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A method for spatially and temporally resolved protein network interrogation in living cells

ABSTRACT: Cells operate through protein interaction networks organized in space and in time, but current methods to interrogate such biology typically resolve only one of these dimensions. Here, we describe a method to resolve both dimensions simultaneously using proximity labeling mediated by engineered ascorbic acid peroxidase (APEX). APEX has been used to label organelle proteomes with high temporal resolution, but its spatial resolution is generally thought to be limited because of its larger labeling radius. We describe an analytical pipeline, based on quantitative proteomics using spatial references, which overcomes this barrier. As proof-of-principle, we apply the methodology to interrogate protein interaction networks of G protein coupled receptors both temporally and spatially. Using this approach, we identify known binding partners as well as novel receptor regulators. Validating the methodology as a discovery tool, we demonstrate that two of these components drive ubiquitin-linked down-regulation of opioid receptors.

INSTRUMENT(S): Orbitrap Fusion, LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Ruth Huttenhain

LAB HEAD: Ruth Huttenhain

PROVIDER: PXD005758 | Pride | 2017-03-03

REPOSITORIES: pride

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			Action	DRS
	Experimental_design_B2AR_DOR_ENDO_PM_shotgun.txt	Txt
	Experimental_design_DOR_location_shotgun.txt	Txt
	FU20170107-01.raw	Raw
	FU20170107-03.raw	Raw
	FU20170107-05.raw	Raw

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A method for spatially and temporally resolved protein network interrogation in living cells

Project description:Cells operate through protein interaction networks organized in space and in time, but current methods to interrogate such biology typically resolve only one of these dimensions. Here, we describe a method to resolve both dimensions simultaneously using proximity labeling mediated by engineered ascorbic acid peroxidase (APEX). APEX has been used to label organelle proteomes with high temporal resolution, but its spatial resolution is generally thought to be limited because of its larger labeling radius. We describe an analytical pipeline, based on quantitative proteomics using spatial references, which overcomes this barrier. As proof-of-principle, we apply the methodology to interrogate protein interaction networks of G protein coupled receptors both temporally and spatially. Using this approach, we identify known binding partners as well as novel receptor regulators. Validating the methodology as a discovery tool, we demonstrate that two of these components drive ubiquitin-linked down-regulation of opioid receptors.

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