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

Abstract

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 labelfree 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, IC 50 and K i), as well as inhibition-reactivation assays. We anticipate that the expansion of this platform has high potential impact in labelfree enzymology as well as in drug discovery.

Current Status and Future Perspectives of Supports and Protocols for Enzyme Immobilization

“…The electrospraying technique, popularly known as electrohydrodynamic atomization (EHDA), is a potential technology similar to electrospinning to synthesize polymeric nanoparticles or bioactive fiber-based materials used in various processes [233][234][235]. Classically, the electrospraying technique is defined as atomizing the liquid through electrical forces [236][237][238][239].…”

“…A few years ago, coaxial electrospraying emerged as an innovative technology for synthesizing products with two miscible or immiscible core and wall materials [233,238,239,241,242]. Coaxial electrospraying is a more convenient one-step method for manufacturing nanoparticles in dry form, thus expanding its industrial applications [233][234][235][236][237][238]. One of the main advantages of this technique for the immobilization of enzymes is the possibility of designing and adjusting the shape and size of nanoparticles only by modifying the experimental conditions [233,238,239].…”

“…In addition, reducing the size of the carrier matter can also improve the efficiency of immobilized enzymes. Thus, the catalytic capacity of enzymes is usually optimized [235,[238][239][240]. However, despite all these advantages mentioned above, there is a scarcity of studies related to this area specifically, to a large degree justified by its only recent discovery [235][236][237][238].…”

Glass surface as strong base, ‘green’ heterogeneous catalyst and degradation reagent

“…First, we used the high-throughput system which is capable of screening thousands of reaction conditions per day [16][17][18] to test how various base-catalyzed chemical reactions, including elimination, solvolysis, imine formation, Katritzky reaction and Knoevenagel condensation, are affected by addition of glass microspheres. Methanol was added to containers containing glass microspheres (diameter: 32.5 µm, 0.02 equivalents) and then reactant solutions in methanol were added in stoichiometric amounts to reach a final concentration of 1 mM.…”

“…In this study we use a desorption electrospray ionization mass spectrometry (DESI-MS) 14,15 based high-throughput system 16 with hardware and software features that enable reproducible quantitative data to be generated from minimal amounts (50 nL) of sample solution. The integrated automatic platform allows both synthetic reactions 17 and enzymatic reactions 18 to be studied at a throughput of 1 s per sample. Therefore, we performed a single-day high-throughput experiment using the HTE DESI-MS system to screen a large set of glass-catalyzed reactions.…”