Time-resolved proteomics vs. ribosome profiling reveals translation dynamics under stress
Tzu-Yu Liu, Hector H. Huang,
Diamond Wheeler, Yichen Xu,
James A. Wells, Yun S. Song,
Arun P. Wiita
- Organism: Homo Sapiens
- Instrument: QTRAP 5500 (SCIEX)
Many small molecule chemotherapeutics induce stresses that globally inhibit mRNA translation, remodeling the cancer proteome and governing response to treatment.
Here we measured protein synthesis in multiple myeloma cells treated with low-dose bortezomib by coupling pulsed-SILAC (pSILAC) with high-accuracy targeted quantitative proteomics. We found that direct measurement of protein synthesis by pSILAC correlated well with the indirect measurement of protein synthesis by ribosome profiling under conditions of robust translation. By developing a statistical model integrating longitudinal proteomic and mRNA-seq measurements, we found that proteomics could directly detect global alterations in translational rate as a function of therapy-induced stress after prolonged bortezomib exposure. Finally, the model we develop here, in combination with our experimental data including both protein synthesis and degradation, predicts changes in proteome remodeling under a variety of cellular perturbations. pSILAC therefore provides an important complement to ribosome profiling in directly measuring proteome dynamics under conditions of cellular stress.
Time course SILAC experiment in multiple myeloma cell line (MM1.s) under low dose Bortezomib treatment. Eukaryotic protein translation model is developed from abundance measurements of light and heavy peptides for 272 highly abundant proteins over a 48 hour period. Heavy isotope incorporation beginning at time 0 hr of drug treatment measures newly synthesized proteins, while the decrease in light isotopes over the time course reflect the degradation of proteins over the same period. SRM was applied to an untreated Epstein-Barr Virus transformed B-cell line as well with 168 protein targets to compare.
SILAC light and heavy whole cell lysate from human MM1.s cell line and EBV-transfromed human B-cells
Created on 11/15/16 8:38 AM