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

Area of Science:

• Bacterial toxins
• Protein modification
• Cellular signaling

Background:

• Mono ADP-ribosylation by bacterial toxins alters protein function.
• Clostridium botulinum C3 exoenzyme (C3bot) inactivates Rho GTPases (RhoA, RhoB, RhoC), disrupting downstream signaling.
• Current ADP-ribosylation quantification methods are limited.

Purpose of the Study:

• To establish a novel liquid chromatography-mass spectrometry (LC-MS)-based method for specific determination and quantification of Rho GTPase ADP-ribosylation.
• To enable in vivo kinetic analysis of Rho GTPase ADP-ribosylation.

Main Methods:

• Development of a novel LC-MS assay for ADP-ribosylation.
• Utilized a heavy-labeled protein standard for relative quantification of ADP-ribosylated and non-ADP-ribosylated peptides in vivo.
• Proof-of-principle experiment involving C3bot treatment of HT22 cells.

Main Results:

• Successfully established and validated an LC-MS method for quantifying ADP-ribosylation of Rho GTPases.
• Demonstrated the ability to perform relative quantification in vivo using a labeled standard.
• Determined the kinetics of RhoA, RhoB, and RhoC ADP-ribosylation in HT22 cells following C3bot treatment.

Conclusions:

• The novel LC-MS method provides a sensitive and specific approach for quantifying Rho GTPase ADP-ribosylation.
• This method facilitates in vivo kinetic studies of toxin-mediated protein modification.
• The findings contribute to understanding the mechanisms of bacterial toxin action and cellular regulation.