Isolation of the Ubiquitin-Proteome from Tumor Cell Lines and Primary Cells Using TUBEs.
Tandem ubiquitin-binding entities (TUBEs) act as molecular traps to isolate polyubiquitylated proteins facilitating the study of this highly reversible posttranslational modification. We provide here sample preparation and adaptations required for TUBE-based enrichment of the ubiquitin proteome from tumor cell lines or primary cells. Our protocol is suitable to identify ubiquitin substrates, enzymes involved in the ubiquitin proteasome pathway, as well as proteasome subunits by mass spectrometry. This protocol was adapted to prepare affinity columns, reduce background, and improve the protein recovery depending on the sample source and necessities.
Proteomics Platform CICbioGUNE, CIBERehd, ProteoRed-ISCIII, Parque Tecnologico de Bizkaia, Derio, Spain.
Wendy
W
Xolalpa
Ubiquitylation and Cancer Molecular Biology, Inbiomed, San Sebastian, Spain.
Lydia
L
Mata-Cantero
Ubiquitylation and Cancer Molecular Biology, Inbiomed, San Sebastian, Spain.
Fabienne
F
Aillet
IPBS, Université de Toulouse, CNRS, UPS and ITAV, Université de Toulouse, CNRS, UPS, 1 Place Pierre Potier, Oncopole entrée B, Toulouse, 31106, France. manuel.rodriguez@itav.fr.
Manuel S
MS
Rodriguez
D000818:Animals; D045744:Cell Line, Tumor; D006801:Humans; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D011499:Protein Processing, Post-Translational / Q000235:genetics / Q000502:physiology; D020543:Proteome / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism*; D054875:Ubiquitination / Q000235:genetics / Q000502:physiology
D020543:Proteome; D025801:Ubiquitin; D046988:Proteasome Endopeptidase Complex
Isolation; Posttranslational modifications; Purification; TUBEs; Ubiquitylation