Accelerating Covalent Binding Studies: Direct mass shift measurement with acoustic ejection and TOF-MS

Tracking chemical reactions by measuring incurred mass shifts upon successful binding is a direct and attractive alternative to existing assays based on chemical tags. Traditional methods use liquid chromatography-mass spectrometry (LC-MS), and because the required buffers are not amenable to direct MS injection, sample pre-treatment is needed to desalt. This leads to analysis times from ten seconds to minutes per sample, limiting throughput and preventing widespread application. Combining an acoustic ejection (AE) interface with a time-of-flight mass spectrometer (MS) removes this bottleneck, as samples can be directly introduced at rates of up to one second per sample. This article describes a complete workflow for measuring the covalent binding of compounds to proteins in real-time, from assay to data evaluation. It is noteworthy that this is the first instance of using SCIEX Echo® MS+ system with ZenoTOF 7600 system to study the kinetic regimes of covalent binding.