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

Autor: Hannah B. L. Jones, Raphael Heilig, Simon Davis, Roman Fischer, Benedikt M. Kessler, Adán Pinto-Fernández
Jazyk: angličtina
Rok vydání: 2022
Předmět:
Zdroj: International Journal of Molecular Sciences; Volume 23; Issue 6; Pages: 3263
ISSN: 1422-0067
DOI: 10.3390/ijms23063263
Popis: 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.
Databáze: OpenAIRE
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