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Mass spectrometry analysis of SMAD7 interactors and SMURF1 ubiquitination via VPRBP

ABSTRACT: We performed mass spectrometry analysis of Flag-tagged SMURF1 in the presence or absence of VPRBP. Analysis of the SMURF1 interactome and modification by mass spectroscopy examination of the co-immunoprecipitants of Flag-tagged SMURF1. We also applied tandem affinity purification (TAP) assay of the whole proteome to identify interacting proteins of SMAD7. HEK293T cells were transfected with TAP-tagged Smad7, followed by immunoprecipitation, SDS–PAGE, and coomassie staining. SDS–PAGE gels were minimally stained with Coomassie brilliant blue, cut into six molecular weight ranges based on heavy-chain IgG bands, and digested with trypsin. Immunocomplexes were identiﬁed on a Thermo Fisher LTQ mass spectrometer. Spectral data were then searched against the human protein RefSeq database in BioWorks or the Proteome Discoverer Suites using either SeQuest (for LTQ data) or Mascot (Orbitrap data) software. The immunoprecipitation–mass spectrometry results were transferred into a FileMaker-based relational database generated in-house,where protein identiﬁcation numbers (protein GIs) were converted to GeneID identiﬁers according to the NCBI ‘gene accession’ table.

ORGANISM(S): Homo Sapiens

SUBMITTER: Fangfang Zhou

PROVIDER: PXD013384 | iProX | Wed Apr 10 00:00:00 BST 2019

REPOSITORIES: iProX

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