ABSTRACT: We developed a pulse-chase variant of SILAC (stable isotope labeling by amino acids in cell culture pcSILAC). 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. Methods : two cultures of Jurkat cells to be compared were fully labeled with, respectively, “medium” (13C6-L-arginine, R6) and “heavy” (13C615N4-L-arginine, R10) Arg, while Lys was left unlabeled (“light”, K0) for both. At the start of the experiment, both cultures were transferred to new media containing light Arg (R0) to perform the chase of R-labeled pre-existing proteins. In turn, the new media contained, respectively, “medium” (“M”, 2H4-Lysine, K4) and “heavy” (“H”, 13C615N2-L-lysine, K8) Lys, to label all newly synthesized proteins for the pulse measurements. At the same time or after medium exchange, one of the cultures was subjected to a perturbation (here, treatment with 1 uM Geldanamycin ). Extracts from control and drug-treated cells were then harvested at defined time points and mixed equimolarly. Proteins were digested with the FASP protocol, separated into 24 fractions by off-gel isoelectric focusing and analysed by nano-LC-MS/MS on an Orbitrap Velos Instrument. Data were analysed as triplex SILAC using MaxQuant and peptide spectrum matches were filtered at 1% FDR. Output tables were processed further with custom-made perl scripts and then used as input for the mathematical model. All subsequent data treatment steps were done with MatLab as described in the article. RAW DATA FILES: 1) pcSILAC experiment 2, replicate 2 (data presented in article) : 3 time points t=6,12,20h ID Description 4957 Mix= M/L (DMSO) + H/L(Geldanamycin); t=6h 5052 Mix= M/L (DMSO) + H/L(Geldanamycin); t=12h 4884 Mix= M/L (DMSO) + H/L(Geldanamycin); t=20h 2) pcSILAC experiment 1, replicate 2 (data presented in supplementary files to article) ID Description 4733 Mix= M/L (DMSO) + H/L(Geldanamycin); t=6h 4912 Mix= M/L (DMSO) + H/L(Geldanamycin); t=12h 4803 Mix= M/L (DMSO) + H/L(Geldanamycin); t=20h 3) stSILAC experiment 1 (internal stSILAC control done within pcSILAC exp. 1 ; data presented in supplementary files to article) ID Description 4731 Mix= M (DMSO) + H(Geldanamycin); t=6h 4734 Mix= M (DMSO) + H(Geldanamycin); t=20h
Manfredo Quadroni 
