High-Throughput Label-Free Enzymatic Assays Using Desorption Electrospray-Ionization Mass Spectrometry

High-throughput (HT) enzymatic assays, which typically rely on labeled compounds and plate readers, are important for drug discovery. Mass spectrometry (MS) provides an alternative method of performing HT label-free assays. Here we demonstrate the use of a HT platform based on desorption electrospray ionization (DESI) MS for the label-free study of enzymatic reactions directly from the bioassay matrix with an effective analysis time of 0.3 s per sample. This system allows for thorough analysis of the enzymatic process through monitoring of its substrate and product after an external calibration. We show the platform capabilities by an in-depth study of the acetylcholinesterase assay, including kinetic parameter determination, rapid inhibitor screening, and further characterization of positive hits (that is, IC₅₀ and K_i), as well as inhibition–reactivation assays. We anticipate that the expansion of this platform has high potential impact in label-free enzymology as well as in drug discovery.