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Protein Samples (protein + sample)
Selected AbstractsAberrant protein expression is associated with decreased developmental potential in porcine cumulus,oocyte complexesMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 1 2010Melissa Paczkowski Oocyte developmental competence is progressively obtained during pubertal development in females. Poor developmental potential in oocytes derived from prepubertal females suggests that essential processes required for oocyte development have not been fulfilled. The objective of this experiment was to analyze the protein profiles of porcine cumulus,oocyte complexes (COC) derived from cyclic and prepubertal females to identify alterations in protein abundance that correlate with developmental potential. COC complexes, aspirated from prepubertal and cyclic ovaries, were pooled into three replicates of 400 COCs each per treatment in ,100,µl SOF-HEPES medium. Protein samples were extracted and analyzed by two-dimensional differential in gel electrophoresis (2D-DIGE). Over 1,600 proteins were resolved on each of the three replicate gels. Sixteen protein spots were identified by mass spectrometry, representing 14 unique, differentially expressed proteins (volume ratio greater than 1.3). Glutathione- S -transferase and pyruvate kinase 3 were more abundant in COCs derived from cyclic females, whereas soluble epoxide hydrolase and transferrin were more abundant in prepubertal derived COCs. Abundance of several glycolytic enzymes (enolase 1, pyruvate kinase 3, and phosphoglycerate kinase) was increased in COCs derived from cyclic females, suggesting glucose metabolism is decreased in prepubertal derived COCs. We conclude that the abundance of proteins involved in metabolism and oxidative stress regulation is significantly altered in prepubertal derived COCs and may play a role in the mechanisms resulting in developmental competence. Mol. Reprod. Dev. 77: 51,58, 2010. © 2009 Wiley-Liss, Inc. [source] Molecular targets of botulinum toxin at the mammalian neuromuscular junctionMOVEMENT DISORDERS, Issue S8 2004Dorothy D. Whelchel MS Abstract The molecular targets of botulinum neurotoxins (BoNTs) are SNARE (soluble N -ethylmaleimide-sensitive factor- attachment protein- receptor) proteins necessary for neurotransmitter release. BoNT are powerful therapeutic agents in the treatment of numerous neurological disorders. The goals of this study were to (1) assess toxin diffusion by measuring substrate cleavage in adjacent and distant muscles, and (2) characterize the clinical course using SNARE protein chemistry. A small volume of BoNT/A was injected unilaterally into the mouse gastrocnemius muscle. Motor impairment was limited to the toxin-treated limb. No systemic illness or deaths occurred. At five time points, a subset of mice were killed, and muscles from both hindlimbs, and the diaphragm, were collected. Protein samples were examined for changes in SNAP-25 (synaptosomal-associated protein of Mr = 25 kDa) using immunochemistry. SNAP-25 cleavage product was noted in the toxin-treated limb as early as 1 day postinjection and continued through day 28. Onset and peak levels of substrate cleavage corresponded to the onset and peak clinical response. Cleavage was observed in adjacent and distant muscles, demonstrating that substrate cleavage is a sensitive indicator of toxin diffusion. Significant increases in full-length SNAP-25 and vesicle-associated membrane protein II were evident early in the impaired limb and continued through day 28. The increased SNARE protein most likely originates from nerve terminal sprouts. © 2004 Movement Disorder Society [source] Discovering robust protein biomarkers for disease from relative expression reversals in 2-D DIGE data.PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 8 2007Troy J. Anderson Abstract This study assesses the ability of a novel family of machine learning algorithms to identify changes in relative protein expression levels, measured using 2-D DIGE data, which support accurate class prediction. The analysis was done using a training set of 36 total cellular lysates comprised of six normal and three cancer biological replicates (the remaining are technical replicates) and a validation set of four normal and two cancer samples. Protein samples were separated by 2-D DIGE and expression was quantified using DeCyder-2D Differential Analysis Software. The relative expression reversal (RER) classifier correctly classified 9/9 training biological samples (p<0.022) as estimated using a modified version of leave one out cross validation and 6/6 validation samples. The classification rule involved comparison of expression levels for a single pair of protein spots, tropomyosin isoforms and ,-enolase, both of which have prior association as potential biomarkers in cancer. The data was also analyzed using algorithms similar to those found in the extended data analysis package of DeCyder software. We propose that by accounting for sources of within- and between-gel variation, RER classifiers applied to 2-D DIGE data provide a useful approach for identifying biomarkers that discriminate among protein samples of interest. [source] Simulations of IEF in microchannel with variable cross-sectional areaELECTROPHORESIS, Issue 5 2009Yin Chou Abstract This study develops a 1-D mass transport model to describe the electrophoresis transport behavior within a microchannel with a variable cross-sectional area. Utilizing three different numerical schemes, simulations are performed to investigate the IEF of proteins in ampholyte-based pH gradients within both a planar microchannel and a contraction,expansion microchannel, respectively. The simulation results obtained using the modified 1-D mass transport model and the finite-volume method (FVM) for the IEF separation of a single protein sample in a ten-ampholyte-based pH gradient within a planar microchannel are consistent with those presented by Shim et al. [Electrophoresis 2007, 28, 572,586] using a 2-D FVM scheme. In addition, the Courant,Friedrichs,Lewy number insensitive conservation element and solution element (CNI-CESE) method is found to be both more robust and more computationally efficient than the conventional CESE scheme when modeling IEF phenomena within a contraction,expansion microchannel. In modeling the IEF separation of four sample ampholytes in a 20-ampholtye-based pH gradient within a contraction,expansion microchannel, the results obtained using the CNI-CESE scheme are in good agreement with those published in literature. Moreover, the simulations can be performed significantly faster with the new 1-D model and the CNI-CESE scheme. Finally, the results obtained using the modified 1-D mass transport model and the CNI-CESE scheme demonstrate that the concentration of the focused test sample and the resolution of the pH gradient within the microchannel increase as the number of ampholytes used to accomplish the IEF separation process is increased. [source] Quantitative Determination of Surface Concentration of Human Apolipoprotein H with Capillary ElectrophoresisIUBMB LIFE, Issue 5 2000Shao-xiong Wang Abstract The phospholipid monolayer at an air/water interface is widely used to mimic the biological membrane. The dynamic process of the protein or peptide interacting with lipid molecules can be reflected in the change in surface pressure of the monolayer. But the conventional method used to measure the surface pressure change gives results that cannot easily be correlated with the contribution of a single protein molecule. Previously, measuring the surface concentration of the protein molecules at the air/water interface has required the protein to be labeled with radioactivity or fluorescence. Here, a new method using capillary electrophoresis is introduced to measure the surface concentration of the protein. The results show at least two advantages of the new method: The numerical results of protein concentration can be obtained in a more precise and rapid way; and there is no need to label the protein sample or to build a special monolayer setup. [source] Using enrichment index for quality control of secretory protein sample and identification of secretory proteinsJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 3 2009Yong Chen Abstract Analysis of secretory proteins is an important area in proteomic research. We propose that a good secretory protein sample should be enriched with known secretory proteins, and a secretory protein should be enriched in the secretory protein sample compared with its corresponding soluble cell lysate. Positive identifications of proteins were subjected to quantitation of spectral counts, which reflect relative protein abundance. Enrichment index of the sample (EIS) and the enrichment index for protein (EIP) were obtained by comparing proteins identified in the secretory protein sample and those in the soluble cell lysate sample. The quality of the secretory protein sample can be represented by EIS. EIP was used to identify the secretory proteins. The secretory proteins from mouse dendritic cell sarcoma (DCS) were analyzed by MS. The EISs of two samples were 75.4 and 84.65, respectively. 72 proteins were significantly enriched in secretory protein samples, of which 42 proteins were either annotated in Swiss-Prot and/or predicted by signal peptides to be secretory. In the remaining 30 proteins, 12 and 15 proteins were positively predicted by SecretomeP and ProP, respectively, and 5 proteins were positive by both methods. Furthermore, 11 proteins were found to be present in exosome in other studies that involved mice dendritic cell lines. We suggest that this assessment method is helpful for systemic research of secretory proteins and biomarker discovery for diseases such as cancer. Copyright © 2008 John Wiley & Sons, Ltd. [source] Crystallizing proteins on the basis of their precipitation diagram determined using a microfluidic formulatorJOURNAL OF SYNCHROTRON RADIATION, Issue 6 2005Morten O. A. Sommer Crystallization of proteins from a purified protein solution remains a bottleneck in the structure determination pipeline. In this paper the crystallization problem is addressed using a microfluidic device capable of determining detailed protein precipitation diagrams using less than 10,µL of protein sample. Based on the experimentally determined protein phase behavior, a crystallization screen can be designed to accommodate the physical chemistry of the particular protein target. Such a tailor-made crystallization screen has a high probability of yielding crystallization hits. The approach is applied to two different proteins: the calcium pump (SERCA), an eukaryotic integral membrane protein, and UMP kinase, a prokaryotic soluble kinase. Protein phase behavior is mapped for both proteins and tailor-made crystallization screens are designed for the two proteins resulting in about 50% crystallization probability per experiment. This illustrates the power of using microfluidic devices for detailed characterization of protein phase behavior prior to crystallization trials. [source] Clean SEA-TROW experiments to map solvent exposed amides in large proteinsCHINESE JOURNAL OF CHEMISTRY, Issue 12 2004Dong-Hai Lin Abstract It is well known that the SEA-TROSY experiment could alleviate some of the problems of resonance overlap in 15N/2H labeled proteins as it was designed to selectively map solvent exposed amide protons. However, SEA-TROSY spectra may be contaminated with exchange-relayed NOE contributions from fast exchanged hydroxyl or amine protons and contributions from longitudinal relaxation. Also, perdeuteration of the protein sample is a prerequisite for this experiment. In this communication, a modified version, clean SEA-TROSY, was proposed to eliminate these artifacts and to allow the experiment to be applied to protonated or partially deuterated proteins and protein complexes. [source] Cover Picture: Electrophoresis 14'09ELECTROPHORESIS, Issue 14 2009Article first published online: 28 JUL 200 Issue no. 14 is an Emphasis Issue with 9 articles on various aspects of "Proteins and Proteomics" while the remaining 14 articles are arranged into 4 different parts including "Microfluidics and Miniaturization", "Genotyping and Transcriptomics", "Enantioseparations", and "Nanoparticles and Abused Drugs Analyses". Selected articles are: Effective elimination of nucleic acids from bacterial protein samples for optimized blue native polyacrylamide gel electrophoresis ((10.1002/elps.200900026)) 2-D difference in gel electrophoresis combined with Pro-Q Diamond staining: A successful approach for the identification of kinase/phosphatase targets ((10.1002/elps.200800780)) Microvalves actuated sandwich immunoassay on an integrated microfluidic system ((10.1002/elps.200800818)) Chemical gradient-mediated melting curve analysis for genotyping of SNPs ((10.1002/elps.200800729)) [source] Effective elimination of nucleic acids from bacterial protein samples for optimized blue native polyacrylamide gel electrophoresisELECTROPHORESIS, Issue 14 2009Jingdan Liang Abstract Nucleic acids remaining within bacterial protein samples from Streptomyces lividans and Escherichia coli were found to interfere significantly with blue native polyacrylamide gel electrophoresis (BN-PAGE), a technique used frequently for analyzing bacterial protein complexes in proteomics studies. We have used ultracentrifugation and/or precipitation of cell lysates with streptomycin sulfate to eliminate nucleic acids from total and/or membrane protein samples. Nucleic acid-binding proteins were first enriched by precipitation with streptomycin sulfate, and contaminating nucleic acids were then eliminated by precipitation by adding polyethyleneimine. The performance of BN-PAGE was found to be dramatically improved by these sample preparation steps. [source] On-line concentration of peptides and proteins with the hyphenation of polymer monolithic immobilized metal affinity chromatography and capillary electrophoresisELECTROPHORESIS, Issue 11 2005Lingyi Zhang Abstract An iminodiacetic acid (IDA)-type adsorbent is prepared at the one end of a capillary by covalently bonding IDA to the monolithic rods of macroporous poly(glycidyl methacrylate,co -ethylene dimethacrylate). Cu(II) is later introduced to the support via the interaction with IDA. By this means, polymer monolithic immobilized metal affinity chromatography (IMAC) materials are prepared. With such a column, IMAC for on-line concentration and capillary electrophoresis (CE) for the subsequent analysis are hyphenated for the analysis of peptides and proteins. The reproducibility of such a column has been proved good with relative standard deviations (RSDs) of dead time of less than 5% for injection-to-injection and 12% for column-to-column (n = 3). Through application on the analysis of standard peptides and real protein samples, such a technique has shown promising in proteome study. [source] A microfabricated capillary electrophoresis chip with multiple buried optical fibers and microfocusing lens for multiwavelength detectionELECTROPHORESIS, Issue 6 2005Suz-Kai Hsiung Abstract We present a new microfluidic device utilizing multiwavelength detection for high-throughput capillary electrophoresis (CE). In general, different fluorescent dyes are only excited by light sources with appropriate wavelengths. When excited by an appropriate light source, a fluorescent dye emits specific fluorescence signals of a longer wavelength. This study designs and fabricates plastic micro-CE chips capable of performing multiple-wavelength fluorescence detection by means of multimode optic fiber pairs embedded downstream of the separation channel. For detection purposes, the fluorescence signals are enhanced by positioning microfocusing lens structures at the outlets of the excitation fibers and the inlets of the detection fibers, respectively. The proposed device is capable of detecting multiple samples labeled with different kinds of fluorescent dyes in the same channel in a single run. The experimental results demonstrate that various proteins, including bovine serum albumin and ,-casein, can be successfully injected and detected by coupling two light sources of different wavelengths to the two excitation optic fibers. Furthermore, the proposed device also provides the ability to measure the speed of the samples traveling in the microchannel. The developed multiwavelength micro-CE chip could have significant potential for the analysis of DNA and protein samples. [source] Dental pulp fibroblasts express neuropeptide Y Y1 receptor but not neuropeptide YINTERNATIONAL ENDODONTIC JOURNAL, Issue 10 2010S. A. Killough Killough SA, Lundy FT, Irwin CR. Dental pulp fibroblasts express neuropeptide Y Y1 receptor but not neuropeptide Y. International Endodontic Journal, 43, 835,842, 2010. Abstract Aim, To investigate whether dental pulp fibroblasts express neuropeptide Y (NPY) and NPY-Y1 in vitro and to determine the effects of the cytokines including interlukin-1, (IL-1,), TGF- ,1, substance P and NPY on the expression of NPY Y1. Methodology, Three primary fibroblast cell strains were obtained from freshly extracted human third molar teeth. RT-PCR was utilized to detect expression of NPY and mRNA expression. Membrane protein samples were isolated, and protein expression was determined by Western blotting. Radioimmunoassay was used to quantify NPY expression in healthy (n = 35) and carious (n = 39) whole pulp samples, and the student's t -test was used to test for statistical significance. In addition, the 3-(4,5-Dimethylthiazol,2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to assay fibroblast cell growth. Results, mRNA transcripts were found in all three fibroblast cell populations with the cytokines having a stimulatory effect on its expression (P < 0.05). NPY mRNA was not detected in the cell strains. NPY-Y1 receptor protein expression was visualized by Western blotting, and there was no effect of IL-1, or TGF- ,1 on its expression. The mean concentration of NPY-Ir determined by radioimmunoassay in non-carious teeth was 19.40 ng g,1 (±17.03 SD) compared to 29.95 ng g,1 (±20.99 SD) in carious teeth (P < 0.05). Conclusion, Human dental pulp fibroblasts express, but do not synthesize, NPY, demonstrating that the fibroblast is a target cell for NPY. The effect of proinflammatory cytokines suggests that fibroblasts play a neuroimmunomodulatory role in the pulpal response to dental caries and injury. [source] Rigorous filtration for protein crystallizationJOURNAL OF APPLIED CRYSTALLOGRAPHY, Issue 4 2009Naomi E. Chayen Centrifugation or filtration of protein samples through 0.22,µm mesh size filters is standard procedure before setting up trials for crystallization. This note concerns other, mostly unused filters, namely 0.1,µm and 100,000,300,000 molecular weight cut-off filters, which have proved to aid in obtaining single high-quality crystals. It serves to pool together information concerning filtration which has been scattered, and somewhat concealed, in various publications over the years and to add information that has not yet been published. Filtration is relevant to all methods of crystallization for both screening and optimization. [source] Using enrichment index for quality control of secretory protein sample and identification of secretory proteinsJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 3 2009Yong Chen Abstract Analysis of secretory proteins is an important area in proteomic research. We propose that a good secretory protein sample should be enriched with known secretory proteins, and a secretory protein should be enriched in the secretory protein sample compared with its corresponding soluble cell lysate. Positive identifications of proteins were subjected to quantitation of spectral counts, which reflect relative protein abundance. Enrichment index of the sample (EIS) and the enrichment index for protein (EIP) were obtained by comparing proteins identified in the secretory protein sample and those in the soluble cell lysate sample. The quality of the secretory protein sample can be represented by EIS. EIP was used to identify the secretory proteins. The secretory proteins from mouse dendritic cell sarcoma (DCS) were analyzed by MS. The EISs of two samples were 75.4 and 84.65, respectively. 72 proteins were significantly enriched in secretory protein samples, of which 42 proteins were either annotated in Swiss-Prot and/or predicted by signal peptides to be secretory. In the remaining 30 proteins, 12 and 15 proteins were positively predicted by SecretomeP and ProP, respectively, and 5 proteins were positive by both methods. Furthermore, 11 proteins were found to be present in exosome in other studies that involved mice dendritic cell lines. We suggest that this assessment method is helpful for systemic research of secretory proteins and biomarker discovery for diseases such as cancer. Copyright © 2008 John Wiley & Sons, Ltd. [source] Biochemical and molecular studies using human autopsy brain tissueJOURNAL OF NEUROCHEMISTRY, Issue 3 2003Matthew R. Hynd Abstract The use of human brain tissue obtained at autopsy for neurochemical, pharmacological and physiological analyses is reviewed. RNA and protein samples have been found suitable for expression profiling by techniques that include RT-PCR, cDNA microarrays, western blotting, immunohistochemistry and proteomics. The rapid development of molecular biological techniques has increased the impetus for this work to be applied to studies of brain disease. It has been shown that most nucleic acids and proteins are reasonably stable post-mortem. However, their abundance and integrity can exhibit marked intra- and intercase variability, making comparisons between case-groups difficult. Variability can reveal important functional and biochemical information. The correct interpretation of neurochemical data must take into account such factors as age, gender, ethnicity, medicative history, immediate ante-mortem status, agonal state and post-mortem and post-autopsy intervals. Here we consider issues associated with the sampling of DNA, RNA and proteins using human autopsy brain tissue in relation to various ante- and post-mortem factors. We conclude that valid and practical measures of a variety of parameters may be made in human brain tissue, provided that specific factors are controlled. [source] Optimizing protein extraction from plant tissues for enhanced proteomics analysisJOURNAL OF SEPARATION SCIENCE, JSS, Issue 11 2008Wei Wang Abstract Plant tissues usually contain high levels of proteases and secondary metabolites that severely interfere with protein extraction, separation, and identification. Preparation of high-quality protein samples from plant tissues for proteomic analysis represents a great challenge. This article briefly describes the critical points in protein separation, especially secondary metabolites in plant tissues, and removal strategy. It provides an updated overview of three total protein extraction methods and their applications in proteomic analysis of various recalcitrant tissues. [source] Gel-free sample preparation for the nanoscale LC-MS/MS analysis and identification of low-nanogram protein samplesJOURNAL OF SEPARATION SCIENCE, JSS, Issue 14 2007Marco Gaspari Abstract Protein identification at the low nanogram level could in principle be obtained by most nanoscale LC-MS/MS systems. Nevertheless, the complex sample preparation procedures generally required in biological applications, and the consequent high risk of sample losses, very often hamper practical achievement of such low levels. In fact, the minimal amount of protein required for the identification from a gel band or spot, in general, largely exceeds the theoretical limit of identification reachable by nanoscale LC-MS/MS systems. A method for the identification of low levels of purified proteins, allowing limits of identification down to 1 ng when using standard bore, 75 ,m id nanoscale LC-MS/MS systems is here reported. The method comprises an offline two-step sample cleanup, subsequent to protein digestion, which is designed to minimize sample losses, allows high flexibility in the choice of digestion conditions and delivers a highly purified peptide mixture even from "real world" digestion conditions, thus allowing the subsequent nanoscale LC-MS/MS analysis to be performed in automated, unattended operation for long series. The method can be applied to the characterization of low levels of affinity purified proteins. [source] Physical and chemical considerations of damage induced in protein crystals by synchrotron radiation: a radiation chemical perspectiveJOURNAL OF SYNCHROTRON RADIATION, Issue 6 2002Peter O'Neill Radiation-induced degradation of protein or DNA samples by synchrotron radiation is an inherent problem in X-ray crystallography, especially at the `brighter' light sources. This short review gives a radiation chemical perspective on some of the physical and chemical processes that need to be considered in understanding potential pathways leading to the gradual degradation of the samples. Under the conditions used for X-ray crystallography at a temperature of <100,K in the presence of cryoprotectant agents, the majority of radiation damage of the protein samples arises from direct ionization of the amino acid residues and their associated water molecules. Some of the chemical processes that may occur at these protein centres, such as bond scission, are discussed. Several approaches are discussed that may reduce radiation damage, using agents known from radiation chemistry to minimize radical-induced degradation of the sample. [source] Methyl TROSY: explanation and experimental verificationMAGNETIC RESONANCE IN CHEMISTRY, Issue 10 2003Jason E. Ollerenshaw Abstract In TROSY experiments, relaxation interference effects are exploited to produce spectra with improved resolution and signal-to-noise. Such experiments cannot be explained using the standard product operator formalism, but must instead be analyzed at the level of individual density matrix elements. Herein we illustrate this point using an example from our recent work on a TROSY 1H,13C correlation experiment for methyl groups in large proteins. Methyl groups are useful spectroscopic probes of protein structure and dynamics because they are found throughout the critical core region of a folded protein and their resonances are intense and well dispersed. Additionally, it is relatively easy to produce highly deuterated protein samples that are 1H,13C labeled at selected methyl positions, facilitating studies of high molecular weight systems. Methyl groups are relaxed by a network of 1H,1H and 1H,13C dipolar interactions, and in the macromolecular limit the destructive interference of these interactions leads to unusually slow relaxation for certain density matrix elements. It is this slow relaxation that forms the basis for TROSY experiments. We present a detailed analysis of evolution and relaxation during HSQC and HMQC pulse schemes for the case of a 13C1H3 spin system attached to a macromolecule. We show that the HMQC sequence is already optimal with respect to the TROSY effect, offering a significant sensitivity enhancement over HSQC at any spectrometer field strength. The gain in sensitivity is established experimentally using samples of two large proteins, malate synthase G (81.4 kDa) and ClpP protease (305 kDa), both highly deuterated and selectively 1H,13C-labeled at isoleucine , methyl positions. Copyright © 2003 John Wiley & Sons, Ltd. [source] Effect of protein structure on deamidation rate in the Fc fragment of an IgG1 monoclonal antibody,PROTEIN SCIENCE, Issue 8 2009Sandipan Sinha Abstract The effects of secondary structure on asparagine (N) deamidation in a 22 amino acid sequence (369-GFYPSDIAVEWESNGQPENNYK-390) of the crystallizable (Fc) fragment of a human monoclonal antibody (Fc IgG1) were investigated using high-resolution ultra performance liquid chromatography with tandem mass spectrometry (UPLC/MS). Samples containing either the intact Fc IgG (,50 kD) ("intact protein"), or corresponding synthetic peptides ("peptide") were stored in Tris buffer at 37°C and pH 7.5 for up to forty days, then subjected to UPLC/MS analysis with high energy MS1 fragmentation. The peptide deamidated only at N382 to form the isoaspartate (isoD382) and aspartate (D382) products in the ratio of ,4:1, with a half-life of ,3.4 days. The succinimide intermediate (Su382) was also detected; deamidation was not observed for the other two sites (N387 and N388) in peptide samples. The intact protein showed a 30-fold slower overall deamidation half-life of ,108 days to produce the isoD382 and D387 products, together with minor amounts of D382. Surprisingly, the D382 and isoD387 products were not detected in intact protein samples and, as in the peptide samples, deamidation was not detected at N388. The results indicate that higher order structure influences both the rate of N-deamidation and the product distribution. [source] Exploring the priming mechanism of liver regeneration: proteins and protein complexesPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 8 2009Xinyu Deng Abstract The liver has the ability to restore its functional capacity following injury or resection and the priming of liver regeneration is a complex process that has not been completely elucidated. In the current research, to further reveal the priming mechanism of liver regeneration, hepatocyte total protein and hepatocyte cytosol of the rats at 4,h after 2/3 partial hepatectomy (PHx) were studied, respectively, by 2-DE and 2-D blue native gel electrophoresis. Seventeen unique differential proteins were identified in hepatocyte total protein samples. Nine differential protein complexes containing 41 protein components were identified in hepatocyte cytosol samples. For the first time, at the priming stage of liver regeneration, the variations of serine protease inhibitor 2c, sulfite oxidase and valosin-containing protein (VCP) were presented and validated by Western blotting, and the VCP complex was further validated by antibody super-shift experiments. The current results suggested that at 4,h after PHx, VCP complex was down-regulated in hepatocyte cytosol, apoptosis pathways were inhibited, nuclear factor-,B and interleukin 6 pathways worked together and triggered the liver regeneration. [source] Analysis of DIGE data using a linear mixed model allowing for protein-specific dye effectsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 23 2007Morten Krogh Dr. Abstract Differential in-gel electrophoresis (DIGE) experiments allow three protein samples to be run per gel. The three samples are labeled with the spectrally resolvable fluorescent dyes, Cy2, Cy3, and Cy5, respectively. Here, we show that protein-specific dye effects exist, and we present a linear mixed model for analysis of DIGE data which takes dye effects into account. A Java implementation of the model, called DIGEanalyzer, is freely available at http://bioinfo.thep.lu.se/digeanalyzer.html. Three DIGE experiments from our laboratory, with 173, 64, and 24 gels, respectively, were used to quantify and verify the dye effects. DeCyder 5.0 and 6.5 were used for spot detection and matching. The fractions of proteins with a statistically significant (0.001 level) dye effect were 19, 34, and 23%, respectively. The fractions of proteins with a dye effect above 1.4-fold change were 1, 4, and 6%, respectively. The median magnitude of the dye effect was 1.07-fold change for Cy5 versus Cy3 and 1.16-fold change for Cy3 versus Cy2. The maximal dye effect was a seven-fold change. The dye effects of spots corresponding to the same protein tend to be similar within each of the three experiments, and to a smaller degree across experiments. [source] Direct analysis of the extracellular proteome from two strains of Helicobacter pyloriPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 13 2007Todd G. Smith Abstract Helicobacter pylori extracellular proteins are of interest because of possible roles in pathogenesis, host recognition, and vaccine development. We utilized a unique approach by growing two strains (including one nonsequenced strain) in a defined serum-free medium and directly analyzing the proteins present in the culture supernatants by LC-MS/MS. Over 125 proteins were identified in the extracellular proteomes of two H. pylori strains. Forty-five of these proteins were enriched in the extracellular fraction when compared to soluble cell-associated protein samples. Our analysis confirmed and expanded on the previously reported H. pylori extracellular proteome. Extracellular proteins of interest identified here included cag pathogenicity island protein Cag24 (CagD); proteases HP0657 and HP1012; a polysaccharide deacetylase, HP0310, possibly involved in the hydrolysis of acetyl groups from host N -acetylglucosamine residues or from residues on the cell surface; and HP0953, an uncharacterized protein that appears to be restricted to Helicobacter species that colonize the gastric mucosa. In addition, our analysis found eight previously unidentified outer membrane proteins and two lipoproteins that could be important cell surface proteins. [source] Discovering robust protein biomarkers for disease from relative expression reversals in 2-D DIGE data.PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 8 2007Troy J. Anderson Abstract This study assesses the ability of a novel family of machine learning algorithms to identify changes in relative protein expression levels, measured using 2-D DIGE data, which support accurate class prediction. The analysis was done using a training set of 36 total cellular lysates comprised of six normal and three cancer biological replicates (the remaining are technical replicates) and a validation set of four normal and two cancer samples. Protein samples were separated by 2-D DIGE and expression was quantified using DeCyder-2D Differential Analysis Software. The relative expression reversal (RER) classifier correctly classified 9/9 training biological samples (p<0.022) as estimated using a modified version of leave one out cross validation and 6/6 validation samples. The classification rule involved comparison of expression levels for a single pair of protein spots, tropomyosin isoforms and ,-enolase, both of which have prior association as potential biomarkers in cancer. The data was also analyzed using algorithms similar to those found in the extended data analysis package of DeCyder software. We propose that by accounting for sources of within- and between-gel variation, RER classifiers applied to 2-D DIGE data provide a useful approach for identifying biomarkers that discriminate among protein samples of interest. [source] Proteomic study of human hepatocellular carcinoma using two-dimensional difference gel electrophoresis with saturation cysteine dyePROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 5 2005Kazuyasu Fujii Abstract To identify the proteomic alterations associated with carcinogenesis of hepatocellular carcinoma (HCC), we compared the protein expression profiles of nine HCC cell lines with those of primary cultured hepatocytes established from five individuals. A differential proteomic study was performed by two-dimensional difference gel electrophoresis, in which protein samples are labeled with different fluorescent dyes and separated according to their isoelectric point and molecular weight. To label the protein samples, we used a newly developed and highly sensitive fluorescent dye, which reacts with all reduced cysteine residues of proteins. Principal component analysis based on the intensity of 1238,protein spots indicated that the HCC cells and the normal hepatocytes had distinct proteomic profiles. The Wilcoxon test was used to determine the protein spots whose intensity was differentially regulated in the HCC cells compared with the normal hepatocytes, and mass spectrometric analysis was used to identify the proteins corresponding to the spots. The proteins identified are involved in cell cycle regulation, binding to a tumor-suppressor gene product, fatty acid binding, and regulation of translation. Western blotting with specific antibodies revealed the overexpression of PCNA, EB1 and E-FABP in HCC tissues compared with noncancerous tissues. Aberrant regulation of EB1 and E-FABP has not previously been implicated in the development of HCC. [source] Dihydropyrimidinase related protein-2 as a biomarker for temperature and time dependent post mortem changes in the mouse brain proteomePROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 10 2003Bo Franzén Abstract Proteome analysis in the central nervous system area represents a large and important challenge in drug discovery. One major problem is to obtain representative and well characterized tissues of high quality for analysis. We have used brain tissues from normal mice to study the effect of post mortem time (up to 32 h) and temperature (4°C and room temperature) on protein expression patterns. A number of proteins were identified using mass spectrometry and potential markers were localized. One of the proteins identified, dihydropyrimidinase related protein-2 (DRP-2), occurs as multiple spots in two-dimensional electrophoresis gels. The ratio between the truncated form of DRP-2 (fDRP-2) and full length DRP-2 is suggested as an internal control that can be used as a biomarker of post mortem time and post mortem temperature between unrelated brain protein samples. Results of this study may be useful in future efforts to detect disease specific alterations in proteomic studies of human post mortem brain tissues. [source] Multiple approaches to data-mining of proteomic data based on statistical and pattern classification methodsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 9 2003Jacob W. Tatay Abstract The data-mining challenge presented is composed of two fundamental problems. Problem one is the separation of forty-one subjects into two classifications based on the data produced by the mass spectrometry of protein samples from each subject. Problem two is to find the specific differences between protein expression data of two sets of subjects. In each problem, one group of subjects has a disease, while the other group is nondiseased. Each problem was approached with the intent to introduce a new and potentially useful tool to analyze protein expression from mass spectrometry data. A variety of methodologies, both conventional and nonconventional were used in the analysis of these problems. The results presented show both overlap and discrepancies. What is important is the breadth of the techniques and the future direction this analysis will create. [source] Powder crystallography on macromoleculesACTA CRYSTALLOGRAPHICA SECTION A, Issue 1 2008I. Margiolaki Following the seminal work of Von Dreele, powder X-ray diffraction studies on proteins are being established as a valuable complementary technique to single-crystal measurements. A wide range of small proteins have been found to give synchrotron powder diffraction profiles where the peak widths are essentially limited only by the instrumental resolution. The rich information contained in these profiles, combined with developments in data analysis, has stimulated research and development to apply the powder technique to microcrystalline protein samples. In the present work, progress in using powder diffraction for macromolecular crystallography is reported. [source] Cysteine-reactive covalent capture tags for enrichment of cysteine-containing peptidesRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 21 2009Priscille Giron Considering the tremendous complexity and the wide dynamic range of protein samples from biological origin and their proteolytic peptide mixtures, proteomics largely requires simplification strategies. One common approach to reduce sample complexity is to target a particular amino acid in proteins or peptides, such as cysteine (Cys), with chemical tags in order to reduce the analysis to a subset of the whole proteome. The present work describes the synthesis and the use of two new cysteinyl tags, so-called cysteine-reactive covalent capture tags (C3T), for the isolation of Cys-containing peptides. These bifunctional molecules were specifically designed to react with cysteines through iodoacetyl and acryloyl moieties and permit efficient selection of the tagged peptides. To do so, a thioproline was chosen as the isolating group to form, after a deprotection/activation step, a thiazolidine with an aldehyde resin by the covalent capture (CC) method. The applicability of the enrichment strategy was demonstrated on small synthetic peptides as well as on peptides derived from digested proteins. Mass spectrometric (MS) analysis and tandem mass spectrometric (MS/MS) sequencing confirmed the efficient and straightforward selection of the cysteine-containing peptides. The combination of C3T and CC methods provides an effective alternative to reduce sample complexity and access low abundance proteins. Copyright © 2009 John Wiley & Sons, Ltd. [source] | |||||||||||||||||||||||||||||||