Target Discovery Institute, Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7FZ, UK.
Chinese Academy for Medical Sciences Oxford Institute, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7FZ, UK.
Int J Mol Sci. 2022 Mar 17;23(6):3263. doi: 10.3390/ijms23063263.
Activity-based protein profiling (ABPP) uses a combination of activity-based chemical probes with mass spectrometry (MS) to selectively characterise a particular enzyme or enzyme class. ABPP has proven invaluable for profiling enzymatic inhibitors in drug discovery. When applied to cell extracts and cells, challenging the ABP-enzyme complex formation with a small molecule can simultaneously inform on potency, selectivity, reversibility/binding affinity, permeability, and stability. ABPP can also be applied to pharmacodynamic studies to inform on cellular target engagement within specific organs when applied to in vivo models. Recently, we established separate high depth and high throughput ABPP (ABPP-HT) protocols for the profiling of deubiquitylating enzymes (DUBs). However, the combination of the two, deep and fast, in one method has been elusive. To further increase the sensitivity of the current ABPP-HT workflow, we implemented state-of-the-art data-independent acquisition (DIA) and data-dependent acquisition (DDA) MS analysis tools. Hereby, we describe an improved methodology, ABPP-HT* (enhanced high-throughput-compatible activity-based protein profiling) that in combination with DIA MS methods, allowed for the consistent profiling of 35-40 DUBs and provided a reduced number of missing values, whilst maintaining a throughput of 100 samples per day.
基于活性的蛋白质谱分析（ABPP）将基于活性的化学探针与质谱（MS）结合使用，以选择性地表征特定的酶或酶类。ABPP 已被证明在药物发现中对酶抑制剂的分析非常有价值。当应用于细胞提取物和细胞时，用小分子挑战 ABP-酶复合物的形成可以同时提供效力、选择性、可逆性/结合亲和力、通透性和稳定性方面的信息。ABPP 还可以应用于药效动力学研究，当应用于体内模型时，提供特定器官内细胞靶标结合的信息。最近，我们为去泛素化酶（DUB）的分析建立了单独的高深度和高通量 ABPP（ABPP-HT）方案。然而，将两者（深度和快速）结合在一种方法中一直难以实现。为了进一步提高当前 ABPP-HT 工作流程的灵敏度，我们实施了最先进的无数据依赖性采集（DIA）和数据依赖性采集（DDA）MS 分析工具。在此，我们描述了一种改进的方法，ABPP-HT*（增强的高通量兼容基于活性的蛋白质谱分析），它与 DIA MS 方法相结合，允许对 35-40 个 DUB 进行一致的分析，并提供了较少的缺失值，同时保持每天 100 个样本的通量。
