Use of high-throughput mass spectrometry to reduce false positives in protease uHTS screens
| Autor: | Paul Zuck, Juncai Meng, Daniel Riley, Eric N. Johnson, Rachel Hunt, Jeffrey D. Hermes, Adam Amoss, Victor N. Uebele, Jeffrey W. Lusen, Joseph M. Rizzo, Amita Patel, Gregory C. Adam |
|---|---|
| Rok vydání: | 2014 |
| Předmět: |
Chromatography
Protease Dose-Response Relationship Drug Chemistry medicine.medical_treatment Reproducibility of Results Mass spectrometry Biochemistry Mass Spectrometry Analytical Chemistry High-Throughput Screening Assays Substrate Specificity Cycle time Aspartate protease Drug Discovery False positive paradox medicine Molecular Medicine Humans Protease Inhibitors Biotechnology Enzyme Assays Peptide Hydrolases |
| Zdroj: | Journal of biomolecular screening. 20(2) |
| ISSN: | 1552-454X |
| Popis: | As a label-free technology, mass spectrometry (MS) enables assays to be generated that monitor the conversion of substrates with native sequences to products without the requirement for substrate modifications or indirect detection methods. Although traditional liquid chromatography (LC)-MS methods are relatively slow for a high-throughput screening (HTS) paradigm, with cycle times typically ≥ 60 s per sample, the Agilent RapidFire High-Throughput Mass Spectrometry (HTMS) System, with a cycle time of 5-7 s per sample, enables rapid analysis of compound numbers compatible with HTS. By monitoring changes in mass directly, HTMS assays can be used as a triaging tool by eliminating large numbers of false positives resulting from fluorescent compound interference or from compounds interacting with hydrophobic fluorescent dyes appended to substrates. Herein, HTMS assays were developed for multiple protease programs, including cysteine, serine, and aspartyl proteases, and applied as a confirmatory assay. The confirmation rate for each protease assay averaged30%, independent of the primary assay technology used (i.e., luminescent, fluorescent, and time-resolved fluorescent technologies). Importantly,99% of compounds designed to inhibit the enzymes were confirmed by the corresponding HTMS assay. Hence, HTMS is an effective tool for removing detection-based false positives from ultrahigh-throughput screening, resulting in hit lists enriched in true actives for downstream dose response titrations and hit-to-lead efforts. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|