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

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...

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Autores principales: Jones, Hannah B. L., Heilig, Raphael, Davis, Simon, Fischer, Roman, Kessler, Benedikt M., Pinto-Fernández, Adán
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8955990/
https://www.ncbi.nlm.nih.gov/pubmed/35328685
http://dx.doi.org/10.3390/ijms23063263
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author Jones, Hannah B. L.
Heilig, Raphael
Davis, Simon
Fischer, Roman
Kessler, Benedikt M.
Pinto-Fernández, Adán
author_facet Jones, Hannah B. L.
Heilig, Raphael
Davis, Simon
Fischer, Roman
Kessler, Benedikt M.
Pinto-Fernández, Adán
author_sort Jones, Hannah B. L.
collection PubMed
description 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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spelling pubmed-89559902022-03-26 ABPP-HT*—Deep Meets Fast for Activity-Based Profiling of Deubiquitylating Enzymes Using Advanced DIA Mass Spectrometry Methods Jones, Hannah B. L. Heilig, Raphael Davis, Simon Fischer, Roman Kessler, Benedikt M. Pinto-Fernández, Adán Int J Mol Sci Article 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. MDPI 2022-03-17 /pmc/articles/PMC8955990/ /pubmed/35328685 http://dx.doi.org/10.3390/ijms23063263 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Jones, Hannah B. L.
Heilig, Raphael
Davis, Simon
Fischer, Roman
Kessler, Benedikt M.
Pinto-Fernández, Adán
ABPP-HT*—Deep Meets Fast for Activity-Based Profiling of Deubiquitylating Enzymes Using Advanced DIA Mass Spectrometry Methods
title ABPP-HT*—Deep Meets Fast for Activity-Based Profiling of Deubiquitylating Enzymes Using Advanced DIA Mass Spectrometry Methods
title_full ABPP-HT*—Deep Meets Fast for Activity-Based Profiling of Deubiquitylating Enzymes Using Advanced DIA Mass Spectrometry Methods
title_fullStr ABPP-HT*—Deep Meets Fast for Activity-Based Profiling of Deubiquitylating Enzymes Using Advanced DIA Mass Spectrometry Methods
title_full_unstemmed ABPP-HT*—Deep Meets Fast for Activity-Based Profiling of Deubiquitylating Enzymes Using Advanced DIA Mass Spectrometry Methods
title_short ABPP-HT*—Deep Meets Fast for Activity-Based Profiling of Deubiquitylating Enzymes Using Advanced DIA Mass Spectrometry Methods
title_sort abpp-ht*—deep meets fast for activity-based profiling of deubiquitylating enzymes using advanced dia mass spectrometry methods
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8955990/
https://www.ncbi.nlm.nih.gov/pubmed/35328685
http://dx.doi.org/10.3390/ijms23063263
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