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Submitted on April 23, 2002
Revised on June 29, 2002
Accepted on July 22, 2002

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

Dept. of Cell Biology, Institute of Cancer Research, University of Vienna, Vienna A-1090

Corresponding Author: Christopher.Gerner{at}univie.ac.at

2D 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 rubyÔ staining and subsequent autoradiography of the same gels, thereby quantifying protein amounts and 35S incorporation. In order to calibrate calculation of absolute synthesis rates, we determined the amount and autoradigraph intensity of radiolabeled haptoglobin secreted by IL-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 2D 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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