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Gel Bands (gel + bands)
Selected AbstractsThe Identification of an Adenovirus Receptor by Using Affinity Capture and Mass SpectrometryCHEMBIOCHEM, Issue 8 2004Sunia A. Trauger Dr. Abstract A tandem mass spectrometry-based approach is demonstrated for detecting a receptor for Ad37, one of the causative agents for epidemic keratoconjunctivitis. Partial purification of membrane glycoproteins was performed by using lectin-affinity chromatography and SDS-PAGE. Gel bands that were shown to bind Ad37 by using Viral Overlay Protein Blot Assay (VOPBA) were excised, proteolyzed and analyzed by using nanoLC-MS/MS to identify putative receptors contained in a mixture of proteins. Four candidate receptors were identified among approximately 50 proteins based on a search against a protein database. Inhibition of gene delivery mediated by an Ad37 vector, with antibodies against the glycoproteins identified by tandem mass spectrometry, strongly indicated that Membrane Cofactor Protein (MCP), a member of the complement regulatory family of proteins, is the receptor. This rapid and sensitive MS/MS-based strategy is perceived to have wide potential applications for the detection of viral receptors. [source] Identification of rat urinary glycoproteome captured by three lectins using gel and LC-based proteomicsELECTROPHORESIS, Issue 21 2008Pyong-Gon Moon Abstract Many different types of urine proteome studies have been done, but urine glycoprotein studies are insufficient. Therefore, we studied the glycoproteins from rat urine, which could be used to identify biomarkers in an animal model. First, urinary proteins were prepared by using the dialysis and lyophilizing methods from rat urine. Glycoproteins enriched with lectin affinity purification, concanavalin A, jacalin and wheat germ agglutinin from the urinary proteins were separated by means of reverse-phase fast protein LC (FPLC) or 1-D PAGE. Each FPLC fraction and 1-D PAGE gel band were trypsin-digested and analyzed by means of nanoLC-MS/MS. LC-MS/MS analyses were carried out by using linear ion trap MS. A total of 318 rat urinary glycoproteins were identified from the FPLC fractions and gel bands; approximately 90% of identified proteins were confirmed as glycoproteins in Swiss-Prot. Many glycoproteins, known as biomarkers, including C-reactive protein, uromodulin, amyloid beta A4 protein, alpha-1-inhibitor 3, vitamin D-binding protein, kallikrein 3 and fetuin-A were identified in this study. By studying urinary glycoproteins collected from rat, these results may help to assist in identifying urinary biomarkers regarding various types of disease models. [source] Altered expression of transcripts for ,-tubulin and an unidentified gene in the spinal cord of phenyl saligenin phosphate treated hens (Gallus gallus)JOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 5 2003Jonathan H. Fox Abstract Phenyl saligenin phosphate (PSP) induces a central-peripheral distal axonopathy in domestic fowl that develops 7,21 days after a single exposure. Neurotoxic esterase (NTE) is the initial molecular target for this neurotoxicity. PSP has to covalently bind to NTE and chemically "age" for induction of axonopathy. It was hypothesized that exposure to PSP results in early changes in spinal cord gene expression that do not occur with phenylmethylsulfonyl fluoride, a non-neuropathic compound that also inhibits NTE, or DMSO controls. Targeted display was used to screen ,15,000 gel bands. Three candidate genes were identified, but only the transcript designated P1 showed decreased expression following PSP exposure (2 mg/kg i.m.) in subsequent Northern blot and in situ hybridization experiments in samples taken <48 h after exposure. Additional experiments revealed that a ,2.5 kb ,-tubulin transcript had decreased expression at 12,48 h after PSP exposure, with maximum change at 48 h (33%, p = 0.0479). A ,4.5 kb ,-tubulin transcript had increased expression at 12 h (38%, p = 0.0125) and decreased expression at 48 h (28%, p = 0.0576). In situ hybridization on spinal cord revealed neuronal expression of P1 and ,-tubulin transcripts. Decreased expression of transcripts for P1 and ,-tubulin was present at 12 and 48 h, respectively. This decrease occurred in all neurons, not just those whose axons degenerate. Results suggest that (1) in PSP-induced OPIDN (organophosphorus-induced delayed neurotoxicity) some gene transcript expression changes are associated with initiation of axonopathy, and (2) PSP modulates spinal cord gene expression in neuronal types that do not undergo axonal degeneration. © 2003 Wiley Periodicals, Inc. J Biochem Mol Toxicol 17:263,271, 2003; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.10088 [source] PROTEOLYTIC ACTIVITY OF LACTOBACILLUS SAKEI, LACTOBACILLUS FARCIMINIS AND LACTOBACILLUS PLANTARUM ON SARCOPLASMIC PROTEINS OF PORK LEANJOURNAL OF FOOD BIOCHEMISTRY, Issue 3 2004ANNA LISA BASSO The aim of this study was to assess the proteolytic activity of Lactobacillus sakei (DSM 6333), L. plantarum (B21), and to a lesser extent, L. farciminis (DSM 20184) on meat sarcoplasmic proteins. The protein composition was assayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and capillary electrophoresis after incubation of meat extract inoculated with bacteria. All strains showed proteolytic activity: a band about 94 kDa disappeared in samples inoculated with L. farciminis and L. plantarum and strongly decreased in those inoculated with L. sakei. The intensity of the bands with a molecular weight between 94 and 38 kDa decreased in all samples. Capillary electrophoresis analysis ascertained the disappearance of the fractions corresponding to 8.64 and 8.66 min retention time in all samples. The bands corresponding to 94 kDa and 38 kDa were, respectively, identified as glycogen phosphorylase muscle isoform and glyceraldehyde 3-phosphate dehydrogenase, by in situ digestion of protein gel bands and peptide map analysis using Matrix Assisted Laser Desorption/Ionization - Time of Flight Mass Spectrometry (MALDI-TOF MS). [source] Island clustering analysis for the comparison of the membrane and the soluble protein fractions of human brain proteomePROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 6 2008Kyung-Hoon Kwon Abstract A protein identified in multiple separate bands of a 1-D gel reflects variation in the molecular weight caused by alternative splicing, endoproteolytic cleavage, or PTMs, such as glycosylation or ubiquitination. To characterize such a protein distribution over the bands, we defined an entity called an ,island' as the band region including the bands of the same protein identified sequentially. We quantified the island distribution using a new variable called an Iscore. Previously, as described in Park et al.. (Proteomics 2006, 6, 4978,4986.), we analyzed human brain tissue using a multidimensional MS/MS separation method. Here, the new method of island analysis was applied to the previous proteome data. The soluble and membrane protein fractions of human brain tissue were reanalyzed using the island distribution. The proteome of the soluble fraction exhibited more variation in island positions than that of the membrane fraction. Through the island analysis, we identified protein modifications and protein complexes over the 1-D gel bands. [source] Strategic shotgun proteomics approach for efficient construction of an expression map of targeted protein families in hepatoma cell linesPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 12 2003Chih-Lei Lee Abstract An expression map of the most abundant proteins in human hepatoma HepG2 cells was established by a combination of complementary shotgun proteomics approaches. Two-dimensional liquid chromatography (LC)-nano electrospray ionization (ESI) tandem mass spectrometry (MS/MS) as well as one-dimensional LC-matrix-assisted laser desorption/ionization MS/MS were evaluated and shown that additional separation introduced at the peptide level was not as efficient as simple prefractionation of protein extracts in extending the range and total number of proteins identified. Direct LC-nanoESI MS/MS analyses of peptides from total solubilized fraction and the excised gel bands from one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis fractionated insolubilized fraction afforded the best combination in efficient construction of a nonredundant cell map. Compiling data from multiple variations of rapid shotgun proteomics analyses is nonetheless useful to increase sequence coverage and confidence of hits especially for those proteins identified primarily by a single or two peptide matches. While the returned hit score in general reflects the abundance of the respective proteins, it is not a reliable index for differential expression. Using another closely related hepatoma Hep3B as a comparative basis, 16 proteins with more than two-fold difference in expression level as defined by spot intensity in two-dimensional gel electrophoresis analysis were identified which notably include members of the heat shock protein (Hsp) and heterogeneous nuclear ribonucleoprotein (hnRPN) families. The observed higher expression level of hnRNP A2/B1 and Hsp90 in Hep3B led to a search for reported functional roles mediated in concert by both these multifunctional cellular chaperones. In agreement with the proposed model for telomerase and telomere bound proteins in promoting their interactions, data was obtained which demonstrated that the expression proteomics data could be correlated with longer telomeric length in tumorigenic Hep3B. This biological significance constitutes the basis for further delineation of the dynamic interactions and modifications of the two protein families and demonstrated how proteomic and biological investigation could be mutually substantiated in a productive cycle of hypothesis and pattern driven research. [source] Nano-high-performance liquid chromatography in combination with nano-electrospray ionization Fourier transform ion-cyclotron resonance mass spectrometry for proteome analysisRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 12 2003Christian Ihling Fourier transform ion-cyclotron resonance (FTICR) mass spectrometry offers several advantages for the analysis of biological samples, including excellent mass resolution, ultra-high mass measurement accuracy, high sensitivity, and wide mass range. We report the application of a nano-HPLC system coupled to an FTICR mass spectrometer equipped with nanoelectrospray source (nano-HPLC/nano-ESI-FTICRMS) for proteome analysis. Protein identification in proteomics is usually conducted by accurately determining peptide masses resulting from enzymatic protein digests and comparing them with theoretically digested protein sequences from databases. A tryptic in-solution digest of bovine serum albumin was used to optimize experimental conditions and data processing. Spots from Coomassie Blue and silver-stained two-dimensional (2D) gels of human thyroid tissue were excised, in-gel digested with trypsin, and subsequently analyzed by nano-HPLC/nano-ESI-FTICRMS. Additionally, we analyzed 1D-gel bands of membrane preparations of COS-6 cells from African green monkey kidney as an example of more complex protein mixtures. Nano-HPLC was performed using 1-mm reverse-phase C-18 columns for pre-concentration of the samples and reverse-phase C-18 capillary columns for separation, applying water/acetonitrile gradient elution conditions at flow rates of 200,nL/min. Mass measurement accuracies smaller than 3,ppm were routinely obtained. Different methods for processing the raw data were compared in order to identify a maximum number of peptides with the highest possible degree of automation. Parallel identification of proteins from complex mixtures down to low-femtomole levels makes nano-HPLC/nano-ESI-FTICRMS an attractive approach for proteome analysis. Copyright © 2003 John Wiley & Sons, Ltd. [source] Placental endothelial nitric oxide synthase localization and expression in normal human pregnancy and pre-eclampsiaCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 5-6 2003SJ Orange Summary 1.,The aim of the present study was to investigate whether pre-eclampisa, a state of placental hypoxia, is associated with placental abnormalities in the amount, distribution and expression of enothelial nitric oxide synthase (eNOS). 2.,Localization and intensity of eNOS was determined by immunohistochemistry using an antibody specific for eNOS. The amount of eNOS mRNA expression was determined by reverse transcription,polymerase chain reaction (RT-PCR) and the densitometry of gel bands was expressed as a ratio of the band density of the housekeeping gene ,2 -microglobulin. 3.,Endothelial NOS staining was localized to syncytiotrophoblast cells within the villi and decidual trophoblast cells. It was not present in the endothelium of terminal villous vessels. There was no significant difference in eNOS villous or decidual staining intensity between normal pregnancy (NP; n = 12), pre-eclampsia (n = 14), or gestational hypertension (GH; n = 4). Staining for eNOS was not significantly different in the decidua compared with the villi in NP, GH or pre-eclampsia. Within the decidua, the depth of eNOS staining was similar in NP, pre-eclampisa and GH. 4.,There was no significant difference in eNOS mRNA expression between NP (0.70 ± 0.11), pre-eclampsia (0.5 ± 0.07) or GH (0.69 ± 0.26). 5.,These findings suggest that the amount of eNOS in the placenta is not deficient in pre-eclampsia, excluding a possible pathogenic role for eNOS in this disease. Furthermore, placental hypoxia, which is associated with pre-eclampsia, did not induce an upregulation of eNOS [source] | ||||||||||