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):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Christof Lenz  

LAB HEAD: Christof Lenz

PROVIDER: PXD009783 | Pride | 2020-04-15

REPOSITORIES: Pride