Label Free Screening of Enzyme Inhibitors at Femtomole Scale Using Segmented Flow Electrospray Ionization Mass Spectrometry

Abstract

Droplet-based microfluidics is an attractive platform for screening and optimizing chemical reactions. Using this approach, it is possible to reliably manipulate nanoliter volume samples and perform operations such as reagent addition with high precision, automation, and throughput. Most studies using droplet microfluidics have relied on optical techniques to detect the reaction; however, this requires engineering color or fluorescence change into the reaction being studied. In this work, we couple electrospray ionization mass spectrometry (ESI-MS) to nanoliter scale segmented flow reactions to enable direct (label-free) analysis of reaction products. The system is applied to a screen of inhibitors for cathepsin B. In this approach, solutions of test compounds (including three known inhibitors) are arranged as an array of nanoliter droplets in a tube segmented by perfluorodecalin. The samples are pumped through a series of tees to add enzyme, substrate (peptides), and quenchant. The resulting reaction mixtures are then infused into a metal-coated, fused silica ESI emitter for MS analysis. The system has potential for high-throughput as reagent addition steps are performed at 0.7 s per sample and ESI-MS at up to 1.2 s per sample. Carryover is inconsequential in the ESI emitter and between 2 and 9% per reagent addition depending on the tee utilized. The assay was reliable with a Z-factor of ~0.8. The method required 0.8 pmol of test compound, 1.6 pmol of substrate, and 5 fmol of enzyme per reaction. Segmented flow ESI-MS allows direct, label free screening of reactions at good throughput and ultralow sample consumption.

A label-free Sirtuin 1 assay based on droplet-electrospray ionization mass spectrometry

“…To make the SIRT1 reaction directly analyzable by ESI-MS, we developed a reaction buffer containing a volatile salt ammonium formate and a small molecule reducing agent DTT which would keep the enzyme in the reduced state and at the same time not interfere with ESI-MS signals. 18 …”

“…14–16 Previously, screens for modulators of acetylcholinesterase and cathepsin B have been carried out using a droplet-ESI-MS system. 17, 18 …”

Microfluidics-to-mass spectrometry: A review of coupling methods and applications

“…The authors found that high viscosity oils, like FC-40 and perfluorodecalin, did not generate signal at low spray voltages, but that the electrospray current and signal rapidly stabilized as each new aqueous droplet entered the emitter so a series of droplets segmented by selected oil plugs could be analyzed without an extraction step. This simple coupling method was applied in the screening of enzyme inhibitors [47]. Solutions of 25 test compounds were introduced as an array of droplets separated by perfluorodecalin in a tube.…”

Advances in capillary electrophoresis and the implications for drug discovery

“…In one approach, samples in MWPs are reformatted into segmented droplets of nanoliter volume inside tubing by using aspiration to sequentially draw up plugs of sample and carrier fluid. [15,16,33–36] Microfluidic droplets can then undergo further manipulations such as mixing,[37–41] merging,[42–46] splitting,[43,47–50] addition,[35,37,38,51,52] incubation and extraction,[15,53–59] which can enable entire assays to be performed at small scale. Indeed, this approach has been used with fluorescence detection to screen 704 compounds against protein tyrosine phosphatase and high-resolution dose response curves were obtained.…”