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Comparison of Quantitative Mass Spectrometric Methods for Drug Target Identification by Thermal Proteome Profiling

Lookup NU author(s): Amy George, Frances Sidgwick, Dr Jessica WattORCiD, Dr Mathew Martin, Professor Matthias TrostORCiD, Dr Jose Luis Marin-RubioORCiD, Dr Maria Duenas FadicORCiD



This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).


© 2023 The Authors. Published by American Chemical SocietyThermal proteome profiling (TPP) provides a powerful approach to studying proteome-wide interactions of small therapeutic molecules and their target and off-target proteins, complementing phenotypic-based drug screens. Detecting differences in thermal stability due to target engagement requires high quantitative accuracy and consistent detection. Isobaric tandem mass tags (TMTs) are used to multiplex samples and increase quantification precision in TPP analysis by data-dependent acquisition (DDA). However, advances in data-independent acquisition (DIA) can provide higher sensitivity and protein coverage with reduced costs and sample preparation steps. Herein, we explored the performance of different DIA-based label-free quantification approaches compared to TMT-DDA for thermal shift quantitation. Acute myeloid leukemia cells were treated with losmapimod, a known inhibitor of MAPK14 (p38α). Label-free DIA approaches, and particularly the library-free mode in DIA-NN, were comparable of TMT-DDA in their ability to detect target engagement of losmapimod with MAPK14 and one of its downstream targets, MAPKAPK3. Using DIA for thermal shift quantitation is a cost-effective alternative to labeled quantitation in the TPP pipeline.

Publication metadata

Author(s): George AL, Sidgwick FR, Watt JE, Martin MP, Trost M, Marin-Rubio JL, Duenas ME

Publication type: Article

Publication status: Published

Journal: Journal of Proteome Research

Year: 2023

Volume: 22

Issue: 8

Pages: 2629–2640

Print publication date: 04/08/2023

Online publication date: 13/07/2023

Acceptance date: 17/02/2023

Date deposited: 08/09/2023

ISSN (print): 1535-3893

ISSN (electronic): 1535-3907

Publisher: American Chemical Society


DOI: 10.1021/acs.jproteome.3c00111

PubMed id: 37439223


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Funder referenceFunder name
215542/Z/19/ZWellcome Trust
European Union Horizon 2020
Marie Skłodowska-Curi
Wellcome Trust