ABPP-HT - High-Throughput Activity-Based Profiling of Deubiquitylating Enzyme Inhibitors in a Cellular Context

Abstract

The potency and selectivity of a small molecule inhibitor are key parameters to assess during the early stages of drug discovery. In particular, it is very informative for characterizing compounds in a relevant cellular context in order to reveal potential off-target effects and drug efficacy. Activity-based probes are valuable tools for that purpose, however, obtaining cellular target engagement data in a high-throughput format has been particularly challenging. Here, we describe a new methodology named ABPP-HT (high-throughput-compatible activity-based protein profiling), implementing a semi-automated proteomic sample preparation workflow that increases the throughput capabilities of the classical ABPP workflow approximately ten times while preserving its enzyme profiling characteristics. Using a panel of deubiquitylating enzyme (DUB) inhibitors, we demonstrate the feasibility of ABPP-HT to provide compound selectivity profiles of endogenous DUBs in a cellular context at a fraction of time as compared to previous methodologies.

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

“…It is of note that DIA-NN recommends the match-between-runs (MBR) setting where the library-free mode is being applied, and this should be considered when interpreting this dataset. The samples used for these comparisons were comprised of MCF-7 cell lysates that were probe-labelled and immunoprecipitated by the HA tag of the HA-Ub-PA probe in triplicate using the high-throughput method of combining Agilent’s liquid handling platform, Evosep liquid chromatography and a timsTOF mass spectrometer as outlined previously [ 17 ].…”

“…FT827 is a highly selective USP7 inhibitor, HBX41108 is a cross-reactive USP7 inhibitor, P22077 is a selective USP7 inhibitor with known cross-reactivity to USP47, and PR619 is a pan-DUB inhibitor [ 12 , 17 , 28 ]. As a proof-of-concept experiment, inhibitor profiles were assessed across the identified DUBomes by DDA/Fragpipe and DIA/DIA-NN.…”

“…We have recently developed a high-throughput compatible activity-based protein profiling (ABPP-HT) method that allows the semi-automated analysis of multiple samples in a microplate format. To achieve this, we automated the affinity purification and proteomic sample preparation steps via a liquid handling robot combined with the high-throughout-compatible LC-MS/MS platform Evosep/tims TOF [ 17 ].…”

“…To overcome this, we recently developed a methodology that employed the use of the Agilent Bravo AssayMAP liquid handling platform to improve the ABPP’s immunoprecipitation and sample preparation throughput, and applied this in combination with runtimes of 15 min per sample on an Evosep/Bruker timsTOF LC-MS/MS [ 17 ]. This methodology, termed ABPP-HT, enabled a 10-fold increase in throughput, allowing for the analysis of 100 samples per day.…”

ABPP-HT* - Deep meets fast for activity-based profiling of deubiquitylating enzymes using advanced DIA mass spectrometry methods

“…To overcome this, we recently developed methodology that employed the use of the Agilent Bravo AssayMAP liquid handling platform to improve the ABPP's immunoprecipitation and sample preparation throughput, and applied this in combination with runtimes of 15 minutes per sample on a Evosep/Bruker timsTOF LC-MS/MS [17]. This methodology, termed ABPP-HT, enabled a 10-fold increase in throughput, allowing for the analysis of 100 samples per day.…”