Direct screening of enzyme activity using infrared matrix‐assisted laser desorption electrospray ionization

Abstract

Rationale High-throughput screening (HTS) is a critical step in the drug discovery process. However, most mass spectrometry-based HTS methods require sample cleanup steps prior to analysis. In this work we present the utility of infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) for monitoring an enzymatic reaction directly from a biological buffer system with no sample cleanup and at high throughput. Methods IR-MALDESI was used to directly analyze reaction mixtures from a well plate at different time points after reaction initiation. The percent conversion of precursors to products was used to screen the enzyme activity. The reaction was performed with two different concentrations of precursors and enzyme in order to assess the dynamic range of the assay. Eventually, a pseudo-HTS study was designed to investigate the utility of IR-MALDESI screening enzyme activity in a high-throughput manner. Results IR-MALDESI was able to readily monitor the activity of IDH1 over time at two different concentrations of precursors and enzyme. The calculated Z-factors of 0.65 and 0.41 confirmed the suitability of the developed method for screening enzyme activity in HTS manner. Finally, in a single-blind pseudo-HTS analysis IR-MALDESI was able to correctly predict the identity of all samples, where 8/10 samples were identified with high confidence and the other 2 samples with lower confidence. Conclusions The enzymatic activity of IDH1 was screened by directly analyzing the reaction content from the buffer in well plates with no sample cleanup steps. This proof-of-concept study demonstrates the robustness of IR-MALDESI for direct analysis of enzymatic reactions from biological buffers with no sample cleanup and its immense potential for HTS applications.

Laser Desorption/Ablation Postionization Mass Spectrometry: Recent Progress in Bioanalytical Applications

“…These reports demonstrate the feasibility of IR‐MALDESI to high throughput identification/quantification of biomolecules in complex matrixes. A further example of the use of IR‐MALDESI in this area is the report of high‐throughput screening of enzymatic activity of IDH1 from a biological buffer system (Nazari et al, 2017). By screening reaction mixtures across a well plate with a throughput of 2.5 s per sample, this methodology enabled the monitoring of enzymatic reactions from estimated percent conversion of precursors to products.…”

Normalization techniques for high‐throughput screening by infrared matrix‐assisted laser desorption electrospray ionization mass spectrometry

“…Infrared matrix‐assisted laser desorption electrospray ionization (IR‐MALDESI) is a hybrid, ambient ionization source that combines the benefits of electrospray Ionization (ESI) and MALDI 15–18 . IR‐MALDESI is commonly used for MS imaging applications but has also been characterized as a high‐throughput, direct analysis platform to analyze a diverse range of biochemical assays at a sub‐second per sample rate 15,16,19–22 . With IR‐MALDESI, neutral molecules are desorbed from a sample surface by a mid‐IR laser that resonantly excites the O‐H stretching bands of water, the sample matrix.…”

Ultrahigh-Throughput Ambient MS: Direct Analysis at 22 Samples per Second by Infrared Matrix-Assisted Laser Desorption Electrospray Ionization Mass Spectrometry

“…Recently, we have developed IR-MALDESI as an alternative MS interface technology capable of high throughput direct analysis from any conventional microtiter plate format 8, 9 . Using a Q Exactive HF-X mass spectrometer, our group found a maximum potential spectra scan rate of 33 Hz at an MS resolving power (RP) of 7500 at m/z=200 9 .…”