Revealing nascent proteomics in signaling pathways and cell differentiation

Proceedings of the National Academy of Sciences of the United States of America
Craig M ForesterAlma L Burlingame

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 approache...Continue Reading

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Oct 20, 2018·Molecular Omics·Indrek Koppel, Mike Fainzilber
Apr 23, 2020·The Journal of International Medical Research·Liang MoYong You
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Jul 15, 2021·Journal of the American Society for Mass Spectrometry·Lei ChenQian Zhao
Aug 9, 2021·Current Opinion in Chemical Biology·Wouter van BergenMarc P Baggelaar

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Methods Mentioned

BETA
Affinity Purification
pull-down
RNAseq
Fluorescence
acetylation
flow cytometry

Software Mentioned

DAVID
Primer Blast
Protein Prospector
BONCAT
OPP
ID

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