A Proteomics Approach to Profiling the Temporal Translational Response to Stress and Growth
Daniel A. Rothenberg,
J. Matthew Taliaferro,
Sabrina M. Huber,
Thomas J. Begley,
Peter C. Dedon,
Forest M. White
Affiliations
Daniel A. Rothenberg
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; The Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
J. Matthew Taliaferro
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, CO 80045, USA; RNA Bioscience Initiative, University of Colorado School of Medicine, Aurora, CO 80045, USA
Sabrina M. Huber
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
Thomas J. Begley
College of Nanoscale Science and Engineering, State University of New York, Albany, NY 12203, USA
Peter C. Dedon
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Infectious Disease IRG, Singapore-MIT Alliance for Research and Technology, Singapore, Singapore
Forest M. White
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; The Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Center for Precision Cancer Medicine, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Corresponding author
Summary: To quantify dynamic protein synthesis rates, we developed MITNCAT, a method combining multiplexed isobaric mass tagging with pulsed SILAC (pSILAC) and bio-orthogonal non-canonical amino acid tagging (BONCAT) to label newly synthesized proteins with azidohomoalanine (Aha), thus enabling high temporal resolution across multiple conditions in a single analysis. MITNCAT quantification of protein synthesis rates following induction of the unfolded protein response revealed global down-regulation of protein synthesis, with stronger down-regulation of glycolytic and protein synthesis machinery proteins, but up-regulation of several key chaperones. Waves of temporally distinct protein synthesis were observed in response to epidermal growth factor, with altered synthesis detectable in the first 15 min. Comparison of protein synthesis with mRNA sequencing and ribosome footprinting distinguished protein synthesis driven by increased transcription versus increased translational efficiency. Temporal delays between ribosome occupancy and protein synthesis were observed and found to correlate with altered codon usage in significantly delayed proteins. : Functional Aspects of Cell Biology; Methodology in Biological Sciences; Proteomics Subject Areas: Functional Aspects of Cell Biology, Methodology in Biological Sciences, Proteomics
