MS-based quantification of RhoA/B and RhoC ADP-ribosylation.

Mono ADP-ribosylation is a common characteristic of bacterial toxins resulting to aberrant activation or inactivation of target proteins. The C3 exoenzyme of Clostridium botulinum (C3bot) ADP-ribosylates the small GTPases RhoA, RhoB and RhoC, leading to inactivation of these important regulators and impaired down-stream signaling. Quantification of ADP-ribosylation using gel migration assays, antibodies, and radioactivity-based methods are limited. Therefore a novel LC-MS-based method to specifically determine and quantify ADP-ribosylation of Rho GTPases was established. A heavy labeled protein standard that contained ADP-ribosylation specific peptides in similar amounts in ADP ribosylated and non ADP ribosylated form was used for relative quantification in vivo. In a proof of principle experiment HT22 cells were treated with C3bot and the kinetics of RhoA/B and RhoC ADP-ribosylation were determined in vivo.