Biomedical subjects

Editha Bayer

Publications and source records attributed to Editha Bayer.

4 recordsLinked to original sources

PubMed2006

Knowledge-based proteome profiling: considering identified proteins to evaluate separation efficiency by 2-D PAGE.

Proteome profiling techniques rely on the separation of proteins or peptides and their subsequent quantification. The reliability of this technique is still limited because a proteome profiling result does not necessarily represent the true protein composition of the analysed sample, thus seriously hampering proper data interpretation. Many experimentally observed proteome alterations are biologically not significant. It was the aim of this study to use the knowledge of the biological context of proteins in order to establish optimised proteome profiling protocols. While 2-D spot patterns of total cell protein fractions were found to poorly represent the true protein composition, purified subcellular protein fractions were found to better represent the protein composition of the analysed sample. The application of a standardised protocol to different kinds of cells revealed several striking observations. Firstly, the protein composition of cultured cells of various origins is very similar. Secondly, proteome alterations observed with the described protocols do make sense from a biologic point of view and may thus be considered as truly representative for the analysed samples. Thirdly, primary white blood cells isolated from different donors were found to show minor, but reproducible and significant individual differences. We designate the consideration of known properties of identified proteins in proteome profiles as a knowledge-based approach. The present data suggest that this approach may tremendously help to improve the applied techniques and assess the results. We demonstrate that the fulfilment of well-defined criteria of proteome profiles eventually results in reliable and biologically relevant data.

Amino Acid Sequence↗

PubMed2004

Towards a standardized human proteome database: quantitative proteome profiling of living cells.

Comparative proteome profiling, performed by two-dimensional polyacrylamide gel electrophoresis or multidimensional protein identification technology, usually relies on the relative comparison of samples of interest with respect to a reference. Currently, no standardized quantitative protein expression database of human cells, facilitating data comparisons between different laboratories, exists. Recently, we have published two-dimensional polyacrylamide gel electrophoresis-based techniques to assess absolute protein data comprising protein amounts, synthesis rates and biological half-lives (Mol. Cell. Proteomics 2002, 1, 528-537). Determination of protein amounts by fluorography of two-dimensional gels was followed by the exact quantification of the amount of incorporated (35)S radiolabel. Here we demonstrate an application of this highly standardized method to quiescent human T cells, phythaemagglutinin-stimulated T cells and Jurkat cells, a human T lymphoblast cell line. While the protein composition of quiescent T cells differed significantly compared to that of Jurkat cells, it was only slightly different compared to the activated T cells. Synthesis profile analyses demonstrated that activated T cells clearly differed from the quiescent cells, performing apparently almost like lymphoblast cells. The great sensitivity of this approach was further demonstrated with human umbilical vein endothelial cells treated for six hours with vascular endothelial growth factor. While no significant alteration of protein amounts was detected at all upon activation, the synthesis rate of several proteins was found to be more than doubled.

Autoradiography↗

PubMed2002

Plasma from cancer patients featuring a characteristic protein composition mediates protection against apoptosis.

By comparative proteome analysis we searched for characteristic alterations of human plasma accompanying neoplastic disease. We identified protein alterations in plasma of prostate-, lung-, and breast-cancer patients in comparison to controls, comprising elevated levels of fibrinogen gamma-chain dimer, degradation products of antiplasmin and laminin gamma-chain, and elevated levels of acute phase proteins. The latter proteins and laminin fragments have been described as anti-apoptotic factors. We raised the question whether these alterations may have any relevance for the regulation of apoptosis. In contrast to plasma derived from healthy donors, samples from prostate-, lung-, and breast-cancer patients selectively inhibited Fas- and staurosporine-induced apoptosis in Jurkat cells but remained ineffective upon UV light-induced apoptosis. These data suggested that inhibition occurred by extracellular interference with apoptosis induction. Supporting this hypothesis, we found that formation of the CD95 death-inducing signal complex was strongly inhibited in the presence of plasma from cancer patients.

Apoptosis↗

PubMed2002

Concomitant determination of absolute values of cellular protein amounts, synthesis rates, and turnover rates by quantitative proteome profiling.

Two-dimensional gel electrophoresis of protein fractions isolated from (35)S-radiolabeled cells provides qualitative information on intracellular amounts, (35)S incorporation rates, protein modifications, and subcellular localizations of up to thousands of individual proteins. In this study we extended proteome profiling to provide quantitative data on synthesis rates of individual proteins. We combined fluorescence detection of radiolabeled proteins with SYPRO ruby(TM) staining and subsequent autoradiography of the same gels, thereby quantifying protein amounts and (35)S incorporation. To calibrate calculation of absolute synthesis rates, we determined the amount and autoradiograph intensity of radiolabeled haptoglobin secreted by interleukin-6 pretreated HepG2 cells. This allowed us to obtain a standard calibration value for (35)S incorporation per autoradiograph intensity unit. This value was used to measure protein synthesis rates during time course experiments of heat-shocked U937 cells. We measured the increasing amounts of hsp70 and calculated it by integration of the determined hsp70 synthesis rates over time. Similar results were obtained by both methods, validating our standardization procedure. Based on the assumption that the synthesis rate of proteins in a steady state of cell metabolism would essentially compensate protein degradation, we calculated biological half-lives of proteins from protein amounts and synthesis rates determined from two-dimensional gels. Calculated protein half-lives were found close to those determined by pulse-chase experiments, thus validating this new method. In conclusion, we devised a method to assess quantitative proteome profiles covering determination of individual amounts, synthesis, and turnover rates of proteins.

Cell Fractionation↗