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

Abstract

Thermal 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.

Stability-based approaches in chemoproteomics

“…As outlined in our previous work, different label-free DIA approaches within a 1D-TPP workflow perform comparably to TMT-DDA (Ref. 53 ). It offers experimental flexibility with no restriction by the number of available TMT channels.…”

High-throughput drug target discovery using a fully automated proteomics sample preparation platform

“…In comparison, label free quantification (LFQ) with DIA is unlimited in sample numbers and suitable for large-scale drug target identification. Although the use of DIA in TPP analysis has been documented at the time of this writing, 30,31 no studies have systematically evaluated the performance of TMT and DIA methods for single temperature TPP analysis. Here, we conducted a comparison between the TMT and DIA methods, referred to as tmtCETSA and diaCETSA, respectively.…”

Chemoproteomics validates selective targeting of Plasmodium M1 alanyl aminopeptidase as a cross-species strategy to treat malaria

“…In contrast, using DIA analyses in thermal proteome pro ling was recently shown to be an effective and economical alternative to traditional DDA-based thermal shift quantitation work ows. 52 DIA-based protein quanti cation also results in high run-to-run reproducibility and a comprehensive dataset that is not biased towards highly abundant proteins. 43,59 Our DIA-based thermal stability proteomics work ow identi ed approximately 47% of the detectable P. falciparum blood stage proteome, 51 with the undetected proteins primarily comprised of membrane-bound proteins that are not readily extracted with detergentfree buffers required for stability assays of soluble proteins.…”

“…We initially developed a streamlined thermal stability proteomics work ow that combined traditional thermal proteome pro ling methods with an optimised data-independent acquisition (DIA)-LC-MS/MS approach. 51,52 To identify the binding target/s of MIPS2673 in P. falciparum asexual blood stages in a proteome-wide manner, we used native parasite lysates, as direct drug-protein interactions are more selectively identi ed in cellular lysates, rather than live cells that are susceptible to downstream effects of drug action. The parasite lysates were exposed to 1 µM or 4 µM of MIPS2673 or vehicle (DMSO control) for 3 minutes prior to heating at 60 °C, a temperature that should allow detection of most drug-induced protein stabilisation events in an untargeted manner with wide proteome coverage.…”