Dataset Information

Proteomic proximity mapping by rapamycin-dependent targeting of APEX2

ABSTRACT: Enhanced ascorbate peroxidase 2 (APEX2) can be used as a genetic tag that produces short-lived yet highly reactive biotin species, allowing the modification of proteins that interact with or are in very close proximity to the tagged protein, ideally within a confined compartment. Biotinylated proteins can be isolated using immobilized streptavidin and analyzed by mass spectrometry. Here we used rapamycin-dependent targeting of APEX2 to tail-anchored proteins of the endoplasmic reticulum and of the inner nuclear membrane (INM). In combination with stable isotope labeling with amino acids in cell culture (SILAC), our novel approach (rapamycin- and APEX-dependent identification of proteins by SILAC; RAPID-SILAC or RAPIDS) allowed the identification of proteins that are in close proximity to the prototypic INM-protein emerin and the vesicle-trafficking protein VAPB. In addition to well-known interaction partners, several new potential binding partners were identified. In RAPIDS, the comparison of plus/minus-rapamycin conditions allows a clear distinction between specific and non-specific hits.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Christof Lenz

LAB HEAD: Christof Lenz

PROVIDER: PXD009783 | Pride | 2020-04-15

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20170208_UP_Reference_HOMSA.fasta	Fasta
	Figs.1-6S.pdf	Pdf
	M_Mueller_080317_120517_2a_R1_01.raw	Raw
	M_Mueller_080317_120517_2a_R1_02.raw	Raw
	M_Mueller_080317_120517_2a_R1_03.raw	Raw

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Publications

Probing the Environment of Emerin by Enhanced Ascorbate Peroxidase 2 (APEX2)-Mediated Proximity Labeling.

Müller Marret M James Christina C Lenz Christof C Urlaub Henning H Kehlenbach Ralph H RH

Cells 20200303 3

Emerin is one of the best characterized proteins of the inner nuclear membrane, but can also occur at the level of the endoplasmic reticulum. We now use enhanced ascorbate peroxidase 2 (APEX2) to probe the environment of emerin. APEX2 can be used as a genetic tag that produces short-lived yet highly reactive biotin species, allowing the modification of proteins that interact with or are in very close proximity to the tagged protein. Biotinylated proteins can be isolated using immobilized strepta ...[more]

PMID: 32138363

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