present such a reference map with more than 300 entries for Bacillus subtilis proteins with an isoelectric point (pI) between 4 and 7. The most abundant proteins of exponentially growing cells were compiled and shown to perform mainly housekeeping functions in glycolysis, tricarboxylic acid cycle (TCC), amino acid biosynthesis and translation as well as protein quality control. Furthermore, putative post-translational modifications were shown at a large scale, with 47 proteins in total forming more than one spot. In a few selected cases evidence for phosphorylation of these proteins is presented. The proteome analysis in the standard pI range was complemented by either stretching the most crowded regions in a narrow pH gradient 4.5-5.5, or by adding other fractions of the total B. subtilis proteome such as alkaline proteins as well as extracellular proteins. A big challenge for future studies is to provide an experimental protocol covering the fraction of intrinsic membrane proteins that almost totally escaped detection by the experimental procedure used in this study.     

液相等电聚焦结合双向凝胶电泳分离碱性蛋白

在蛋白组学研究中, 经典的双向凝胶电泳法(2-DE)对碱性蛋白及低丰度蛋白的分离存在技术障碍, 但预分离技术的应用可弥补其缺陷. 液相等电聚焦可有效地分离富集复杂蛋白样品. 碱性胶条用于2-DE可极大地提高蛋白上样量和凝胶分辨率. 将上述两种技术相结合用于碱性蛋白质和低丰度蛋白质的分离鉴定, 可使碱端区域双向凝胶图谱质量显著提高, 蛋白点更清晰且点数增多, 质谱鉴定确信度提高, 碱性蛋白和低丰度蛋白质谱鉴定成功率提高, 对于蛋白组学研究具有一定的意义.     

Lung alveolar proteomics of bronchoalveolar lavage from a pulmonary alveolar proteinosis patient using high-resolution FTICR mass spectrometry

High-resolution Fourier transform ion cyclotron resonance (FTICR) mass spectrometry was developed and applied to the proteome analysis of bronchoalveolar lavage fluid (BALF) from a patient with pulmonary alveolar proteinosis. With use of 1-D and 2-D gel electrophoresis, surfactant protein A (SP-A) and other surfactant-related lung alveolar proteins were efficiently separated and identified by matrix-assisted laser desorption/ionization FTICR mass spectrometry . Low molecular mass BALF proteins were separated using a gradient 2-D gel. An efficient extraction/precipitation system was developed and used for the enrichment of surfactant proteins. The result of the BALF proteome analysis show the presence of several isoforms of SP-A, in which an N-non-glycosylierte form and several proline hydroxylations were identified. Furthermore, a number of protein spots were found to contain a mixture of proteins unresolved by 2-D gel electrophoresis, illustrating the feasibility of high-resolution mass spectrometry to provide identifications of proteins that remain unseparated in 2-D gels even upon extended pH gradients. Yu Bai and Dmitry Galetskiy both contributed equally to this work.     

Zooming-in on the proteome: very narrow-range immobilised pH gradients reveal more protein species and isoforms

Two-dimensional gel electrophoresis (2-DE) enables separation of complex mixtures of proteins on a single polyacrylamide gel according to isoelectric point, molecular weight, solubility, and relative abundance. For this reason, 2-DE together with mass spectrometry (MS) has become a key technology in proteome analysis. The introduction of immobilised pH gradients (IPGs) for isoelectric focusing of proteins affords improved reproducibility and permits full-scale proteome analyses to be undertaken. Whilst broad-range IPGs are useful for investigating simple proteomes (e.g. Mycoplasma genitalium) it is becoming clear that additional resolving power is needed for separating the more complex proteomes of eukaryotic organisms. The use of narrow-range and very narrow-range IPGs provides the means with which to dissect a complex proteome. We have compared very narrow-range IPGs (3.5-4.5L, 4-5L, 4.5-5.5L, 5-6L, and 5.5-6.7L) with broad- (3-10NL) and narrow-range IPGs (4-7L and 6-9L) for the visualisation of the human heart proteome. The superior ability of very narrow-range IPGs to separate different protein species and isoforms, compared with 3-10NL and 4-7L 2-D gels is demonstrated. The results are supported by MS identifications which further show that reduction of the number of comigrating protein species results in less ambiguous and more reliable database search results.     

Spot volume vs. amount of protein loaded onto a gel: a detailed,statistical comparison of two gel electrophoresis systems

The long-term goal of this research program is to clarify the molecular mechanisms that participate in the formation of human pituitary macroadenomas. One approach to that goal is to characterize the differentially expressed proteins that are found by a comparison of the proteomes of control pituitary vs. macroadenoma tissues. In order to accurately perform a comparative proteomics study, based on the combination of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and PDQuest 2-D analysis software, a reproducible 2-DE separation system with a wide linear dynamic measure range is needed. A typical horizontal system is the Multiphor II system that analyzes one gel at a time, using a precast gradient gel (180 x 245 x 0.5 mm); a typical vertical system is the Dodeca system that analyzes up to 12 gels at a time on a single-concentration gel (190 x 205 x 1.0 mm). We have evaluated (Zhan and Desiderio, Electrophoresis 2003, 24, 1834-1846) the spatial and quantitative reproducibility of the two second-dimensional gel systems to separate a human pituitary proteome; that study showed a higher reproducibility for the Dodeca gel system. This present study investigated the relationship between the spot volume and the amount of protein loaded onto the gel for those two 2-D systems. The results demonstrated that the Dodeca gel system provides a wider linear dynamic range to measure the changes in the protein abundance in pituitary proteome.     

Two-dimensional electrophoresis and peptide mass fingerprinting of bacterial outer membrane proteins.

Many bacterial outer membrane proteins (OMPs) are missing from two-dimensional (2-D) gel proteome maps. Recently, we developed a technique for 2-D electrophoresis (2-DE) of Escherichia coli OMPs using alkaline pH incubation for isolation of OMPs, followed by improved solubilization conditions for array by 2-DE using immobilized pH gradients. In this report, we expanded our study, examining protein components from the outer membranes of two enteric bacteria, Salmonella typhimurium and Klebsiella pneumoniae (also known as Klebsiella aerogenes), as well as the unrelated, free-living alpha-proteobacteria Caulobacter crescentus. Patterns of OMPs expression appeared remarkably conserved between members of the Enterobacteriaceae, while C. crescentus was unique, displaying a greater number of clusters of higher-molecular-weight proteins (>80 kDa). Peptide mass fingerprinting (PMF) was used for protein identification, and despite matching across-species boundaries, proved useful for first-pass protein assignment of enteric OMPs. In contrast, identification of C. crescentus OMPs was successful only when searching against its recently completed genome. For all three microorganisms examined, the majority of proteins identified on the 2-D gel appear localized to the outer membrane, a result consistent with our previous finding in Escherichia coli. In addition, we discuss some of the benefits and limitations of PMF in cross-species searching.     

A two-dimensional gel protein database of noncultured total normal human epidermal keratinocytes: identification of proteins strongly up-regulated in psoriatic epidermis

A two-dimensional (2-D) gel database of proteins from noncultured total normal human epidermal keratinocytes has been established. A total of 1449 [35S]methionine labelled proteins (1112 isoelectric focusing, 337 nonequilibrium pH gradient electrophoresis) were resolved and recorded using computer assisted (PDQ-SCAN and PDQUEST software) 2-D gel electrophoresis. By matching the protein patterns of total keratinocytes and transformed human amnion cells (master database; Celis et al., Leukemia 1988, 2, 561-602) as well as by 2-D immunoblotting and microsequencing of keratinocyte proteins, it was possible to identify 72 polypeptides in the keratinocyte database. The database also includes data on polypeptides that are synthesized at a higher level by keratinocytes enriched in basal cells, and on six secreted proteins which are produced, albeit at a reduced rate, by normal keratinocytes and that are strongly up-regulated in psoriatic epidermis (Celis et al., FEBS Letters, in press).     

A turning point in proteome analysis: sample prefractionation via multicompartment electrolyzers with isoelectric membranes

Sample prefractionation, as obtained via multicompartment electrolyzers with isoelectric membranes, greatly enhanced the load ability, resolution and detection sensitivity of two-dimensional (2-D) maps in proteome analysis. This was demonstrated with different samples. In an Escherichia coli total cell extract, analysis by a 2-D map run in a pH 4-5 gradient showed many more spots when prefractionated, as compared with standard maps available in databases such as SWISS-2DPAGE. Analysis of human plasma in the pH 3-6 range showed an increase in the number of highly acidic proteins in the fractionated sample compared to whole plasma. With both samples no protein precipitation or smears occurred and much larger sample amounts could be loaded upon prefractionation, so that a large number of spots could be visualized by Coomassie staining, which is fully compatible with subsequent matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis.     

Towards a two-dimensional proteome map of Mycoplasma pneumoniae

A Proteome map of the bacterium Mycoplasma pneumoniae was constructed using two-dimensional (2-D) gel electrophoresis in combination with mass spectrometry (MS). M. pneumoniae is a human pathogen with a known genome sequence of 816 kbp coding for only 688 open reading frames, and is therefore an ideal model system to explore the scope and limits of the current technology. The soluble protein content of this bacterium grown under standard laboratory conditions was separated by 1-D or 2-D gel electrophoresis applying various pH gradients, different acrylamide concentrations and buffer systems. Proteins were identified using liquid chromatography-electrospray ionization ion trap and matrix-assisted laser desorption/ionization-MS. Mass spectrometric protein identification was supported and controlled using N-terminal sequencing and immunological methods. So far, proteins from about 350 spots were characterized with MS by determining the molecular weights and partial sequences of their tryptic peptides. Comparing these experimental data with the DNA sequence-derived predictions it was possible to assign these 350 proteins to 224 genes. The importance of proteomics for genome analysis was shown by the identification of four proteins, not annotated in the original publication. Although the proteome map is still incomplete, it is already a useful reference for comparative analyses of M. pneumoniae cells grown under modified conditions.     

Reduction and alkylation of proteins in preparation of two-dimensional map analysis: why, when, and how?

The standard procedure adopted up to the present in proteome analysis calls for just reduction prior to the isoelectric focusing/immobilized pH gradient (IEF/IPG) step, followed by a second reduction/alkylation step in between the first and second dimension, in preparation for the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) step. This protocol is far from being optimal. It is here demonstrated, by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry, that failure to reduce and alkylate proteins prior to any electrophoretic step (including the first dimension) results in a large number of spurious spots in the alkaline pH region, due to "scrambled" disulfide bridges among like and unlike chains. This series of artefactual spots comprises not only dimers, but an impressive series of oligomers (up to nonamers) in the case of simple polypeptides such as the human alpha- and beta-globin chains, which possess only one (alpha-) or two (beta-) -SH groups. As a result, misplaced spots are to be found in the resulting two-dimensional (2-D) map, if performed with the wrong protocol. The number of such artefactual spots can be impressively large. In the case of analysis of complex samples, such as human plasma, it is additionally shown that failure to alkylate proteins results in a substantial loss of spots in the alkaline gel region, possibly due to the fact that these proteins, at their pI, regenerate their disulfide bridges with concomitant formation of macroaggregates which become entangled with and trapped within the polyacrylamide gel fibers. This strongly quenches their transfer in the subsequent SDS-PAGE step.     

Using 2-D electrophoresis followed by nano HPLC in nuclear protein analysis of MCF-7 breast cancer cell line by MALDI-TOF/TOF

The biomarker identification is an important tool in early cancer detection. The MCF-7 breast cancer cell line was chosen as a model system. The nuclear proteins were extracted utilizing a commercially manufactured kit and separated on two dimensional (2-D) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). The first dimension was performed on isoelectric focusing strips with pH range 4–7. Afterwards the proteins were tryptic digested and identification was performed by matrix assisted laser desorption technique with time of flight mass analyzer (MALDI-TOF/TOF). For unambiguous identification proteins with too low concentration or spots contains protein mixture the nano high performance liquid chromatography (HPLC) was used. The 2-D gel electrophoresis (2-DGE) seems to be a good tool to separate large amount of proteins using relatively simple procedure and its hyphenation with HPLC can create the perfect analytical solution for proteome identification. About 150 nuclei protein spots were visualized and the most abundant of them were identified.      

A two-dimensional proteome map of Shigella flexneri

Shigella flexneri is a Gram-negative facultatively intracellular pathogen responsible for bacillary dysentery in humans. In this study, extracellular proteins from the culture medium and whole cell proteins in cellular extracts of S. flexneri 2a strain 2457T were examined by two-dimensional (2-D) gel electrophoresis using immobilized pH gradient (IPG) technology. Proteins were identified by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) in combination with Mascot search program. In total, among the 488 proteins spots processed, 388 proteins were identified. The identified proteins represented 169 genes. By comparing results of Mascot search against databases of Escherichia coli and genomes of S. flexneri 2a, one S. flexneri-specific protein was identified and one possible gap was found in 2457T genome sequences. Although this proteome map is still incomplete, it is already a useful reference for future studies involving pathogenicity, vaccine development, design of novel antibacterial drugs, etc. Proteome maps and a table of all identified proteins are available on the internet at www.proteomics.com.cn.     

Comprehensive proteome analysis of mouse liver by ampholyte-free liquid-phase isoelectric focusing

In this study, ampholyte-free liquid-phase IEF (LIEF) was combined with narrow pH range 2-DE and SDS-PAGE RP-HPLC for comprehensive analysis of mouse liver proteome. Because LIEF prefractionation was able to reduce the complexity of the sample and enhance the loading capacity of IEF strips, the number of visible protein spots on subsequent 2-DE gels was significantly increased. A total of 6271 protein spots were detected after integrating five narrow pH range 2-DE gels following LIEF prefractionation into a single virtual 2-DE gel. Furthermore, the pH 3-5 LIEF fraction and the unfractionated sample were separated by pH 3-6 2-DE and identified by MALDI-TOF/TOF MS, respectively. In parallel, the pH 3-5 LIEF fraction was also analyzed by SDS-PAGE RP-HPLC MS/MS. LIEF-2-DE and LIEF-HPLC could obviously improve the separation efficiency and the confidence of protein identification, which identified a higher number of low-abundance proteins and proteins with extreme physicochemical characteristics or post-translational modifications compared to conventional 2-DE method. Furthermore, there were 207 proteins newly identified in mouse liver in comparison with previously reported large-scale datasets. It was observed that the combination of LIEF-2-DE and LIEF-HPLC was effective in promoting MS-based liver proteome profiling and could be applied on similar complex tissue samples.     

Towards high performance two-dimensional gel electrophoresis using ultrazoom gels

Proteomics is the analysis of protein expression in cells or tissues, e.g., to study cellular processes at the molecular level. Ultimately, a proteome analysis should encompass most if not all protein species in a biological sample, including those present in low copy numbers. We are developing two-dimensional gel electrophoresis technology by applying narrow pH range ultrazoom gels to enhance resolution and to improve the detection of low abundance proteins. Ultrazoom gels in the acidic pH range allow the detection of proteins down to 300 copies per cell of a B-lymphoma cell line. Protein separation in the alkaline pH range, however, still requires optimization, especially in conjunction with high sample loads.     

Sample complexity reduction for two-dimensional electrophoresis using solution isoelectric focusing prefractionation

Despite its excellent resolving power, 2-DE is of limited use when analyzing cellular proteomes, especially in differential expression studies. Frequently, fewer than 2000 protein spots are detected on a single 2-D gel (a fraction of the total proteome) regardless of the gel platform, sample, or detection method used. This is due to the vast number of proteins expressed and their equally vast dynamic range. To exploit 2-DE unique ability as both an analytical and a preparative tool, the significant sample prefractionation is necessary. We have used solution isoelectric focusing (sIEF) via the ZOOM IEF Fractionator (Invitrogen) to generate sample fractions from complex bacterial lysates, followed by parallel 2-DE, using narrow-range IPG strips that bracket the sIEF fractions. The net result of this process is a significant enrichment of the bacterial proteome resolved on multiple 2-D gels. After prefractionation, we detected 5525 spots, an approximate 3.5-fold increase over the 1577 spots detected in an unfractionated gel. We concluded that sIEF is an effective means of prefractionation to increase depth of field and improve the analysis of low-abundance proteins.     

New ligand, N-(2-pyridylmethyl)aminoacetate, for use in the immobilised metal ion affinity chromatographic separation of proteins.

A new chelating compound has been developed for use in the immobilised metal ion affinity chromatographic separation of proteins. The tridentate ligand, sodium N-(2-pyridylmethyl)aminoacetate (carbpyr), 1, was prepared via a one-step synthesis from 2-picolylamine, 3 and then immobilised onto Sepharose CL-4B through the epoxide coupling procedure. The binding behaviour of the resulting IMAC sorbent, following chelation with Cu2+ ions to a density of 152 micromol Cu2+ ions/g gel was characterised by frontal analysis experiments using horse heart myoglobin (HMYO) at pH 7.0 and pH 9.0. From the derived isotherms, the adsorption capacity, q(m), for the binding of HMYO to immobilised Cu2+-N-(2-pyridylmethyl)aminoacetate (im-Cu2+-carbpyr)-Sepharose CL-4B at these pH values was found to be 1.92 and 1.91 micromol/g sorbent, respectively, whilst the dissociation constants K(D) were 0.0092 x 10(-6) M and 0.0062 x 10(-6) M at pH 7.0 and pH 9.0, respectively, indicating that the HMYO-im-Cu2+-N-(2-pyridylmethyl)aminoacetate complex was more stable under alkaline conditions, although the binding capacity in terms of micromol protein/g gel remained essentially unchanged. The selectivity features of the im-Cu2+-carbpyr-Sepharose CL-4B sorbent were further characterised in terms of the binding properties with several human serum proteins at pH 5.0, pH 7.0 and pH 9.0.     

Proteome analysis of human stomach tissue: separation of soluble proteins by two-dimensional polyacrylamide gel electrophoresis and identification by mass spectrometry

Two-dimensional gel electrophoresis (2-DE) maps for human stomach tissue proteins have been prepared by displaying the protein components of the tissue by 2-DE and identifying them using mass spectrometry. This will enable us to present an overview of the proteins expressed in human stomach tissues and lays the basis for subsequent comparative proteome analysis studies with gastric diseases such as gastric cancer. In this study, 2-DE maps of soluble fraction proteins were prepared on two gel images with partially overlapping pH ranges of 4-7 and 6-9. On the gels covering pH 4-7 and pH 6-9, about 900 and 600 protein spots were detected by silver staining, respectively. For protein identification, proteins spots on micropreparative gels stained with colloidal Coomassie Brilliant Blue G-250 were excised, digested in-gel with trypsin, and analyzed by peptide mass fingerprinting with delayed extraction-matrix assisted laser desorption/ionization-mass spectrometry (DE-MALDI-MS). In all, 243 protein spots (168 spots in acidic map and 75 spots in basic map) corresponding to 136 different proteins were identified. Besides these principal maps, overview maps (displayed on pH 3-10 gels) for total homogenate and soluble fraction, are also presented with some identifications mapped on them. Based on the 2-DE maps presented in this study, a 2-DE database for human stomach tissue proteome has been constructed and is available at http://proteome.gsnu.ac.kr/DB/2DPAGE/Stomach/. The 2-DE maps and the database resulting from this study will serve important resources for subsequent proteomic studies for analyzing the normal protein variability in healthy tissues and specific protein variations in diseased tissues.     

富硒螺旋藻光谱特性及其含硒蛋白质组分析

以富硒螺旋藻( Selenium enriched spirulina platensis, Se-SP)为材料,采用红外、紫外-可见吸收光谱分析Se-SP的光谱特征,并利用二维电泳技术(Two-dimensional electrophoresis,2DE)分离、飞行时间质谱(Matrix assisted laser desorption ionization time of flight, MALDI-TOF)鉴定和电感耦合等离子体质谱( Inductively coupled plasma mass spectrometry, ICP-MS)检测硒含量,对Se-SP蛋白质组及其含硒蛋白质分布进行初步鉴定和分析。结果显示,硒对螺旋藻蛋白质组中多种蛋白质表达有一定影响,观察到对捕光色素蛋白如藻蓝蛋白( Phycocya-nin, PC)、别藻蓝蛋白( Allophycocyanin , APC)和 Robisco大亚基及超氧化物歧化酶( Superoxide Dismutase, SOD)等蛋白质具有明显上调作用。硒在Se-SP蛋白质组中存在较为普遍,但分布相对集中在pH 4~7、分子质量14.4~43 kDa的蛋白质中。硒在藻体特定蛋白质中的存在形式还有待深入研究和鉴定。     

Microscale solution IEF combined with 2-D DIGE substantially enhances analysis depth of complex proteomes such as mammalian cell and tissue extracts

Current gel-based protein profiling methods such as 2-DE and fluorescent 2-D difference in gel electrophoresis (DIGE) evaluate small portions of complex proteomes. Hence, sample prefractionation is essential for more comprehensive proteome coverage and detection of low-abundant proteins. In this study, we describe the combination of DIGE labeling with microscale solution IEF (MicroSol-IEF) fractionation and subsequent analysis on slightly overlapping narrow pH range 2-D gels. By fluorescently tagging and mixing samples and controls prior to prefractionation, complications resulting from minor run-to-run variations during MicroSol-IEF separations of multiple samples are avoided. This greatly improves the reliability of quantitative comparisons. To illustrate its utility, this 3-D DIGE strategy was applied to analysis of human melanoma cells and mouse lung tissue extracts. Approximately 1000 reproducible spots can be obtained from narrow range 2-D gels of individual MicroSol-IEF fractions, and approximately 6000 spots can be obtained from entire proteomes. Quantitative changes in closely related samples could be more reliably detected and the method has a greatly increased capacity to distinguish between closely related protein isoforms. Thus the 3-D DIGE strategy produces a powerful method for more comprehensive and more reliable quantitative comparisons of protein profiles of very complex proteomes.     

Virtual two-dimensional gel electrophoresis of high-density lipoproteins

High-density lipoproteins (HDLs) isolated by immunoaffinity chromatography and separated by immobilized pH gradient-isoelectric focusing (IPG-IEF) were examined by mass spectrometry directly, applying a new proteomics technology, virtual two-dimensional (2-D) gel electrophoresis. A preliminary examination of HDL particles has revealed at least 42 unique masses for protein species with isoelectric points between pH 5.47-5.04, some of which have not been observed previously. By delivering masses of intact proteins from complex cellular mixtures in a format that correlates directly to classical 2-D gel analyses, virtual 2-D gel electrophoresis constitutes a general discovery tool to expose and monitor protein isoforms and post-translational modifications. Furthermore, its general ability to deliver ions from sub-picomole level proteins enmeshed in complex cellular mixtures potentially fulfills the need of top-down proteomics to obtain intact protein ions from microscale samples. Additional comparison of such data to 2-D gel analyses and their identified proteins may elucidate the functions of the individual apolipoprotein components and the cardioprotective effects of HDL.