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.

单ADP-核糖基化是导致目标蛋白异常激活或失活的细菌毒素的共同特征。肉毒梭菌（C3bot）ADP的C3外切酶使小的GTPases RhoA，RhoB和RhoC核糖基化，从而导致这些重要调控因子的失活和下游信号传导受损。使用凝胶迁移分析，抗体和基于放射性的方法对ADP-核糖基化的定量分析是有限的。因此，建立了一种新颖的基于LC-MS的方法来特异性确定和定量Rho GTPases的ADP-核糖基化。使用重标记的蛋白质标准品，其中包含相似量的ADP核糖基化和非ADP核糖基化形式的ADP-核糖基化特异性肽，用于体内相对定量。在原理验证实验中，用C3bot处理HT22细胞，并在体内测定RhoA / B和RhoC ADP-核糖基化的动力学。