Extended Data Figure 9. Detection of the reaction intermediates in SdeA-catalyzed ubiquitination.

a, Controls were analyzed by HPLC of NAD alone and in the presence of SdeA, Ub, and SdeA and Ub. In these reactions, AMP and NAD were identified with retention times of 3.6 and 6.8 min, respectively. b, Both AMP (left) and NAD (right) were additionally identified by ESI mass spectrometry. Both NAD and a product in which the nicotinamide group has been lost were observed in these experiments. c, To determine whether other fragments are generated in this reaction, retention time for nicotinamide mononucleotide (NMN, left) and nicotinamide (Nic, right) was determined by HPLC to be 5.6 and 2.6 min respectively. d, To identify additional components, a reaction was set up and the individual components were identified by HPLC. In the reaction mixture, AMP (3.5 min), nicotinamide (Nic 5.5 min), and NAD (6.5 min) were observed. An additional component to the reaction mixture (labeled X) was observed (6.1 min), but could not be further identified by mass spectrometry. Data in all panels are one representative from three independent experiments with similar results.