Monitoring structural modulation of redox-sensitive proteins in cells with MS-CETSA

Area of Science:

• Cellular Biology
• Biochemistry
• Proteomics

Background:

• Reactive oxygen species (ROS) are crucial in cellular signaling and stress responses, linked to aging, cancer, and neurological disorders.
• ROS significantly impact drug efficacy and adverse effects, yet direct studies of ROS-modulated protein function are challenging.
• Understanding specific protein roles in cellular ROS responses remains limited.

Purpose of the Study:

• To establish Mass Spectrometry-Cellular Thermal Shift Assay (MS-CETSA) as a method for directly monitoring ROS and redox modulations of protein structure at the proteome level.
• To identify novel ROS-sensitive proteins and understand their roles in cellular processes.
• To investigate the utility of MS-CETSA in studying drug effects on cellular redox states.

Main Methods:

• Utilized MS-CETSA to analyze protein structure changes in response to altered ROS levels in cultured human hepatocellular carcinoma cell lysates and intact cells.
• Applied hydrogen peroxide and sulfasalazine (a ROS-modulating drug) treatments to intact cells.
• Performed proteome-level analysis to detect CETSA responses and identify redox-sensitive proteins.

Main Results:

• MS-CETSA successfully detected ROS and redox modulations of protein structure across the proteome.
• Identified numerous known redox-sensitive proteins and novel candidate ROS-sensitive proteins.
• Revealed direct protein modifications and downstream cellular effects of ROS, including modulation of ATP levels and key cellular processes like epigenetic control and protein translation.

Conclusions:

• MS-CETSA is a powerful new method for studying cellular redox modulations of proteins.
• The method facilitates the discovery of proteins implicated in diverse cellular processes and provides insights into drug efficacy and adverse effects.
• CETSA-based biomarkers can be developed for monitoring drug responses.