Identification of RNF111 substrates by integrative ubiquitome and proteome analysis

ABSTRACT: In the present work, we sought to identify exhaustively the endogenous substrates ubiquitinated and degraded by the E3 ubiquitin ligase RNF111 in presence of TGF-β signaling by performing label free quantitative proteomics after enrichment of ubiquitinated proteins (ubiquitome) in parental U2OS cell line compared to U2OS CRISPR engineered clones expressing a truncated form of RNF111 devoid of C-terminal Ring domain. We compare two methods of enrichment for ubiquitinated proteins prior to mass spectrometry proteomics analysis, the diGly remnant peptide immunoprecipitation with a K-e-GG antibody and a novel approach using protein immunoprecipitation with an ubiquitin pan (Pan UB) nanobody that recognizes all ubiquitin chains and mono-ubiquitination. These ubiquitomes were compared to the corresponding proteome to identify proteins ubiquitinated and degraded by RNF111 upon TGF-β signaling pathway.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Osteosarcoma Cell Line

SUBMITTER: Victor Laigle  

LAB HEAD: Damarys Loew

PROVIDER: PXD025890 | Pride | 2021-11-17

REPOSITORIES: Pride