4.7 Article

ABPP-HT*-Deep Meets Fast for Activity-Based Profiling of Deubiquitylating Enzymes Using Advanced DIA Mass Spectrometry Methods

期刊

出版社

MDPI
DOI: 10.3390/ijms23063263

关键词

activitomics; activity-based probes; chemical biology; data-independent acquisition mass spectrometry (DIA); deubiquitylating enzymes; drug discovery; mass spectrometry; proteomics; ubiquitin

资金

  1. Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Science (CIFMS), China [2018-I2M-2-002]
  2. Pfizer
  3. Brystol-Meyers Sqibb award

向作者/读者索取更多资源

Activity-based protein profiling (ABPP) is an important method for characterizing specific enzymes, and has proven invaluable in drug discovery and pharmacodynamic studies. In this study, an improved ABPP-HT method was developed, combining state-of-the-art MS analysis tools to achieve high throughput and improved data accuracy.
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.

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