Enabling Protein Degradation Drug Discovery

Validation and Utility of the Ubiselect™ Kit

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The post-translational modification of proteins by ubiquitin is involved in a wide range of cellular processes (Kirkin and Dikic, 2007). In any given cell the proportion of protein modified by ubiquitin is very small therefore it has been difficult to isolate and identify this post translational modification from mammalian whole cell lysates. Ubiquitin proteomics remains challenging even though the sensitivity of Mass Spectrometry (MS) has improved dramatically through the use of innovative techniques (Sylvestersen et al., 2013). Various approaches employing tagged ubiquitin or ubiquitin like molecules have been used with varying degrees of success (Peng et al., 2003; Tirard et al., 2012; Tsirigotis et al., 2001).

In recent years, the isolation of ubiquitylated proteins from neurons of Drosophila melanogaster using a tagged ubiquitin with a 15 amino acid long biotin accepting peptide has been described (Franco et al., 2011). This was the first demonstration that proteomics could be used to identify neuronal targets of the ubiquitinproteasome system. This novel technique allowed for the solation and enrichment of ubiquitin conjugates from neurons using a relatively small sample up to levels that allowed direct detection by MS and Western Blotting. In addition, di-glycine signatures indicative of the ubiquitin attachment sites could also be detected on ubiquitin conjugates. Where antibodies were available for specific substrates of ubiquitylation it was also possible to determine whether a substrate was mono- or polyubiquitylated (Franco et al., 2011). Using a similar approach, hemizygous BiotinylatedUbiquitin (Bio-Ub) and control BirA transgenic mouse models have been created (see Figure 1) which express either three moieties of Biotin-Accepting Peptide (BAP tag)-ubiquitin plus the E. coli enzyme BirA or the E.coli enzyme BirA enzyme alone (Lectez et al., 2014). The Ubiselect kit includes both Ubiselect-MEF_Bio-Ub (P0) (Cat# 66-5011-001) and control Ubiselect-MEF_BirA (P0) (Cat# 66-5010-001) cell lines derived from 13.5 day old embryos from these transgenic mice through homogenisation and trypsinisation of the embryos minus the head and liver (Lectez et al., 2014).

  • Validation and Utility of the Ubiselect™ Kit