Authors

L Mueller¹; S Peace²; M Leveridge²; M Trost¹; R Peltier-Heap²; M Duenas¹; 
¹ Newcastle University, UK;  ² GlaxoSmithKline, UK

Discussion

MALDI-TOF MS capabilities have been exploited in the high-throughput screening (HTS) environment to provide fast and label-free readouts for in vitro assays. Here, we describe the development of a MALDI-TOF MS-based drug discovery assay for an immuno-oncology target. Enzymatic activity is tightly modulated by substrate properties, thus screening with a label-free technique was crucial. We used substrate, product, and internal standard (IS) peptides from an established RapidFire (RF) MS assay. The workflow consisted of the following: the enzyme was pre-incubated with compound before addition of the substrate to start the reaction. The reaction was quenched by adding the IS in acidic solution. Samples were then mixed with matrix, spotted, and data acquired on a RapifleX MS. To improve limit and linearity of detection, thus increasing assay stability, we incorporated arginine residues in the substrate sequence and used the ¹³C¹⁵N labelled product peptide as IS. Next, we determined the enzyme kinetics for these new peptides and characterised known inhibitors by performing dose-response analysis; IC₅₀ values aligned with the literature. We also performed a screen with an ‘assay robustness’ set which showed no interference. As a proof-of-concept experiment, a duplicate screen of ~10k compounds was conducted, demonstrating HTS compatibility. Screening of an enzyme targeted compound set showed robust identification of hit compounds. To validate the assay, results were compared with the RF MS assay. In single concentration and subsequent dose-response screening, high correlation within and between the platforms was observed. To sum up, we successfully developed a MALDI-TOF MS assay. The MALDI-TOF MS based assay remains the only label-free setup with sufficient throughput to evaluate the target enzyme in HTS. The findings described add to the growing number of targets that can be investigated by MALDI-TOF MS.