Accelerating covalent binding studies: Direct mass shift measurement with acoustic ejection and TOF-MS.

Area of Science:

• Analytical Chemistry
• Biochemistry
• Mass Spectrometry

Background:

• Traditional methods for tracking chemical reactions, like liquid chromatography-mass spectrometry (LC-MS), require extensive sample pre-treatment (desalting) due to incompatible buffers.
• This pre-treatment step is time-consuming, with analysis times ranging from seconds to minutes per sample, limiting overall assay throughput and broad applicability.
• Direct mass spectrometry (MS) injection is desirable for faster analysis but is hindered by buffer incompatibilities in existing assays.

Purpose of the Study:

• To develop and validate a streamlined workflow for real-time measurement of covalent binding kinetics.
• To overcome the throughput limitations of traditional LC-MS methods for chemical reaction monitoring.
• To demonstrate the application of a novel acoustic ejection (AE) interface coupled with a time-of-flight mass spectrometer (MS) for direct sample analysis.

Main Methods:

• Integration of an acoustic ejection (AE) interface with a time-of-flight mass spectrometer (MS) for direct sample introduction.
• Development of a complete workflow encompassing assay setup, sample analysis, and data evaluation for covalent binding studies.
• Utilized the SCIEX Echo® MS+ system with the ZenoTOF 7600 system for kinetic analysis of covalent binding.

Main Results:

• The AE-MS system enables direct sample injection at rates up to one sample per second, drastically reducing analysis time compared to traditional methods.
• Successfully established a complete workflow for real-time measurement of covalent binding of compounds to proteins.
• This represents the first reported use of the SCIEX Echo® MS+ system with the ZenoTOF 7600 system for studying covalent binding kinetics.

Conclusions:

• Combining acoustic ejection with time-of-flight mass spectrometry offers a direct, high-throughput alternative for monitoring chemical reactions, specifically covalent binding.
• The developed workflow eliminates the need for sample pre-treatment, significantly accelerating analysis and enabling real-time kinetic studies.
• This technological advancement holds promise for broader application in drug discovery and chemical biology research due to its speed and efficiency.