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. 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

Craig M Forester  1   2   3 , Qian Zhao  4   5 , Nancy J Phillips  4 , Anatoly Urisman  6 , Robert J Chalkley  4 , Juan A Oses-Prieto  4 , Li Zhang  2   7 , Davide Ruggero  8   3   9 , Alma L Burlingame  8   4
Affiliations

Revealing nascent proteomics in signaling pathways and cell differentiation

Craig M Forester et al. Proc Natl Acad Sci U S A. .

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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
OPP-ID as a tool to identify nascent protein synthesis: By mimicking tyrosine-tRNA, OPP binds to the acceptor (A) site of the ribosome during elongation and leads to subsequent polypeptide chain termination instead of extension of the polypeptide through the peptidyl (P) site and release of tRNA at the exit (E) site. The alkyne group on OPP is conjugated to an azide group on biotin using cycloaddition. Biotin–polypeptide complexes are bound to streptavidin beads followed by on-bead digestion and analysis by LC-MS/MS for identification and quantification.
Fig. 2.
Fig. 2.
OPP labels nascent proteomes with comparable coverage, peptide intensity, and identification to HPG. (A) TAMRA-conjugated proteins labeled by specified concentrations of HPG or OPP in comparison with DMSO vehicle were isolated by SDS/PAGE and analyzed by fluorescence scan at 532 nm. Denoted box indicates overlaid image of molecular weight ladder captured at 647 nm. Representative section of Coomassie-stained gel for loading comparison. (B) Venn diagram of identified proteins in HPG 25 μM or OPP 30 μM after subtraction of overlapping proteins identified in DMSO vehicle. (C) Proteins identified in all labeling schemes were aligned, and mean percentage peptide coverage with 95% confidence interval plotted. ***P < 0.001; ****P < 0.0001. (D) Proteins identified in all labeling schemes were aligned and mean intensity (summed extracted ion chromatograms represented as arbitrary units) 95% confidence interval displayed. **P < 0.01; ****P < 0.0001.
Fig. 3.
Fig. 3.
OPP-ID identifies a specific nascent proteome repressed by an active-site mTOR inhibitor. (A) Schematic for K562 cell-based analysis of the MLN128-repressed nascent proteome. (B) Cell lysates from culture conditions were analyzed for TAMRA-azide conjugated OPP proteins by SDS/PAGE with fluorescence detection at 532 nm. Denoted box indicates overlaid image of molecular weight ladder captured at 647 nm. Coomassie staining of gel for total protein loading shown adjacent to fluorescence image. (C) Plot of number of identified proteins segregated by binned average protein intensities (log2) in each of the four treatment conditions. (D) Fold change (log2) of nascent protein abundance in DMSO vehicle vs. MLN128-treated cells per individual protein. Dotted line denotes cutoff of log2 FC > 1. (E) Fold change analysis between DMSO vs. MLN128 subdivided into groups of log2 FC. (F) Western blot verification of OPP-ID identified nascent proteins with densitometric reduction normalized to beta-actin.
Fig. 4.
Fig. 4.
OPP-ID identifies a specific nascent proteome inherent in maintaining expansion of ESRE erythroid precursors. (A) Schematic for ESRE-based analysis of the nascent proteome associated with either expansion or maturation media. (B) Fold change (log2) of nascent protein abundance in expansion vs. maturation media treated ESRE cells per individual protein. Dotted line denotes log2 FC > 1. (C) Fold change analysis between expansion vs. maturation subdivided into groups of log2 FC. (D) Subclasses of identified genes up-regulated in ESRE expansion media. Genes listed in red have been previously implicated in hematopoiesis.
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