Cargando…
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...
Autores principales: | , , , , , |
---|---|
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 |
_version_ | 1784676471117185024 |
---|---|
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. |
format | Online Article Text |
id | pubmed-8955990 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
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 |
work_keys_str_mv | AT joneshannahbl abpphtdeepmeetsfastforactivitybasedprofilingofdeubiquitylatingenzymesusingadvanceddiamassspectrometrymethods AT heiligraphael abpphtdeepmeetsfastforactivitybasedprofilingofdeubiquitylatingenzymesusingadvanceddiamassspectrometrymethods AT davissimon abpphtdeepmeetsfastforactivitybasedprofilingofdeubiquitylatingenzymesusingadvanceddiamassspectrometrymethods AT fischerroman abpphtdeepmeetsfastforactivitybasedprofilingofdeubiquitylatingenzymesusingadvanceddiamassspectrometrymethods AT kesslerbenediktm abpphtdeepmeetsfastforactivitybasedprofilingofdeubiquitylatingenzymesusingadvanceddiamassspectrometrymethods AT pintofernandezadan abpphtdeepmeetsfastforactivitybasedprofilingofdeubiquitylatingenzymesusingadvanceddiamassspectrometrymethods |