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RPM-1 in vivo affinity-purification proteomics using C. elegans

ABSTRACT: To uncover putative RPM-1 protein-protein interactions, we performed a comprehensive series of in vivo affinity-purification proteomics experiments using C. elegans. We utilized integrated transgenes that express RPM-1 fused with a Protein G::Streptavidin binding peptide (GS) affinity tag expressed using the native rpm-1 promoter on an rpm-1 protein null background. Negative control animals expressed an integrated transgene where the GS tag was fused to GFP (GS::GFP) and expressed by the rpm-1 promoter. We also designed a biochemical “trap” using an RPM-1 ligase-dead (RPM-1 LD) site-directed mutant to identify ubiquitination substrates. RPM-1 LD would enrich substrates by trapping them in stalled ubiquitination complexes, as well as preventing proteasome-mediated degradation and elevating substrate levels. RPM-1-binding proteins that are not ubiquitination substrates would bind to both RPM-1 and RPM-1 LD.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Caenorhabditis Elegans

SUBMITTER: Brock Grill

LAB HEAD: Brock Grill

PROVIDER: PXD051783 | Pride | 2024-11-19

REPOSITORIES: Pride

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			Action	DRS
	042017_MS0698_BG_112-A.mzXML	Mzxml
	042017_MS0698_BG_112-A.raw	Raw
	042017_MS0698_BG_112-A_GFP.msf	Msf
	042017_MS0698_BG_112-A_GFP.mzid.gz	Mzid
	042017_MS0698_BG_112-A_GFP.mzid_042017_MS0698_BG_112-A_GFP.MGF	Mzid

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RPM-1 in vivo affinity-purification proteomics using C. elegans

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