Abstract
Standard proteomics methods allow the relative quantitation of levels of thousands of proteins in two or more samples. While such methods are invaluable for defining the variations in protein concentrations which follow the perturbation of a biological system, they do not offer information on the mechanisms underlying such changes. Expanding on previous work [1], we developed a pulse-chase (pc) variant of SILAC (stable isotope labeling by amino acids in cell culture). pcSILAC can quantitate in one experiment and for two conditions the relative levels of proteins newly synthesized in a given time as well as the relative levels of remaining preexisting proteins. We validated the method studying the drug-mediated inhibition of the Hsp90 molecular chaperone, which is known to lead to increased synthesis of stress response proteins as well as the increased decay of Hsp90 “clients”. We showed that pcSILAC can give information on changes in global cellular proteostasis induced by treatment with the inhibitor, which are normally not captured by standard relative quantitation techniques. Furthermore, we have developed a mathematical model and computational framework that uses pcSILAC data to determine degradation constants kd and synthesis rates Vs for proteins in both control and drug-treated cells. The results show that Hsp90 inhibition induced a generalized slowdown of protein synthesis and an increase in protein decay. Treatment with the inhibitor also resulted in widespread protein-specific changes in relative synthesis rates, together with variations in protein decay rates. The latter were more restricted to individual proteins or protein families than the variations in synthesis. Our results establish pcSILAC as a viable workflow for the mechanistic dissection of changes in the proteome which follow perturbations. Data are available via ProteomeXchange with identifier PXD000538.
Highlights
Monitoring protein abundances and their evolution throughout biological processes is critical for understanding the mechanisms by which cellular functions are achieved
Stable Isotope labeled amino acids were included in the ‘heavy-pcSILAC PEM’ and in the ‘medium-pcSILAC PEM’ (13C6-L-arginine) at 100 mg/l, whereas proline was supplied at 180 mg/l, that is a 9-fold excess over its standard concentration in RPMI medium
The application of this concept using stable isotope labeled amino acids and MS detection has resulted in the pulsed SILAC strategy, which can measure the amount of newly synthesized protein accumulating after a medium exchange or, more precisely, the ratio new/old protein at a given time
Summary
Monitoring protein abundances and their evolution throughout biological processes is critical for understanding the mechanisms by which cellular functions are achieved. The required protein abundances in the cell are maintained through a series of concerted and very tightly regulated processes, including DNA transcription [2], RNA processing and degradation [3] and translation [4] [5], through to the modification [6], localization and degradation (reviewed in [7]) of the protein products. A dynamic balance of protein abundances is accomplished as a result of the coordinated control of all these processes. Strategies for measuring how these processes contribute to attaining an overall dynamic balance of protein abundances have evolved from the quantitation of mRNAs to the direct estimation of protein levels on a high-throughput scale using quantitative proteomics. It is accepted that posttranscriptional and translational control can lead to a poor correlation between mRNA and protein abundances (reviewed in [8,9])
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