Abstract

Studying protein-ligand interactions using mass spectrometry (MS) offers a high-throughput label-free biophysical characterization toolbox. We have now adopted affinity selection mass spectrometry (ASMS) into our daily high-throughput screening (HTS) operations at Pivot Park Screening Centre (PPSC). We selected thrombin, a well-studied protease to establish ASMS-based screening in our lab and compare its output relative to a functional biochemical assay. We screened the robustness set compound collection that is comprised of various classes of compounds with assay interfering and non-drug-like mode-of-action properties as well as so called ‘clean’ diverse drug-like compounds for which no obvious assay interfering properties are expected. In total three out of the five compounds showing significant binding to thrombin were identified as specific binders in the ASMS confirmation assay, of which two were also active in the biochemical MALDI-TOF setup. We established a throughput around 5,000 samples/wells per day that can be drastically increased by compound pooling to screen large compound libraries. During our journey of setting up ASMS for target engagement profiling, we have gained many technical and biological insights about ASMS as a semi-quantitative label-free method that I will highlight in my presentation.