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Two-dimensional Electrophoresis (two-dimensional + electrophoresis)
Selected AbstractsIdentification of RSVP14 and RSVP20 Components by Two-dimensional Electrophoresis and Western-blottingREPRODUCTION IN DOMESTIC ANIMALS, Issue 1 2008JA Cardozo Contents We have already shown that RSVP14 and RSVP20, two ram seminal plasma (SP) proteins postulated to be involved in sperm capacitation and gamete interaction can protect spermatozoa against cold-shock. In this study, we use two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) for the analysis of SP proteins of Rasa Aragonesa rams, using enhanced protein solubilization in the presence of tributyl phosphine (TBP) and a polyacrylamide linear gradient gel with a narrow pH range (4,7). The image analysis of the 2D map detected 195 protein spots, with isoelectric points (pIs) ranging from 4.5 to 6.6, and molecular weight (Mr) from 11.7 to 90.4. Staining of 2D gels with Pro-Q Emerald 300 Glycoprotein Stain revealed that most significant proteins in ram SP are glycosylated. The removing of protein N-linked oligosaccharides improved the gel resolution. 2D-PAGE analysis of the whole fraction 6 (F6) separated from ram SP by exclusion chromatography showed six main protein spots, four (a, b, c, d) in the 14 kDa and two (e, f) in the 20 kDa region. Western-blot analyses indicated that the anti-P14 antibody recognized four spots on the SP map, 4, 5, 6 and 7, that matched with spots a, b, c, d of F6 map. The anti-P20 antibody recognized spots 13 and 14 of SP map that corresponded to spots e, f of F6 map. The deduced sequences by de novo sequencing evidenced that protein spots 7 and 13 have significant similarities to BSP family, while protein spots 4 and 14 did not appear to be homologous with any reported protein in the current mammalian Proteinbank databases. [source] Two-dimensional electrophoresis with cationic detergents, a powerful tool for the proteomic analysis of myelin proteins.JOURNAL OF NEUROSCIENCE RESEARCH, Issue 4 2008Part 1: Technical aspects of electrophoresis Abstract The analysis of proteins in damaged myelin is crucial to clarify the mechanisms of dysmyelination and demyelination. In the present study, proteomic analysis of myelin using a modified two-dimensional electrophoresis (2-DE) method was carried out to obtain a better understanding of myelin biology. Although standard 2-DE (immobilized pH gradient isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis; IPG/SDS-PAGE) methods of analysis provide high resolutions of soluble proteins with isoelectric focusing points in the pH range of 4,8, major myelin components include highly basic proteins are compacted at the basic edge of the 2-DE gels and are not sufficiently separated for satisfactory analysis. In an attempt to improve the separation of these proteins, an alternative 2-DE method using the cationic detergents was applied. In part 1 of this study, we describe technical aspects of conditioning 2-DE using cationic detergent. In the accompanying paper (part 2), practical 2-DE analysis using cationic detergents is described to identify proteins in the purified CNS myelin fraction. We carried out benzyldimethyl- n -hexadecylammonium chloride (16-BAC)/SDS-PAGE 2-DE and tested 2-DE with four other cationic detergents. We found that 16-BAC was the most effective agent for separation of myelin proteins and that hexadecyltrimethylammonium bromide (cetyltrimethylammonium bromide; CTAB) was the most effective agent for solubilization of myelin proteins. The combination of 16-BAC/SDS-PAGE and CTAB/SDS-PAGE is a powerful tool for the analysis of myelin proteins, including highly basic, high-MW (MW > 100K), and integral membrane proteins. © 2007 Wiley-Liss, Inc. [source] Differential display proteomic analysis of Picea meyeri pollen germination and pollen-tube growth after inhibition of actin polymerization by latrunculin BTHE PLANT JOURNAL, Issue 2 2006Yanmei Chen Summary To investigate roles of the actin cytoskeleton in growth of the pollen tube of Picea meyeri, we used the actin polymerization inhibitor latrunculin B (LATB) under quantitatively controlled conditions. At low concentrations, LATB inhibited polymerization of the actin cytoskeleton in the growing pollen tube, which rapidly inhibited tip growth. The proteomic approach was used to analyse protein expression-profile changes during pollen germination and subsequent pollen-tube development with disturbed organization of the actin cytoskeleton. Two-dimensional electrophoresis and staining with Coomassie Brilliant Blue revealed nearly 600 protein spots. A total of 84 of these were differentially displayed at different hours with varying doses of LATB, and 53 upregulated or downregulated proteins were identified by mass spectrometry. These proteins were grouped into distinct functional categories including signalling, actin cytoskeleton organization, cell expansion and carbohydrate metabolism. Moreover, actin disruption affected the morphology of Golgi stacks, mitochondria and amyloplasts, along with a differential expression of proteins involved in their functions. These findings provide new insights into the multifaceted mechanism of actin cytoskeleton functions and its interaction with signalling, cell-expansion machinery and energy-providing pathways. [source] Characterization of the major allergens of Pachycondyla chinensis in ant sting anaphylaxis patientsCLINICAL & EXPERIMENTAL ALLERGY, Issue 4 2009E. K. Lee Summary Background The ant species Pachycondyla chinensis, which has spread from Far Eastern Asia to New Zealand and North America, induces anaphylactic reactions in human with its sting. However, the major allergens of P. chinensis have not yet been characterized. Methods We selected seven patients with histories of anaphylaxis induced by P. chinensis. Two-dimensional electrophoresis (2-DE) was used to identify the major allergens. We subsequently performed Western blots for P. chinensis -specific IgEs, N-terminal amino acid sequencing, ESI-MS/MS, and RT-PCR using primers based on the N-terminal sequence. Results Six of the anaphylactic subjects had an IgE specific to a 23 kDa allergen of P. chinensis. Two candidates for major allergens, 23 kDa (pI 8.7) and 25 kDa (pI 6.2), were revealed by 2-DE using P. chinensis -specific IgE immunoblotting. In N-terminal sequencing and ESI-MS/MS analysis, 23 kDa (pI 8.7) and 25 kDa (pI 6.2) allergens, belonging to the protein families of antigen 5, were identified and share marked amino acid sequence similarity. The 23 kDa allergen is 206 amino acids in length and homology searches showed 54.0% and 50.0% homology with Sol i 3 and Ves v 5, respectively. Conclusion The major allergens of P. chinensis are 23 kDa (pI 8.7) and 25 kDa (pI 6.2) proteins that belong to the antigen 5 family of proteins. [source] Effect of sialic acid content on glycoprotein pI analyzed by two-dimensional electrophoresisELECTROPHORESIS, Issue 17 2010Sílvia Barrabés Abstract 2-DE is broadly used for quantitative analysis of differential protein expression in complex mixtures such as serum samples or cell lysates. PTMs directly influence the 2-DE pattern, and knowledge of the rules of protein separation is required in order to understand the protein distribution in a 2-DE gel. Glycosylation is the most common PTM and can modify both the molecular weight and the pI of a protein. In particular, the effect of charged monosaccharides (mainly sialic acids, SAs) on the 2-DE pattern of a protein is of major interest since changes in sialylation are regularly observed in comparative studies. Little is known about the pI shift of a glycoprotein induced by the presence of SAs, or whether this shift is the same for all glycoproteins. To address this issue, this study examined the influence of SA on the 2-DE pattern of three serum glycoproteins (haptoglobin, ,1-antitrypsin and ribonuclease 1), which N -glycan chains had been previously characterised, and reviewed existing bibliographic data. The SA content of the different glycoforms of a glycoprotein showed a negative linear correlation with the pI, although the slope varied among the studied glycoproteins. We also described a positive correlation between the protein pI and the pI decrease per SA molecule. [source] Cover Picture: Electrophoresis 13'2010ELECTROPHORESIS, Issue 13 2010Article first published online: 30 JUN 2010 Issue no. 13 is a special issue on "Food and Beverage Analysis" comprising 23 contributions distributed over four distinct parts. Part I "presents six review papers, five of them are focused on the use of capillary electromigration techniques for detecting enantiomers, nucleosides, nucleotides, pesticides and contaminants in foods as well as foodborne pathogens". The sixth review deals with the application of two-dimensional electrophoresis to investigate food allergens. A series of original contributions on the use of CE, CEC and MEKC in the analysis of food constituents are grouped in Part II. Part III presents "Works on the detection of genetically modified foods by CGE and the analysis of antibiotics and antioxidants by microemulsion electrokinetic chromatography". Finally, Part IV is on the use of CE-MS to analyze endocrine disruptors and polyphenols in different food matrices, and on the speciation analysis of arsenic and selenium compounds in different foodstuffs by capillary electrophoresis-inductively coupled plasma-mass spectrometry. [source] Cover Picture: Electrophoresis 2'09ELECTROPHORESIS, Issue 2 2009Article first published online: 9 FEB 200 Regular issues provide a wide range of research and review articles covering all aspects of electrophoresis. Here you will find cutting-edge articles on methods and theory, instrumentation, nucleic acids, CE and CEC, miniaturization and microfluidics, proteomics and two-dimensional electrophoresis. Issue no. 2 has a "Fast Track" paper on the attomole protein analysis by capillary isoelectric focusing (CIEF) with LIF detection based on a post-column sheath flow cuvette employing Chromeo P503 as a fluorogenic reagent for protein labeling before CIEF analysis. Further selected topics of issue 2 are: Influence of image-analysis software on quantitation of two-dimensional gel electrophoresis data A PDMS sheath flow cuvette for high-sensitivity LIF measurements in CE [source] Cover Picture: Electrophoresis 22'2008ELECTROPHORESIS, Issue 22 2008Article first published online: 26 NOV 200 Regular issues provide a wide range of research and review articles covering all aspects of electrophoresis. Here you will find cutting-edge articles on methods and theory, instrumentation, nucleic acids, CE and CEC, miniaturization and microfluidics, proteomics and two-dimensional electrophoresis. Selected topics of issue 22 are: Microfluidics: Applications for analytical purposes in chemistry and biochemistry ((http://doi.wiley.com/10.1002/elps.200800121)) Simultaneous laser-induced fluorescence and retro-reflected beam interference detection for CE ((http://doi.wiley.com/10.1002/elps.200800292)) Quantitative Proteomics by Fluorescent Labeling of Cysteine Residues using a Set of Two Cyanine-based or Three Rhodamine-based Dyes ((http://doi.wiley.com/10.1002/elps.200800092)) Chemometric resolution of fully overlapped capillary electrophoresis peaks: quantitation of carbamazepine in human serum in the presence of several interferences ((http://doi.wiley.com/10.1002/elps.200800400)) Identification of inorganic ions in post-blast explosive residues using portable capillary electrophoresis instrumentation and capacitively-coupled contactless conductivity detection ((http://doi.wiley.com/10.1002/elps.200800226)) [source] Cover Picture: Electrophoresis 15/2008ELECTROPHORESIS, Issue 15 2008Article first published online: 24 JUL 200 Regular issues provide a wide range of research and review articles covering all aspects of electrophoresis. Here you will find cutting-edge articles on methods and theory, instrumentation, nucleic acids, CE and CEC, miniaturization and microfluidics, proteomics and two-dimensional electrophoresis. Selected topics of issue 15 are: The application of perfluorooctanoate to investigate trimerization of the human immunodeficiency virus-1 gp41 ectodomain by electrophoresis Metabolic fingerprinting of schistosoma mansoni infection in mice urine with capillary electrophoresis Supercritical fluid extraction as an on-line clean-up technique for determination of riboflavin vitamins in food samples by capillary electrophoresis with fluorimetric detection A two-step electro-dialysis method for DNA purification from polluted metallic environmental samples. [source] Sample complexity reduction for two-dimensional electrophoresis using solution isoelectric focusing prefractionationELECTROPHORESIS, Issue 12 2008Matthew R. Richardson Abstract 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. [source] Cover Picture: Electrophoresis 11/2008ELECTROPHORESIS, Issue 11 2008Article first published online: 11 JUN 200 Regular issues provide a wide range of research and review articles covering all aspects of electrophoresis. Here you will find cutting-edge articles on methods and theory, instrumentation, nucleic acids, CE and CEC, miniaturization and microfluidics, proteomics and two-dimensional electrophoresis. Issue 11 also offers a series of 9 papers on bioanalysis related to proteomics, protein analysis, genes, DNA sequencing, SNP, genotyping and SSCP. [source] Cover Picture: Electrophoresis 10/2008ELECTROPHORESIS, Issue 10 2008Article first published online: 21 MAY 200 Regular issues provide a wide range of research and review articles covering all aspects of electrophoresis. Here you will find cutting-edge articles on methods and theory, instrumentation, nucleic acids, CE and CEC, miniaturization and microfluidics, proteomics and two-dimensional electrophoresis. "The present issue includes 29 manuscripts subdivided into three major parts: one part is devoted to instrumental and methodological advances, and two parts are providing an insight into up-to-date applications from the fields of natural products and food analysis on the one hand and biomedical and pharmaceutical analysis on the other hand. The approaches used comprise different modes of electroseparation methods such as CZE, packed column, monolithic column and open-tubular CEC, MEKC, CIEF, CITP, different modes of ionization such as MALDI, ICP, and ESI, as well as a range of mass analyzers from simple single quadrupole MS to top of the range Q-TOF instruments, providing MS-MS and accurate mass features." [source] Cover Picture: Electrophoresis 9/2008ELECTROPHORESIS, Issue 9 2008Article first published online: 28 APR 200 Regular issues provide a wide range of research and review articles covering all aspects of electrophoresis. Here you will find cutting-edge articles on methods and theory, instrumentation, nucleic acids, CE and CEC, miniaturization and microfluidics, proteomics and two-dimensional electrophoresis. The 20 papers gathered here span areas across miniaturization theory, basic technologies and applications. While not comprehensive, this special issue represents a selection of the state-of-the-art science of miniaturization and its application in various areas of life sciences. We feel to a large degree that this special issue sets a good foundation, and we are looking forward to another thought-provoking update in the near future. Issue 9 also offers one Fast Track article describing particularly important investigations in electrophoresis: "AC Electrokinetic separation and detection of DNA nanoparticles in high conductance solutions." [source] Cover Picture: Electrophoresis 8/2008ELECTROPHORESIS, Issue 8 2008Article first published online: 17 APR 200 Regular issues provide a wide range of research and review articles covering all aspects of electrophoresis. Here you will find cutting-edge articles on methods and theory, instrumentation, nucleic acids, CE and CEC, miniaturization and microfluidics, proteomics and two-dimensional electrophoresis. On April 2 Professor Hjertén celebrated his 80th birthday, and it is an honor to take this opportunity to congratulate him on this special occasion and at the same time on his fruitful work. Stellan Hjertén's distinguished personality in research and life makes this celebration very special. It is therefore appropriate to devote a separate laudation in ELECTROPHORESIS to his achievements through which he has attained renown within the separation science community: indeed, he is considered undoubtedly to be the "Father of Capillary Electrophoresis". Professor Hjertén's preliminary work with Arne Tiselius motivated him to commit his career to electrophoresis: the development of free zone electrophoresis certainly revolutionized separation science, and since the construction of the first "capillary electrophoresis" equipment, one of the most cited works in this field carries his name. His friends were very keen to contribute manuscripts to this Issue, covering almost all areas in which Professor Hjertén has worked in his distinguished career. [source] Cover Picture: Electrophoresis 7/2008ELECTROPHORESIS, Issue 7 2008Article first published online: 2 APR 200 Regular issues provide a wide range of research and review articles covering all aspects of electrophoresis. Here you will find cutting-edge articles on methods and theory, instrumentation, nucleic acids, CE and CEC, miniaturization and microfluidics, proteomics and two-dimensional electrophoresis. Issue 7 also offers one Fast Track article describing particularly important investigations in electrophoresis: "Electrokinetic Analyte Transport Assay" for alpha - phetoprotein immunoassay integrates mixing, reaction and separation on-chip Tomohisa Kawabata, Henry G. Wada, Mitsuo Watanabe, Shinji Satomura [source] Cover Picture: Electrophoresis 6/2008ELECTROPHORESIS, Issue 6 2008Article first published online: 18 MAR 200 Regular issues provide a wide range of research and review articles covering all aspects of electrophoresis. Here you will find cutting-edge articles on methods and theory, instrumentation, nucleic acids, CE and CEC, miniaturization and microfluidics, proteomics and two-dimensional electrophoresis. In addition, issue no. 6 features a series of 7 important papers on "Microfluidics and Miniaturization" dealing with LCD-based optoelectronic tweezers, cell sorting, single cell clone analysis and cultivation, integrated ITP stacking and gel electrophoresis, floating injection, electrokinetic flows on thermosensitive surfaces and flow velocity measurement in CE chip instruments. [source] Evaluation of two-dimensional electrophoresis and liquid chromatography , tandem mass spectrometry for tissue-specific protein profiling of laser-microdissected plant samplesELECTROPHORESIS, Issue 17 2005Martina Schad See original http://dx.doi.org/10.1002/elps.200410399 [source] Characterization of glyco isoforms in plasmaderived human antithrombin by on-line capillary zone electrophoresis-electrospray ionization-quadrupole ion trap-mass spectrometry of the intact glycoproteinsELECTROPHORESIS, Issue 13 2004Uwe M. Demelbauer Abstract The carbohydrate structures of five isoforms of ,-AT and two isoforms of ,-AT were determined by applying capillary zone electrophoresis (CZE) on-line coupled to electrospray ionization-mass spectrometry (ESI-MS) using an ion-trap analyzer. For the AT preparations gained from a plasma pool at least semiquantitative information on the isoform-distributions could be gained. Unlike to the commonly used approaches starting from enzymatically treated glycoproteins, this approach deals with intact proteins. The high accuracy of the molecular mass determination obtained by the ion-trap analyzer allows one to calculate and ascertain the carbohydrate composition assuming no variations in the protein moiety of AT and to exclude or confirm the presence of the potential post-translational or other modifications. Therefore, the direct coupling of CZE with ESI-MS does not only represent a fast alternative technique to two-dimensional electrophoresis (2-DE) but serves as a method which provides structural information complementary to that gained from peptide mapping methods. [source] Comprehensive proteome analysis by chromatographic protein prefractionationELECTROPHORESIS, Issue 7-8 2004Pierre Lescuyer Abstract Protein copy number is distributed from 7 to 8 orders of magnitude in cells and probably up to 12 orders of magnitude in plasma. Classical silver-stained two-dimensional electrophoresis (2-DE) can only display up to four orders of magnitude. This is a major drawback since it is assumed that most of the regulatory proteins are low-abundance gene products. It is thus clear that the separation of low copy number proteins in amounts sufficient for postseparation analysis is an important issue in proteome studies to complete the comprehensive description of the proteome of any given cell type. The visualization of a polypeptide on a 2-DE gel will depend on the copy number, on the quantity loaded onto the gel and on the method of detection. As the amount of protein that can be loaded onto a gel is limited, one efficient solution is to fractionate the sample prior to 2-DE analysis. Several approaches exist including subcellular fractionation, affinity purification and chromatographic and electrophoretic protein prefractionation. The chromatographic step adds a new dimension in the protein separation using specific protein properties. It allows proteins to be adsorbed to a surface and eluted differentially under certain conditions. This review article presents studies combining chromatography-based methods to 2-DE analysis and draws general conclusions on this strategy. [source] Highly sensitive and simple fluorescence staining of proteins in sodium dodecyl sulfate-polyacrylamide-based gels by using hydrophobic tail-mediated enhancement of fluorescein luminescenceELECTROPHORESIS, Issue 19-20 2003Chulhun Kang Abstract Fluorescein has an extremely low luminescence intensity in acidic aqueous media. However, when it was bound to proteins, subsequent increase of luminescence intensity took place. Furthermore, when a hydrophobic tail, such as aliphatic hydrocarbons, was introduced to fluorescein, more dramatic increase of luminescence intensity was observed upon binding to proteins. In the present study, by utilizing this luminescence enhancement, three hydrophobic fluorescein dyes (5-dodecanoyl amino fluorescein, 5-hexadecanoyl amino fluorescein, and 5-octadecanoyl amino fluorescein) were examined as noncovalent fluorescent stains of protein bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Effective incorporation of the dyes to proteins in gels was accomplished either simply by adding dyes at the protein fixation step, or by treating gels with a staining solution after the fixation. The sensitivity of this staining method using the fluorescein derivatives was approximately 1 ng/band for most proteins. For some cases, protein bands containing as low as 0.1 ng were successfully visualized. In addition, the detection sensitivity showed much less protein-to-protein variation than silver staining. This new staining method was also successfully applied to two-dimensional electrophoresis of rat brain proteins. Its overall sensitivity was comparable to that of silver staining. [source] Quantitative evaluation of sample application methods for semipreparative separations of basic proteins by two-dimensional gel electrophoresisELECTROPHORESIS, Issue 19-20 2003Richard C. Barry Abstract The use of cup-loading for sample application has become widely used in two-dimensional electrophoresis (2-DE) for resolution of basic proteins, but no side-by-side quantitative study has been published which compares cup-loading with the alternative passive and active rehydration methods to fully promote one type of loading method over another. Replicate 2-D gels from each loading method were quantitatively evaluated for gel-to-gel reproducibility using IPG 6,11 strips and semipreparative protein loads (300 ,g). Gels were stained with SYPRO Ruby and analyzed with PDQuest. An inexpensive home-made assembly for cup-loading was used with the Protean IEF Cell for separation of whole cell extracts from the archaeon, Sulfolobus solfataricus. Cup-loading was determined to be far superior for IPG 6,11 separations than active or passive rehydration methods. Cup-loading consistently produced the greatest number of detectable spots, the best spot matching efficiency (56%), lowest spot quantity variations (28% coefficient of variation, CV), and the best-looking gels qualitatively. The least satisfactory results were obtained with active rehydration, followed closely by passive rehydration in off-line tubes. Passive rehydration experiments, performed using an on-line isoelectric focusing (IEF) tray, produced comparable spot numbers to cup-loading (84%), with 55% of the spots having higher intensity but 10% more spot quantity variance than cup-loading. [source] Comparative proteome analysis of culture supernatant proteins from virulent Mycobacterium tuberculosis H37Rv and attenuated M. bovis BCG CopenhagenELECTROPHORESIS, Issue 19-20 2003Jens Mattow Abstract A comprehensive analysis of culture supernatant (CSN) proteins of Mycobacterium tuberculosis H37Rv was accomplished by combination of two-dimensional electrophoresis (2-DE), mass spectrometry, and N -terminal sequencing by Edman degradation. Analytical 2-DE gels resolved approximately 1250 protein spots from CSN of M. tuberculosis H37Rv, 381 of which were identified by mass spectrometry and/or Edman degradation. This study revealed 137 different proteins, 42 of which had previously been described as secreted. Comparative proteome analysis of CSN from virulent M. tuberculosis H37Rv and attenuated Mycobacterium bovis BCG Copenhagen identified 39 M. tuberculosis- specific spots containing 27 different proteins, representing candidate antigens for novel vaccines and diagnostics in tuberculosis. These included five proteins encoded by open reading frames absent from M. bovis BCG, e.g., early secretory antigen target (Esat6), as well as 22 novel differential proteins, such as acetyl-CoA C-acetyltransferase (Rv0243) and two putative Esat6-like proteins (Rv1198, Rv1793). [source] Proteomics of snake venoms from Elapidae and Viperidae families by multidimensional chromatographic methodsELECTROPHORESIS, Issue 16 2003Jiraporn Nawarak Abstract Snake venoms contain a large number of biologically active substances and the venom components are very useful for pharmaceutical applications. Our goal is to separate and identify components of snake venoms in ten snake species from the Elapidae and Viperidae families using multidimensional chromatographic methods. The multidimensional chromatographic methods include reversed-phase high-performance liquid chromatography (RP-HPLC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), lab-on-a-chip, two-dimensional electrophoresis (2-DE), and mass spectrometry. The venoms of eight snake species demonstrated major differences in hydrophobicity, molecular weight separations, and 2-DE protein distribution patterns. The 2-DE images showed major differences between families, within each family and even between the same species. Venoms of the Elapidae family showed many basic proteins with a wide range of molecular weights, while venoms of the Viperidae family showed wide ranges of pI and molecular weights, especially for Trimeresurus sp. The multidimensional chromatographic methods revealed specific differences in venom proteins intra-species as well as between species and families. We have isolated and identified proteins that may be unique for each species for further studies in the proteome of snake venoms and their potentially use in the pharmaceutical applications. [source] Multiple polypeptide forms observed in two-dimensional gels of Methylococcus capsulatus (Bath) polypeptides are generated during the separation procedureELECTROPHORESIS, Issue 4 2003Frode S. Berven Abstract We have examined two-dimensional electrophoresis (2-DE) gel maps of polypeptides from the Gram-negative bacterium Methylococcus capsulatus (Bath) and found the same widespread trains of spots as often reported in 2-DE gels of polypeptides of other Gram-negative bacteria. Some of the trains of polypeptides, both from the outer membrane and soluble protein fraction, were shown to be generated during the separation procedure of 2-DE, and not by covalent post-translational modifications. The trains were found to be regenerated when rerunning individual polypeptide spots. The polypeptides analysed giving this type of trains were all found to be classified as stable polypeptides according to the instability index of Guruprasad et al. (Protein Eng. 1990, 4, 155,161). The phenomenon most likely reflects conformational equilibria of polypeptides arising from the experimental conditions used, and is a clear drawback of the standard 2-DE procedure, making the gel picture unnecessarily complex to analyse. [source] Monitoring of protein profiles for the optimization of recombinant fermentation processes using public domain databasesELECTROPHORESIS, Issue 1-2 2003Karin Dürrschmid Abstract The expression of human superoxide dismutase in fed-batch fermentation of E. coli HMS174(DE3)(pET3ahSOD) was studied as model system. Due to the frequently used strong T7 promoter system a high metabolic load is exerted, which triggers stress response mechanisms and finally leads to the differentiation of the host cell. As a consequence, host cell metabolism is partly shifted from growth to survival accompanied by significant alterations of the protein pattern. In terms of process optimization two-dimensional electrophoresis deserves as a powerful tool to monitor these changes on protein level. For the analysis of samples derived from different states of recombinant protein production wide-range Immobiline Dry Strips pH 3,10 were used. In order to establish an efficient procedure for accelerated process optimization and to avoid costly and time-consuming analysis like mass spectrometry (MS), a database approach for the identification of significant changes of the protein pattern was evaluated. On average, 935 spots per gel were detected, whereby 50 are presumably stress-relevant. Out of these, 24 proteins could be identified by using the SWISS-2DPAGE database (www.expasy.ch/ch2d/). The identified proteins are involved in regulatory networks, energy metabolism, purine and pyrimidine nucleotide synthesis and translation. By this database approach, significant fluctuations of individual proteins in relation to recombinant protein production could be identified. Seven proteins show strong alterations (>100%) directly after induction and can therefore be stated as reliable marker proteins for the assessment of stress response. For distinctive interpretation of this highly specific information, a bioinformatic and statistic tool would be essential in order to perceive the role and contribution of individual proteins in stress response. [source] Proteome analysis of human liver tumor tissue by two-dimensional gel electrophoresis and matrixassisted laser desorption/ionization-mass spectrometry for identification of disease-related proteinsELECTROPHORESIS, Issue 24 2002Jina Kim Abstract Hepatocellular carcinoma (HCC) is a common malignancy worldwide and is a leading cause of death. To contribute to the development and improvement of molecular markers for diagnostics and prognostics and of therapeutic targets for the disease, we have largely expanded the currently available human liver tissue maps and studied the differential expression of proteins in normal and cancer tissues. Reference two-dimensional electrophoresis (2-DE) maps of human liver tumor tissue include labeled 2-DE images for total homogenate and soluble fraction separated on pH 3,10 gels, and also images for soluble fraction separated on pH 4,7 and pH 6,9 gels for a more detailed map. Proteins were separated in the first dimension by isoelectric focusing on immobilized pH gradient (IPG) strips, and by 7.5,17.5% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels in the second dimension. Protein identification was done by peptide mass fingerprinting with delayed extraction-matrix assisted laser desorption/ionization-time of flight-mass spectrometry (DE-MALDI-TOF-MS). In total, 212 protein spots (117 spots in pH 4,7 map and 95 spots in pH 6,9) corresponding to 127 different polypeptide chains were identified. In the next step, we analyzed the differential protein expression of liver tumor samples, to find out candidates for liver cancer-associated proteins. Matched pairs of tissues from 11 liver cancer patients were analyzed for their 2-DE profiles. Protein expression was comparatively analyzed by use of image analysis software. Proteins whose expression levels were different by more than three-fold in at least 30% (four) of the patients were further analyzed. Numbers of protein spots overexpressed or underexpressed in tumor tissues as compared with nontumorous regions were 9 and 28, respectively. Among these 37 spots, 1 overexpressed and 15 underexpressed spots, corresponding to 11 proteins, were identified. The physiological significance of the differential expressions is discussed. [source] Identification of Low Molecular Weight Diapause-Associated Proteins of Two-Spotted Spider Mite, Tetranychus urticaeENTOMOLOGICAL RESEARCH, Issue 4 2005JUNG Duck-Oung ABSTRACT Proteins in whole bodies of two-spotted spider mites, Tetranchus urticae, were compared between non-diapause and diapause conditions using one- and two-dimensional electrophoresis. At one-dimensional analysis diapausing mites synthesized several unique proteins that are higher in their quantity than non-diapausing ones. Overall protein numbers in two-dimensional gel show that diapause mites expressed fewer proteins than non-diapausing ones. However, 25 proteins that have higher quantity in diapause were selected at the range of low molecular weight (5-20 kDa). Among them 13 proteins were only present in diapause mites. Our results thus indicate some proteins uniquely expressed during diapause and may have specifie roles during long overwintering period. [source] Expression of psoriasis-associated fatty acid-binding protein in senescent human dermal microvascular endothelial cellsEXPERIMENTAL DERMATOLOGY, Issue 9 2004Moon Kyung Ha Abstract:, Aging is associated with the progressive pathophysiologic modification of endothelial cells. In vitro endothelial cell senescence is accompanied by proliferative activity failure and by perturbations in gene and protein expressions. Moreover, this cellular senescence in culture has been proposed to reflect processes that occur in aging organisms. In order to observe the changing patterns of protein expression in senescent human dermal microvascular endothelial cells (HDMECs), proteins obtained from both early- and late-passaged HDMECs were separated by two-dimensional electrophoresis, visualized by silver staining, and quantified by image processing. Proteins of interest were extracted by in-gel digestion with trypsin and quantified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), by searching the National Center for Biotechnology Information protein-sequence database. More than 2000 spots were detected by 2D electrophoresis within a linear pH range of 3,10. Twenty-two major differentially expressed spots were observed in serially passaged HDMECs and identified with high confidence by MALDI-TOF-MS. One of these spots was found to be a 14,15 kDa psoriasis-associated fatty acid-binding protein (PA-FABP) with high affinity for long-chain fatty acids. The expression of PA-FABP was confirmed to be elevated in senescent HDMECs (passage 20) by fluorescence-activated cell sorting (FACS), confocal laser microscopy, and by immunohistochemistry in aged human skin tissue. Our results suggest that the overexpression of FABP in cultured senescent HDMECs is closely related to skin aging. [source] A proteomic study of Escherichia coli O157:H7 NCTC 12900 cultivated in biofilm or in planktonic growth modeFEMS MICROBIOLOGY LETTERS, Issue 1 2002Frédéric Trémoulet Abstract Escherichia coli 0157:H7 biofilms were studied by a new method of cultivation in order to identify some of the proteins involved in the biofilm phenotype. A proteomic analysis of sessile or planktonic bacteria of the same age was carried out by two-dimensional electrophoresis, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) and database searching. Comparison of two-dimensional gels showed clear differences between protein patterns of sessile and planktonic cells. Fourteen proteins increased in biofilms, whereas three decreased. From these 17 proteins, 10 were identified by MALDI-TOF-MS and could be classified into four categories according to their function: (1) general metabolism proteins (malate dehydrogenase, thiamine-phosphate pyrophosphorylase), (2) sugar and amino acid transporters (d -ribose-binding periplasmic protein, d -galactose-binding protein, YBEJ), (3) regulator proteins (DNA starvation protein and H-NS) and (4) three proteins with unknown function. The results of this study showed that E. coli O157:H7 modified the expression of several proteins involved in biofilm growth mode. [source] A comparative proteome analysis of human metaphase chromosomes isolated from two different cell lines reveals a set of conserved chromosome-associated proteinsGENES TO CELLS, Issue 3 2007Hideaki Takata A comparative proteome analysis of human metaphase chromosomes between a typical epithelial-like cell, HeLa S3, and a lymphoma-type cell, BALL-1, was performed. One-dimensional (1-D) SDS-PAGE and radical-free and highly reducing two-dimensional electrophoresis (RFHR 2-DE) detected more than 200 proteins from chromosomes isolated from HeLa S3 cells, among which 189 proteins were identified by mass spectrometry (MS). Consistent with our recent four-layer structural model of a metaphase chromosome, all the identified proteins were grouped into four distinct levels of abundance. Both HeLa S3 and BALL-1 chromosomes contained specific sets of abundant chromosome structural and peripheral proteins in addition to less abundant chromosome coating proteins (CCPs). Furthermore, titin array analysis and a proteome analysis of the ultra-high molecular mass region indicated an absence of titin with their molecular weight (MW) more than 1000 kDa. Consequently, the present proteome analyses together with previous information on chromosome proteins provide the comprehensive list of proteins essential for the metaphase chromosome architecture. 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