Revealing nascent proteomics in signaling pathways and cell differentiation

Craig M. Forester, Qian Zhao, Nancy J. Phillips, Anatoly Urisman, Robert J. Chalkley, Juan A. Oses-Prieto, Li Zhang, Davide Ruggero, Alma L. Burlingame

Research output: Journal article publicationJournal articleAcademic researchpeer-review

20 Citations (Scopus)

Abstract

Regulation of gene expression at the level of protein synthesis is a crucial element in driving how the genetic landscape is expressed. However, we are still limited in technologies that can quantitatively capture the immediate proteomic changes that allow cells to respond to specific stimuli. Here, we present a method to capture and identify nascent proteomes in situ across different cell types without disturbing normal growth conditions, using O-propargyl-puromycin (OPP). Cell-permeable OPP rapidly labels nascent elongating polypeptides, which are subsequently conjugated to biotin-azide, using click chemistry, and captured with streptavidin beads, followed by digestion and analysis, using liquid chromatography–tandem mass spectrometry. Our technique of OPP-mediated identification (OPP-ID) allows detection of widespread proteomic changes within a short 2-hour pulse of OPP. We illustrate our technique by recapitulating alterations of proteomic networks induced by a potent mammalian target of rapamycin inhibitor, MLN128. In addition, by employing OPP-ID, we identify more than 2,100 proteins and uncover distinct protein networks underlying early erythroid progenitor and differentiation states not amenable to alternative approaches such as amino acid analog labeling. We present OPP-ID as a method to quantitatively identify nascent proteomes across an array of biological contexts while preserving the subtleties directing signaling in the native cellular environment.
Original languageEnglish
Pages (from-to)2353-2358
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume115
Issue number10
DOIs
Publication statusPublished - 6 Mar 2018

Keywords

  • Erythropoiesis
  • mTOR
  • Proteomics
  • Puromycin
  • Translation

ASJC Scopus subject areas

  • General

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