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On-Chip Preconcentration Microchip Capillary Electrophoresis Based CE-PRM-LIVE for High-Throughput Selectivity Profiling of Deubiquitinase Inhibitors
Analytical Chemistry ( IF 6.7 ) Pub Date : 2022-06-21 , DOI: 10.1021/acs.analchem.2c01337
He Zhu 1 , J Scott Mellors 2 , Wai Cheung Chan 1 , J Will Thompson 2 , Scott B Ficarro 1 , Isidoro Tavares 1 , Ariana S Bratt 1 , Jens Decker 3 , Michael Krause 3 , Gary Kruppa 4 , Sara J Buhrlage 1 , Jarrod A Marto 1
Affiliation  

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.

中文翻译:

基于 CE-PRM-LIVE 的片上预富集微芯片毛细管电泳,用于去泛素酶抑制剂的高通量选择性分析

去泛素酶 (DUB) 家族包含约 100 种酶,可将泛素从底物蛋白上裂解下来,从而调节人类生理学的关键方面。尽管目前还没有批准的 DUB 靶向药物,并且大多数临床前小分子都是低效和/或多靶点的,但 DUB 最近已成为与疾病相关且化学上易于处理的药物。我们将包含集成“片上”C18 床 (SPE-ZipChip) 的新型毛细管电泳微芯片与 timsTOF Pro 质谱仪上我们最近描述的 PRM-LIVE 采集方案的 TMT 版本配对,以促进基于活性的快速蛋白质分析DUB 抑制剂。我们展示了 SPE-ZipChip 提高复杂样品蛋白质组覆盖率的能力,以及 TMT 标记样品的 CE-PRM-LIVE 定量完整性的能力。这些技术提供了一个平台,可在 15 分钟内跨越 49 个内源性 DUB 的多重检测中准确量化共价和可逆抑制剂的竞争性结合。
更新日期:2022-06-21
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