Development of a Novel GC/MS Method for the Detection of Nicotinamide and Activity of ADP-Ribosylating Toxins

Disease-causing bacteria, such as those that cause cholera, diphtheria and other diseases, use toxin proteins to inactivate target proteins using NADH. This reaction releases nicotinamide and irreversibly inactivates the target protein by ADP-ribosylation. Historically, the action of ADP-ribosylating toxins has been detected via radioassays that detect the release of radiotagged nicotinamide; although effective, these methods require radioactive materials. The objective of this study is to create an alternative means to detect nicotinamide release using GC/MS methods. Cholera toxin was chosen as a model enzyme for this study because of its high activity and commercial availability. A system for derivatization of nicotinamide was developed to enhance GC/MS performance and sensitivity. Using this derivatization, nicotinamide detection was achieved at a concentration of 0.5ng/μl. To measure activity of the enzyme via this method, a reaction mixture of cholera toxin, NADH and water was incubated, derivatized and analyzed via GC/MS. Nicotinamide was detected in samples with the enzyme and substrate. However, some nicotinamide was also detected in the absence of enzyme, suggesting low levels of NADH decomposition in water or nicotinamide contamination of the NADH. Further steps are being developed to reduce or overcome the effects of the contamination. In conclusion, a novel GC/MS method for the analysis of nicotinamide has been developed. This method shows promise as an alternative, non-radioactive method for detecting ADP-ribosylation activity