On-Chip Preconcentration Microchip Capillary Electrophoresis Based CE-PRM-LIVE for High-Throughput Selectivity Profiling of Deubiquitinase Inhibitors
| Autor: | He Zhu, J. Scott Mellors, Wai Cheung Chan, J. Will Thompson, Scott B. Ficarro, Isidoro Tavares, Ariana S. Bratt, Jens Decker, Michael Krause, Gary Kruppa, Sara J. Buhrlage, Jarrod A. Marto |
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| Rok vydání: | 2022 |
| Předmět: | |
| Zdroj: | Analytical Chemistry. 94:9508-9513 |
| ISSN: | 1520-6882 0003-2700 |
| Popis: | The family of deubiquitinases (DUBs) comprises ∼100 enzymes that cleave ubiquitin from substrate proteins and thereby regulate key aspects of human physiology. DUBs have recently emerged as disease-relevant and chemically tractable, although currently there are no approved DUB-targeting drugs and most preclinical small molecules are low-potency and/or multitargeted. We paired a novel capillary electrophoresis microchip containing an integrated, "on-chip" C18 bed (SPE-ZipChip) with a TMT version of our recently described PRM-LIVE acquisition scheme on a timsTOF Pro mass spectrometer to facilitate rapid activity-based protein profiling of DUB inhibitors. We demonstrate the ability of the SPE-ZipChip to improve proteome coverage of complex samples as well as the quantitation integrity of CE-PRM-LIVE for TMT labeled samples. These technologies provide a platform to accurately quantify competitive binding of covalent and reversible inhibitors in a multiplexed assay that spans 49 endogenous DUBs in less than 15 min. |
| Databáze: | OpenAIRE |
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