Identification of ubiquitin‐conjugated proteins from mouse heart expressing tagged ubiquitin

Ubiquitination is a novel post‐translational modification, which regulates the stability, activity, localization, and interaction of target substrates. The discovery of a new ubiquitinated proteins as well as its new function seems to be everlasting, which graced ubiquitin (Ub) with Darwin’s phosphate. Proteomics tools have been applied to identify the ubiquitinated proteins systematically from yeast or cultured cells after inducing the expression of tagged ubiquitin. In this study, we generated the transgenic mouse expressing His‐tagged Ub in heart. In order to purify Ub‐conjugated proteins, total proteins were first incubated with CNBr to fragment the polypeptides by cleaving after methionine, while polyUb chains were maintained. PolyUb attached polypeptides were purified by Ni‐affinity chromatography in denatured condition, which were then digested with LysC/trypsin and identified by tandem mass spectrometry coupled with reverse phase chromatography (LC‐MS/MS). Finally, we identified 164 potential Ub‐conjugated proteins and 73 Ub attachment sites by searching the signature peptide of ubiquitination. As far as we know, this is the first trial to identify Ub‐conjugated proteins in tissue or organ level. We believe that transgenic mouse expressing tagged Ub will be a valuable tool not only to identify novel Ub‐conjugates but also to perform the comparative analysis of Ub‐conjugated proteins in different pathological status of heart.