Proteome Profiling (proteome + profiling)

Distribution by Scientific Domains


Selected Abstracts


Indications for cell stress in response to adenoviral and baculoviral gene transfer observed by proteome profiling of human cancer cells

ELECTROPHORESIS, Issue 11 2010
Christopher Gerner
Abstract Gene transfer to cultured cells is an important tool for functional studies in many areas of biomedical research and vector systems derived from adenoviruses and baculoviruses are frequently used for this purpose. In order to characterize how viral gene transfer vectors affect the functional state of transduced cells, we applied 2-D PAGE allowing quantitative determination of protein amounts and synthesis rates of metabolically labeled cells and shotgun proteomics. Using HepG2 human hepatoma cells we show that both vector types can achieve efficient expression of green fluorescent protein, which accounted for about 0.1% of total cellular protein synthesis 72,h after transduction. No evidence in contrast was found for expression of proteins from the viral backbones. With respect to the host cell response, both vectors induced a general increase in protein synthesis of about 50%, which was independent of green fluorescent protein expression. 2-D PAGE autoradiographs identified a 3.6-fold increase of ,-actin synthesis in adenovirus transduced cells. In addition shotgun proteomics of cytoplasmic and nuclear extract fractions identified a slight induction of several proteins related to inflammatory activation, cell survival and chromatin function by both virus types. These data demonstrate that commonly used gene transfer vectors induce a response reminiscent of stress activation in host cells, which needs to be taken into account when performing functional assays with transduced cells. [source]


Comprehensive proteome analysis of mouse liver by ampholyte-free liquid-phase isoelectric focusing

ELECTROPHORESIS, Issue 11 2008
Hua Zhong
Abstract In this study, ampholyte-free liquid-phase IEF (LIEF) was combined with narrow pH range 2-DE and SDS-PAGE RP-HPLC for comprehensive analysis of mouse liver proteome. Because LIEF prefractionation was able to reduce the complexity of the sample and enhance the loading capacity of IEF strips, the number of visible protein spots on subsequent 2-DE gels was significantly increased. A total of 6271 protein spots were detected after integrating five narrow pH range 2-DE gels following LIEF prefractionation into a single virtual 2-DE gel. Furthermore, the pH,3,5 LIEF fraction and the unfractionated sample were separated by pH,3,6 2-DE and identified by MALDI-TOF/TOF MS, respectively. In parallel, the pH 3,5 LIEF fraction was also analyzed by SDS-PAGE RP-HPLC MS/MS. LIEF-2-DE and LIEF-HPLC could obviously improve the separation efficiency and the confidence of protein identification, which identified a higher number of low-abundance proteins and proteins with extreme physicochemical characteristics or post-translational modifications compared to conventional 2-DE method. Furthermore, there were 207 proteins newly identified in mouse liver in comparison with previously reported large-scale datasets. It was observed that the combination of LIEF-2-DE and LIEF-HPLC was effective in promoting MS-based liver proteome profiling and could be applied on similar complex tissue samples. [source]


The proteome of Mannheimia succiniciproducens, a capnophilic rumen bacterium

PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 12 2006
Jeong Wook Lee
Abstract Mannheimia succiniciproducens MBEL55E isolated from bovine rumen is an industrially important bacterium as an efficient succinic acid producer. Recently, its full genome sequence was determined. In the present study, we analyzed the M. succiniciproducens proteome based on the genome information using 2-DE and MS. We established proteome reference map of M. succiniciproducens by analyzing whole cellular proteins, membrane proteins, and secreted proteins. More than 200 proteins were identified and characterized by MS/MS supported by various bioinformatic tools. The presence of proteins previously annotated as hypothetical proteins or proteins having putative functions were also confirmed. Based on the proteome reference map, cells in the different growth phases were analyzed at the proteome level. Comparative proteome profiling revealed valuable information to understand physiological changes during growth, and subsequently suggested target genes to be manipulated for the strain improvement. [source]


Transforming growth factor-,1-regulated proteins in human endothelial cells identified by two-dimensional gel electrophoresis and mass spectrometry

PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 4 2004
Marta Lomnytska
Abstract Transforming growth factor-, (TGF,) is a potent regulator of angiogenesis affecting proliferation, differentiation and migration of endothelial cells. The effect of TGF, on endothelial cells depends on the origin of the cells and on the experimental conditions. Global analysis of TGF, signalling is expected to unveil mechanisms of this variability and identify novel targets of the growth factor. Here, we report proteome profiling of human microvascular endothelial cells obtained from dermis, which were treated with TGF,1 and compared to nontreated cells. We identified 54 proteins affected by TGF,1 using two-dimensional gel electrophoresis and peptide mass fingerprinting. Thirteen of the identified proteins are involved in various signalling processes. Seven proteins are involved in cytoskeleton rearrangements and six are involved in regulation of metabolism. Ten proteins were identical to predicted hypothetical proteins with no assigned functions. In agreement with the effect of TGF,1 on components of the cytoskeleton, TGF,1 induces actin cytoskeleton rearrangements. TGF,1 also affected expression of E2F6, p57Kip2, G(q),, hnRNP A1 and myosin light chain proteins as shown by immunoblotting. Down-regulation of the transcriptional repressor E2F6 by TGF,1 correlated with a weak growth-inhibitory activity of TGF,1 on HMVEC-d cells. Twenty-five of the identified proteins have not previously been described as being regulated by TGF,1, providing new insights into TGF,1 signalling in endothelial cells. [source]


High-resolution biomarker discovery: Moving from large-scale proteome profiling to quantitative validation of lead candidates

PROTEOMICS - CLINICAL APPLICATIONS, Issue 10-11 2008
Johannes A. Hewel
Abstract Diverse proteomic techniques based on protein MS have been introduced to systematically characterize protein perturbations associated with disease. Progress in clinical proteomics is essential for personalized medicine, wherein treatments will be tailored to individual needs based on patient stratification using noninvasive disease monitoring procedures to reveal the most appropriate therapeutic targets. However, breakthroughs await the successful development and application of a robust proteomic pipeline capable of identifying and rigorously assessing the relevance of multiple candidate proteins as informative diagnostic and prognostic indicators or suitable drug targets involved in a pathological process. While steady progress has been made toward more comprehensive proteome profiling, the emphasis must now shift from in depth screening of reference samples to stringent quantitative validation of selected lead candidates in a broader clinical context. Here, we present an overview of the emerging proteomic strategies for high-throughput protein detection focused primarily on targeted MS/MS as the basis for biomarker verification in large clinical cohorts. We discuss the conceptual promise and practical pitfalls of these methods in terms of achieving higher dynamic range, higher throughput, and more reliable quantification, highlighting research avenues that merit additional inquiry. [source]


Cancer biomarker discovery via low molecular weight serum proteome profiling , Where is the tumor?

PROTEOMICS - CLINICAL APPLICATIONS, Issue 12 2007
Michael T. Davis
Abstract Time-course analyses of rapidly processed serum performed in parallel by SELDI and nanoscale LC-MS/MS have revealed the temporal correlation of several literature-based disease markers with ex vivo driven events such that their in vivo existence in healthy subjects is questionable. Identification by MS/MS reveals these putative biomarkers to be byproducts of the coagulation cascade and platelet activation and suggests plasmatic analysis may be preferred. In a pilot plasmatic study, a cohort of naïve prostate cancer (PCa) samples were uniformly distinguished from their age-matched controls (n,=,20) on the basis of multiple peptidic components; most notably by a derivative of complement C4 at 1863,m/z (GLEEELQFSLGSKINVK, C41353,1369). The fully tryptic nature of this and other putative PCa discriminants is consistent with the cleavage specificity of common blood proteases and questions the need for tumor-derived proteolytic activities as has been proposed. In light of the known correlation of disregulated hemostasis with malignant disease, we suggest the underlying differentiating phenomena in these types of analyses may lie in the temporal disparity of sample activation such that the case (patient) samples are preactivated while the control samples are not. [source]