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

Abstract

Two-dimensional gel electrophoresis of protein fractions isolated from35 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™ 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.Molecular & Cellular Proteomics 1: 528 -537, 2002.The ability to monitor variations at the transcriptional and translational levels using DNA microarrays and proteomics is essential to improve our understanding of physiologically relevant processes at a molecular level (1). Current DNA array technologies allow quantitative high throughput analysis of many thousands genes simultaneously. Yet, this analysis is rather confined to steady state mRNA levels, and mRNA abundance does not necessarily reflect the levels of corresponding proteins (2, 3). Protein levels are regulated by posttranscriptional mechanisms including transcript localization (4) and stability (5), translational regulation (6, 7), and protein degradation (8). Therefore, methods to simultaneously determine levels and turnover of the cellular proteins remain an urgent necessity. However, current proteome techniques still lag behind in quantitative high throughput analysis as compared with DNA arrays.A variety of methods have been developed to quantitatively monitor the protein expression profile of cells. Although protein chips and encoded particle-based solution arrays are particularly encouraging as the next generation proteomics platforms (9, 10), current techniques still largely rely on separation o...

A proteomic method for the analysis of changes in protein concentrations in response to systemic perturbations using metabolic incorporation of stable isotopes and mass spectrometry

“…An alternative explanation for this observation could involve PTM of a different form of Hsp70. Using radiolabeling and fluorescence detection of proteins separated by 2-DE, the synthesis rate of Hsp70 was, in contradiction to our result, found to increase and to correlate with the accumulating amount of Hsp70 after a short heat shock treatment of U937 cells [14]. These seemingly contradictory results are most likely due to the significantly different experimental protocols used for heat stress treatment between these two studies.…”

“…One culture was grown in a medium highly enriched in 15 N, resulting in metabolic incorporation of the stable isotope into all proteins, while the other culture was grown in a medium with the natural relative abundance of the 14 N isotope (99.6%). Protein extracts from the two cell cultures were mixed and, after protein separation and proteolytic digestion, analyzed by MS. Based on the signal intensity ratios of the 15 N-and 14 N-incorporated tryptic peptides, the relative abundance of each analyzed protein could be determined with high precision.…”

Monitoring chaperone engagement of substrates in the endoplasmic reticulum of live cells

“…These experiments lead to a view of Crt as a highly dynamic chaperone and the ER as a highly robust folding environment. Despite high concentrations of numerous maturation enzymes (9,38), Crt remained highly mobile. Even when a population of nascent substrate is effectively immobilized (with Chx; see Fig.…”

Post-transcriptional Expression Regulation in the Yeast Saccharomyces cerevisiae on a Genomic Scale

“…In order to improve the precision of PHDs, independent experimental verification of protein levels is particularly important. Recently, other means of measuring protein turnover rates at a larger scale have been suggested (14,18,31), but up to now no half-life dataset of the size presented here has been published.…”

“…Finally, we demonstrate the possibility to calculate a PHD, which relates the steady-state protein level to the synthesis rate (18). We had to combine measurements from different laboratories, where growth conditions might not always be identical.…”