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Proc Natl Acad Sci U S A. 2018 Mar 6;115(10):2353-2358. doi: 10.1073/pnas.1707514115. Epub 2018 Feb 21.

Revealing nascent proteomics in signaling pathways and cell differentiation.

Author information

1
Division of Pediatric Allergy, Immunology & Bone Marrow Transplantation, University of California, San Francisco, CA 94158.
2
Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA 94158.
3
Department of Urology, University of California, San Francisco, CA 94158.
4
Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94158.
5
State Key Laboratory of Chirosciences, Department of Applied Biology and Chemical Technology, Hong Kong Polytechnic University, Hong Kong.
6
Department of Pathology, University of California, San Francisco, CA 94143.
7
Department of Epidemiology and Biostatistics, University of California, San Francisco, CA 94158.
8
Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA 94158; Davide.Ruggero@ucsf.edu alb@cgl.ucsf.edu.
9
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158.

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.

KEYWORDS:

erythropoiesis; mTOR; proteomics; puromycin; translation

PMID:
29467287
PMCID:
PMC5877968
DOI:
10.1073/pnas.1707514115
[Indexed for MEDLINE]
Free PMC Article

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