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Molecular & Cellular Proteomics 1:528-537, 2002.
© 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Concomitant Determination of Absolute Values of Cellular Protein Amounts, Synthesis Rates, and Turnover Rates by Quantitative Proteome Profiling^*

Christopher Gerner, Susanne Vejda, Dieter Gelbmann, Editha Bayer, Josef Gotzmann, Rolf Schulte-Hermann and Wolfgang Mikulits

From the Institute of Cancer Research, University of Vienna, 1090 Vienna, Austria

Two-dimensional gel electrophoresis of protein fractions isolated from ³⁵S-radiolabeled cells provides qualitative information on intracellular amounts, ³⁵S 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 ³⁵S 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 ³⁵S 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.

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