Proteomic analysis of Acinetobacter lwoffii K24 by 2-D gel electrophoresis and electrospray ionization quadrupole-time of flight mass spectrometry

The MS/MS analysis by Electrospray ionization quadrupole-time of flight mass spectrometry (ESI-Q-TOF MS) was applied to identify proteins in proteome analysis of bacteria whose genomes are not known. The protein identification by ESI-Q-TOF MS was performed sequentially by database search and then de novo sequencing using MS/MS spectra. Soil bacteria having unanalyzed genome, Acinetobacter lwoffii K24 is an aniline degrading bacterium. In this report, we present the results of a comparison between the proteome profile of A. lwoffii K24 cultured in aniline- or succinate-containing media. Protein analysis was performed using two-dimensional gel electrophoresis (2-DE) with pH 3-10 immobilized pH gradient (IPG) strips followed by ESI-Q-TOF MS. More than 780 protein spots were detected by 2-DE from the soluble proteome. Forty-eight of these proteins were expressed exclusively in aniline cultured bacteria, and 81 proteins increased and 162 proteins decreased in aniline-cultured versus succinate cultured A. lwoffii K24. Internal amino acid sequences of 43 major protein spots were successfully determined by ESI-Q-TOF MS to try to identify the bacterial proteins responding to aniline culture condition. Since the A. lwoffii K24 genome is not yet sequenced, many proteins were found to be hypothetical. Comparative proteome analysis of the insoluble protein fractions showed that one novel protein that was strongly induced by succinate-cultured A. lwoffii K24 was repressed under aniline culture conditions. These results suggest that comprehensive analysis of bacterial proteomes by 2-DE and amino acid sequence analysis by ESI-Q-TOF MS is useful for understanding induced novel proteins of biodegrading bacteria.     

Qualitative and comparative proteomic analysis of Xanthomonas campestris pv. campestris 17

The bacterium Xanthomonas campestris pathovar campestris (XCC) 17 is a local isolate that causes crucifer black rot disease in Taiwan. In this study, its proteome was separated using 2-DE and the well-resolved proteins were excised, trypsin digested, and analyzed by MS. Over 400 protein spots were analyzed and 281 proteins were identified by searching the MS or MS/MS spectra against the proteome database of the closely related XCC ATCC 33913. Functional categorization of the identified proteins matched 141 (50%) proteins to 81 metabolic pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. In addition, we performed a comparative proteome analysis of the pathogenic strain 17 and an avirulent strain 11A to reveal the virulence-related proteins. We detected 22 up-regulated proteins in strain 17 including the degrading enzymes EngXCA, HtrA, and PepA, which had been shown to have a role in pathogenesis in other bacteria, and an anti-host defense protein, Ohr. Thus, further functional studies of these up-regulated proteins with respect to their roles in XCC pathogenicity are suggested.     

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.     

The extracellular proteome of Bifidobacterium animalis subsp. lactis BB-12 reveals proteins with putative roles in probiotic effects

Probiotics are live microorganisms that exert health-promoting effects on the human host, as demonstrated for numerous strains of the genus Bifidobacterium. To unravel the proteins involved in the interactions between the host and the extensively used and well-studied probiotic strain Bifidobacterium animalis subsp. lactis BB-12, proteins secreted by the bacterium, i.e. belonging to the extracellular proteome present in the culture medium, were identified by 2-DE coupled with MALDI-TOF MS. Among the 74 distinct proteins identified, 31 are predicted to carry out their physiological role either outside the cell or on its surface. These proteins include solute-binding proteins for oligosaccharides, amino acids and manganese, cell wall-metabolizing proteins, and 18 proteins that have been described to interact with human host epithelial cells or extracellular matrix proteins. The potential functions include binding of plasminogen, formation of fimbriae, adhesion to collagen, attachment to mucin and intestinal cells as well as induction of immunomodulative response. These findings suggest a role of the proteins in colonization of the gastrointestinal tract, adhesion to host tissues, or immunomodulation of the host immune system. The identification of proteins predicted to be involved in such interactions can pave the way towards well targeted studies of the protein-mediated contacts between bacteria and the host, with the goal to enhance the understanding of the mode of action of probiotic bacteria.     

Outer membrane vesicles from group B Neisseria meningitidis delta gna33 mutant: proteomic and immunological comparison with detergent-derived outer membrane vesicles

We compared the proteome of detergent-derived group B Neisseria meningitidis (MenB) outer membrane vesicles (DOMVs) with the proteome of outer membrane vesicles (m-OMVs) spontaneously released into culture supernatant by MenB delta gna33, a mutant in which the gene coding for a lytic transglycosylase homologous to the E. coli MltA was deleted. In total, 138 proteins were identified in DOMVs by 1- and 2-DE coupled with MS; 64% of these proteins belonged to the inner membrane and cytoplasmic compartments. By contrast, most of the 60 proteins of m-OMVs were classified by PSORT as outer membrane proteins. When tested for their capacity to elicit bactericidal antibodies, m-OMVs elicited a broad protective activity against a large panel of MenB strains. Therefore, the identification of mutations capable of conferring an OMV-releasing phenotype in bacteria may represent an attractive approach to study bacterial membrane composition and organization, and to design new efficacious vaccine formulations.     

Extensive analysis of the human platelet proteome by two-dimensional gel electrophoresis and mass spectrometry

Platelets play a key role in the control of bleeding and wound healing, contributing to the formation of vascular plugs. Under pathologic circumstances, they are involved in thrombotic disorders, including heart disease. Since platelets do not have a nucleus, proteomics offers a powerful alternative approach to provide data on protein expression in these cells, helping to address their biology. In this publication we extend the previously reported analysis of the pI 4-5 region of the human platelet proteome to the pI 5-11 region. By using narrow pI range two-dimensional electrophoresis (2-DE) for protein separation followed by high-throughput tandem mass spectrometry (MS/MS) for protein identification, we were able to identify 760 protein features, corresponding to 311 different genes, resulting in the annotation of 54% of the pI 5-11 range 2-DE proteome map. We evaluated the physicochemical properties and functions of the identified platelet proteome. Importantly, the main group of proteins identified is involved in intracellular signalling and regulation of the cytoskeleton. In addition, 11 hypothetical proteins are reported. In conclusion, this study provides a unique inventory of the platelet proteome, contributing to our understanding of platelet function and building the basis for the identification of new drug targets.     

The proteome analysis of oleaginous yeast Lipomyces starkeyi

Oleaginous yeast Lipomyces starkeyi, a species in the Saccharomycetales order, has the capability to accumulate over 70% of its cell biomass as lipid under defined culture conditions. In this study, analysis of L. starkeyi AS 2.1560 proteome samples from different culture stages during a typical lipid production process was performed using an online multidimensional μRPLC/MS/MS method. Data searching against the proteome database of the yeast Saccharomyces cerevisiae led to the identification of 289 protein hits. Further comparative and semi-quantitative analysis under more stringent criteria revealed 81 proteins with significant expression-level changes. Among them, 52 proteins were upregulated and 29 proteins were downregulated. Gene ontology annotation indicated that global responses occurred when cells were exposed to the nitrogen deficiency environment for lipid production. Protein hits were annotated and largely concerned metabolic processes for alternative nitrogen sources usage or lipid accumulation. Many of the downregulated proteins were related to glycolysis, whereas the majority of the upregulated proteins were involved in proteolysis and peptidolysis, carbohydrate metabolism and lipid metabolism. Insights were provided in terms of cellular responses to nutrient availability as well as the basic biochemistry of lipid accumulation. This work presented potentially valuable information for understanding the biochemical events related to microbial oleaginity and rational engineering of oleaginous yeasts.     

华根霉固态与液态培养产胞外蛋白的比较蛋白质组学分析

【目的】考察固态和液态两种培养方式对丝状真菌华根霉(Rhizopus chinensis)CCTCC M201021产胞外蛋白的影响,以加深对微生物固、液态培养特异性产酶的认识。【方法】利用成分相同的培养基对华根霉分别进行平板固态培养和液态培养,提取华根霉所产胞外蛋白,采用二维凝胶电泳(2-DE)结合质谱分析,分离鉴定差异蛋白。【结果】固态培养下华根霉所产胞外蛋白的蛋白酶活性是液态培养的9.2倍。胞外蛋白质组数据分析表明,华根霉固态和液态培养所产胞外蛋白存在显著差异,其中约70%是固态和液态培养下各自产生的特有蛋白。质谱鉴定进一步表明,固、液态培养对华根霉产胞外蛋白的种类和表达量都有显著影响,其中水解酶类所占比例较大,主要是与蛋白质降解相关的蛋白。【结论】固态和液态不同培养方式影响了华根霉产胞外蛋白的组成,有些基因只在特定培养方式下表达。固态培养下华根霉产胞外蛋白酶种类相对更多以及大部分蛋白酶的上调表达,可能是固态培养下胞外蛋白酶活性很高的原因。研究结果提示需要注意固液态不同培养方式下丝状真菌胞外酶的筛选和生产可能存在的不一致性。     

Mapping of alkaline proteins in bovine skeletal muscle

In agricultural sciences, proteomics has become the new hope for analyzing the meat quality traits that are closely related to the skeletal muscle traits. 2-DE muscle maps of many species have been recently reported and used to find molecular markers of meat quality traits. However, one limitation of 2-DE based analyses is due to the limited alkaline protein separation. Considering this problem, there is a need to use recent advances that have markedly improved the 2-DE based analysis of alkaline proteins. Hence, the present study provides additional information concerning the alkaline proteome of bovine skeletal muscle by using an appropriate protocol to characterize proteins over the entire range of pH 7-11. A total of 32 distinct gene products corresponding to 60 protein spots were identified by PMF and grouped in seven categories according to their main function. This 2-D map will contribute to muscle proteome studies since a significant portion of proteins is in the alkaline pH range.     

Proteomic analysis of chlorosome-depleted membranes of the green sulfur bacterium Chlorobium tepidum

Green sulfur bacteria are obligate anaerobic phototrophs, which in addition to outer and plasma membranes contain chlorosomes. The analysis of the membrane proteome of Chlorobium tepidum from chlorosome-depleted membranes is described in this study. The membranes were purified by sucrose density centrifugation and characterized by 1-DE and 2-DE coupled with MS, absorption spectroscopy, and electron microscopy. 1-DE and 2-DE were employed to analyze the membrane proteins and to characterize the capabilities of the methods. Solubilization of the membrane proteins prior to 2-DE was improved by using a series of zwitterionic detergents. Based on the resolved spots after 2-DE, the combination of amidosulfobetaine 14 with Triton X-100 is more efficient than the combination of CHAPS, N-decyl-N,N-dimethyl-3-ammonio-1-propane sulfonate, and Triton X-100. From the application of 1-DE and 2-DE, 167 and 202 unique proteins were identified, respectively, using PMF by MALDI-TOF MS. Both methods resulted in the detection of 291 different proteins of which only 88 were predicted membrane proteins, indicating the limitation of membrane protein detection after separation with electrophoresis methods. In addition, 53 of these proteins were identified as outer membrane proteins.     

The proteome of Mannheimia succiniciproducens, a capnophilic rumen bacterium

Mannheimia succiniciproducens MBEL55E isolated from bovine rumen is an industrially important bacterium as an efficient succinic acid producer. Recently, its full genome sequence was determined. In the present study, we analyzed the M. succiniciproducens proteome based on the genome information using 2-DE and MS. We established proteome reference map of M. succiniciproducens by analyzing whole cellular proteins, membrane proteins, and secreted proteins. More than 200 proteins were identified and characterized by MS/MS supported by various bioinformatic tools. The presence of proteins previously annotated as hypothetical proteins or proteins having putative functions were also confirmed. Based on the proteome reference map, cells in the different growth phases were analyzed at the proteome level. Comparative proteome profiling revealed valuable information to understand physiological changes during growth, and subsequently suggested target genes to be manipulated for the strain improvement.     

Complementary analysis of the Mycobacterium tuberculosis proteome by two-dimensional electrophoresis and isotope-coded affinity tag technology

Classical proteomics combined two-dimensional gel electrophoresis (2-DE) for the separation and quantification of proteins in a complex mixture with mass spectrometric identification of selected proteins. More recently, the combination of liquid chromatography (LC), stable isotope tagging, and tandem mass spectrometry (MS/MS) has emerged as an alternative quantitative proteomics technology. We have analyzed the proteome of Mycobacterium tuberculosis, a major human pathogen comprising about 4,000 genes, by (i) 2-DE and mass spectrometry (MS) and by (ii) the isotope-coded affinity tag (ICAT) reagent method and MS/MS. The data obtained by either technology were compared with respect to their selectivity for certain protein types and classes and with respect to the accuracy of quantification. Initial datasets of 60,000 peptide MS/MS spectra and 1,800 spots for the ICAT-LC/MS and 2-DE/MS methods, respectively, were reduced to 280 and 108 conclusively identified and quantified proteins, respectively. ICAT-LC/MS showed a clear bias for high M(r) proteins and was complemented by the 2-DE/MS method, which showed a preference for low M(r) proteins and also identified cysteine-free proteins that were transparent to the ICAT-LC/MS method. Relative quantification between two strains of the M. tuberculosis complex also revealed that the two technologies provide complementary quantitative information; whereas the ICAT-LC/MS method quantifies the sum of the protein species of one gene product, the 2-DE/MS method quantifies at the level of resolved protein species, including post-translationally modified and processed polypeptides. Our data indicate that different proteomic technologies applied to the same sample provide complementary types of information that contribute to a more complete understanding of the biological system studied.     

Analysis of the Pasteurella multocida outer membrane sub-proteome and its response to the in vivo environment of the natural host

This study describes the identification of outer membrane proteins (OMPs) of the bacterial pathogen Pasteurella multocida and an analysis of how the expression of these proteins changes during infection of the natural host. We analysed the sarcosine-insoluble membrane fractions, which are highly enriched for OMPs, from bacteria grown under a range of conditions. Initially, the OMP-containing fractions were resolved by 2-DE and the proteins identified by MALDI-TOF MS. In addition, the OMP-containing fractions were separated by 1-D SDS-PAGE and protein identifications were made using nano LC MS/MS. Using these two methods a total of 35 proteins was identified from samples obtained from organisms grown in rich culture medium. Six of the proteins were identified only by 2-DE MALDI-TOF MS, whilst 17 proteins were identified only by 1-D LC MS/MS. We then analysed the OMPs from P. multocida which had been isolated from the bloodstream of infected chickens (a natural host) or grown in iron-depleted medium. Three proteins were found to be significantly up-regulated during growth in vivo and one of these (Pm0803) was also up-regulated during growth in iron-depleted medium. After bioinformatic analysis of the protein matches, it was predicted that over one third of the combined OMPs predicted by the bioinformatics sub-cellular localisation tools PSORTB and Proteome Analyst, had been identified during this study. This is the first comprehensive proteomic analysis of the P. multocida outer membrane and the first proteomic analysis of how a bacterial pathogen modifies its outer membrane proteome during infection.     

双向电泳法在家蚕蛋白质分离中的应用

家蚕Bombyxmori(L.)既是重要的经济昆虫,又是鳞翅目昆虫研究的典型模式生物。开展家蚕蛋白质组研究,将有助于阐明家蚕绢丝蛋白的分泌机理,也是研究鳞翅目昆虫及其他生物生命本质的需要。双向电泳是蛋白质分离的关键技术。为探讨适宜家蚕蛋白质组研究的双向电泳条件,以家蚕丝腺、丝腺内容物、蚕卵和血液为材料,在不同条件下进行双向电泳,并对分离的蛋白点进行质谱分析。结果表明:通过改进的蛋白质裂解液辅以超声破碎制备的蛋白质,双向电泳后能够得到较好的2-DE图,也能满足进行MALDI-TOFMS分析的需要。因此本研究方法适用于家蚕不同组织中蛋白质的提取和双向电泳。     

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.     

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.     

Proteomic analysis of membrane proteins from Streptococcus pneumoniae with multiple separation methods plus high accuracy mass spectrometry

Abstract Streptococcus pneumoniae is a Gram-positive human pathogen that causes a variety of serious mucosal and invasive diseases in human. Bacterial membrane proteins play crucial roles in host-pathogen interactions and bacterial pathogenesis, and thus are potential drug targets or vaccine candidates. In this study, membranes from Streptococcus pneumoniae D39 were enriched by mechanical grinding and ultracentrifugation, and then the membrane proteins were extracted with trifluroethanol and chloroform. Around 60% of the extracted proteins were identified to be membrane proteins with 2-DE coupled with MALDI-MS/MS and 2D-LC-ESI-MS/MS. These identified membrane proteins can be functionally categorized into various groups involved in nutriment transport, signal transduction, protein folding or secretion, oxidation, carbohydrate metabolism, and other physiological processes. A protein interaction network was constructed for understanding the regulation relationship of the membrane proteins. This study represents the first global characterization of membrane proteome from Gram-positive streptococcus species of bacteria, providing valuable clues for further investigation aiming at identifying drug/vaccine targets for the bacterial infection.     

Benefits of heat treatment to the protease packed neutrophil for proteome analysis: halting protein degradation

Neutrophils, cells of the innate immune system, contain an array of proteases and reactive oxygen species-generating enzymes that assist in controlling the invasion of bacteria and pathogens. The high content of intracellular proteolytic enzymes makes them difficult cells to work with as they can degrade proteins of potential interest. Here, we describe the benefits of heat treatment of neutrophils in reducing protein degradation for subsequent proteome analysis. Neutrophils isolated from four healthy volunteers were each divided into three aliquots and subjected to different preparation methods for 2-DE: (i) Heat treatment, (ii) resuspension in NP40 lysis buffer and (iii) resuspension in standard 2-DE lysis buffer. Representative spots found to be statistically significant between groups (p<0.01) were excised and identified by LC-MS/MS, three of which were validated by immunoblotting. Heat-treated samples contained proteins in the high-molecular-weight range that were absent from NP40-treated samples. Moreover, NP40-treated samples showed an increase in spot number and volume at lower molecular weights suggestive of protein degradation. Incorporating heat treatment into sample preparation resulted in the identification of proteins that may not have previously been detected due to sample degradation, thus leading to a more comprehensive 2-DE map of the human neutrophil proteome.