Top-down analysis of novel synthetic branched proteins.

A strategy for top-down analysis of branched proteins has been reported earlier, which relies on electron transfer dissociation assisted by collisional activation, and software designed for graphic interpretation of tandem mass spectra and adapted for branched proteins. In the present study, the strategy is applied to identify unknown and novel products of reactions in which rationally mutated proteoforms of Rub1 are used to probe the selectivity of E1 and E2 enzymes normally active in ubiquitination. To test and demonstrate this application, components and attachment sites of three branched dimers are deduced and the mutations are confirmed.