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Moerdyk-Schauwecker M.,University of North Carolina at Charlotte | Hwang S.-I.,Proteomics Laboratory for Clinical and Translational Research | Grdzelishvili V.Z.,University of North Carolina at Charlotte
PLoS ONE | Year: 2014

Virus particles (virions) often contain not only virus-encoded but also host-encoded proteins. Some of these host proteins are enclosed within the virion structure, while others, in the case of enveloped viruses, are embedded in the host-derived membrane. While many of these host protein incorporations are likely accidental, some may play a role in virus infectivity, replication and/or immunoreactivity in the next host. Host protein incorporations may be especially important in therapeutic applications where large numbers of virus particles are administered. Vesicular stomatitis virus (VSV) is the prototypic rhabdovirus and a candidate vaccine, gene therapy and oncolytic vector. Using mass spectrometry, we previously examined cell type dependent host protein content of VSV virions using intact ("whole") virions purified from three cell lines originating from different species. Here we aimed to determine the localization of host proteins within the VSV virions by analyzing: i) whole VSV virions; and ii) whole VSV virions treated with Proteinase K to remove all proteins outside the viral envelope. A total of 257 proteins were identified, with 181 identified in whole virions and 183 identified in Proteinase K treated virions. Most of these proteins have not been previously shown to be associated with VSV. Functional enrichment analysis indicated the most overrepresented categories were proteins associated with vesicles, vesicle-mediated transport and protein localization. Using western blotting, the presence of several host proteins, including some not previously shown in association with VSV (such as Yes1, Prl1 and Ddx3y), was confirmed and their relative quantities in various virion fractions determined. Our study provides a valuable inventory of virion-associated host proteins for further investigation of their roles in the replication cycle, pathogenesis and immunoreactivity of VSV. © 2014 Moerdyk-Schauwecker et al. Source

Lee J.-G.,Proteomics Laboratory for Clinical and Translational Research | McKinney K.Q.,Proteomics Laboratory for Clinical and Translational Research | Lee Y.-Y.,Proteomics Laboratory for Clinical and Translational Research | Chung H.-N.,Proteomics Laboratory for Clinical and Translational Research | And 6 more authors.
PLoS ONE | Year: 2015

Though the rhesus monkey is one of the most valuable non-human primate animal models for various human diseases because of its manageable size and genetic and proteomic similarities with humans, proteomic research using rhesus monkeys still remains challenging due to the lack of a complete protein sequence database and effective strategy. To investigate the most effective and high-throughput proteomic strategy, comparative data analysis was performed employing various protein databases and search engines. The UniProt databases of monkey, human, bovine, rat and mouse were used for the comparative analysis and also a universal database with all protein sequences from all available species was tested. At the same time, de novo sequencing was compared to the SEQUEST search algorithm to identify an optimal work flow for monkey proteomics. Employing the most effective strategy, proteomic profiling of monkey organs identified 3,481 proteins at 0.5% FDR from 9 male and 10 female tissues in an automated, high-throughput manner. Data are available via ProteomeXchange with identifier PXD001972. Based on the success of this alternative interpretation of MS data, the list of proteins identified from 12 organs of male and female subjects will benefit future rhesus monkey proteome research. © 2015 Lee et al. Source

Mckinney K.Q.,Proteomics Laboratory for Clinical and Translational Research | Lee J.-G.,Proteomics Laboratory for Clinical and Translational Research | Sindram D.,Liver Biliary Pancreatic Center | Russo M.W.,Liver Biliary Pancreatic Center | And 4 more authors.
Cancer Genomics and Proteomics | Year: 2012

Pancreatic cancer is an aggressive disease with nearly equal yearly rates of diagnosis and death. Current therapies have failed to improve outcomes due to rapid disease progression and late stage at presentation. Recently, pathways involved in progression and metastasis have been elucidated; however, new knowledge has not generated more effective therapies. We report on the use of subcellular fractionation and liquid chromatography (LC)-mass spectrometry to identify 3,907 proteins in four pancreatic cancer cell lines, 540 of which are unique to primary cancer cells, and 487 unique to cells derived from metastatic sites. Statistical analysis identified 134 proteins significantly differentially expressed between the two populations. The subcellular localization of these proteins was determined and expression levels for four targets were validated using western blot techniques. These identified proteins can be further investigated to determine their roles in progression and metastasis and may serve as therapeutic targets in the development of more effective treatments for pancreatic cancer. Source

Lee J.-G.,Proteomics Laboratory for Clinical and Translational Research | McKinney K.Q.,Proteomics Laboratory for Clinical and Translational Research | Pavlopoulos A.J.,Proteomics Laboratory for Clinical and Translational Research | Park J.-H.,Seoul National University | Hwang S.,Proteomics Laboratory for Clinical and Translational Research
Journal of Proteomics | Year: 2015

Therapeutic strategies for cancer treatment often remain challenging due to the cumulative risk derived from metastasis, which has been described as an aggressive state of cancer cell proliferation often resulting in failure of clinical therapy. In the current study, anti-metastatic properties of three chemotherapeutic drugs and three compounds from natural sources were investigated by comparative proteomic analysis. Proteomic profile comparison of the isogenic primary and metastatic colon cancer cell lines SW480 and SW620 identified two potential metastasis related molecular targets: fatty acid synthase and histone H4. To demonstrate their biological roles in cancer metastasis, the expression of these target genes was suppressed by siRNA transfection. Subsequent cell migration assays demonstrated reduced migratory effects. SW620 cells were treated with six anti-cancerous components. Through comprehensive proteomic analysis, three of the tested compounds, oxaliplatin, ginsenoside 20(S)-Rg3 and curcumin, were revealed to have a suppressive effect on FASN and histone H4 expression. SW620 cells treated with these drugs showed significantly reduced migratory activity, which suggests that drug-induced targeted suppression of these genes may affect cell migration. The validity of the proteomic datasets was verified by knowledgebase pathway analysis and immunoblotting assays. The anti-metastatic components revealed by the current proteomic analysis represent promising chemotherapeutic candidates for the treatment of colorectal adenocarcinoma. Biological significance The current study demonstrates anti-metastatic activity of chemotherapeutics and natural components by the suppression of target molecules, fatty acid synthase and histone H4 identified by a comparative proteomic analysis employing the isogenic primary andmetastatic colon cancer cell lines,SW480 and SW620. Three tested drugs, namely, oxaliplatin, ginsenoside 20(S)-Rg3 and curcumin were revealed to possess suppressive effects on fatty acid synthase and histone H4 and reduce metastasis as determined by cell migration assay. Data were confirmed by the correlation between spectral counts from proteomic data and Western blot analysis, which were in good agreement with immunohistochemistry. © 2014. Source

Watts J.A.,Carolinas Medical Center | Lee Y.-Y.,Proteomics Laboratory for Clinical and Translational Research | Gellar M.A.,Carolinas Medical Center | Fulkerson M.-B.K.,Carolinas Medical Center | And 2 more authors.
Thrombosis Research | Year: 2012

Introduction: Microparticles (MPs) are small fragments of apoptotic or activated cells that may contribute to pathological processes in cardiovascular diseases. In studies of MPs in clinical cohorts, it is unclear if observed changes in MP composition are a cause or a result of the cardiovascular disease being studied. The present studies employed a well-characterized rat model of experimental pulmonary embolism (PE) to determine if there were changes in MP characteristics as a result of pulmonary vascular occlusion. Methods: PE was produced by infusing 25 μm polystyrene microspheres into the jugular vein of anesthetized rats. MPs were isolated by differential centrifugation of arterial blood 18 hr after PE. Proteins were separated by 1D gel electrophoresis and identified from tryptic digests by ultraperformance liquid chromatography (UPLC) coupled with tandem mass spectrometry. Statistical analysis was conducted using the Power Law Global Error Model (PLGEM). Changes in two proteins were confirmed by Western blot. Results: Experimental PE produced pulmonary hypertension, mild systemic hypotension, hypoxia, hypercapnia and lactic acidosis. MPs showed significant elevation in proteins involved in clotting (fibronectin precursor, fibrinogen alpha, beta and gamma and von Willebrand factor) and several macroglobulin proteins, such as alpha-2-macroglobulin precursor compared with vehicle-treated control rats. Consistent with recent observations of hemolysis in PE, haptoglobin precursor protein, a major protein of hemoglobin clearance, decreased significantly in the PE animals. Plasma d-Dimer concentrations were significantly elevated, indicating that experimental PE produced a pro-coagulant state. Conclusions: These findings suggest that experimental PE produced significant, changes in MP characteristics to a prothrombotic phenotype. © 2012 Elsevier Ltd. All rights reserved. Source

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