Experimental Strategies to Improve Drug-target Identification in Mass Spectrometry-based Thermal Stability Assays

Significance of the Topic

Thermal stability assays coupled with mass spectrometry (MS-TSA) offer a robust approach to characterize protein–drug interactions by detecting ligand-induced shifts in protein melting temperatures. This technique addresses a critical bottleneck in drug discovery by enabling simultaneous identification of on-target and off-target binding events in complex biological samples.

Objectives and Study Overview

This study evaluates the combined use of three MS acquisition strategies—Phased-constrained Spectral Deconvolution Method (ΦSDM), high-field asymmetric ion mobility spectrometry (FAIMS), and an isobaric carrier channel—to enhance the qualitative and quantitative performance of MS-TSA. Jurkat cells treated with a MEK inhibitor or DMSO control underwent temperature-dependent fractionation, tryptic digestion, and TMT10plex labelling. Half of the samples were spiked with an isobarically labelled whole-cell digest, and eight acquisition modes were compared both individually and in combination.

Methodology and Instrumentation

Sample preparation and labelling:

• Jurkat cell lysates treated with MEK inhibitor or DMSO control
• Temperature gradient from 30 °C to 70 °C with supernatant collection, digestion, and TMT10plex labelling
• Isobaric carrier channel: detergent-aided labelled whole-cell digest to boost low-intensity peptides

Acquisition strategies:

• ΦSDM: Fourier-transform-based deconvolution at 15 K resolution, 22 ms max ion time
• FAIMS: CV settings −35, −50, −65 to reduce spectral interferences
• Combined mode (iMAATSA): simultaneous ΦSDM, FAIMS, and isobaric carrier channel

Main Results and Discussion

Each individual acquisition strategy outperformed the standard approach in terms of the number of high-quality protein melt curves. When all three techniques were combined, the number of unique high-confidence melt-curve comparisons increased substantially—up to 1800 proteins—compared to control workflows. ΦSDM at 15 K resolution delivered accurate melting transitions at high scan rates, while the isobaric carrier did not distort melting profiles or calculated Tm values. The synergistic effect of iMAATSA yielded the most comprehensive target engagement data.

Benefits and Practical Applications

• Enhanced proteome coverage and sensitivity for thermal profiling
• Accurate determination of protein melting temperatures with faster acquisition
• Scalable workflow for high-throughput drug screening campaigns
• Reduced spectral interferences and improved quantitation precision

Future Trends and Opportunities

Advancements in multiplexing, integration with data-independent acquisition, and machine learning–driven data analysis are expected to further elevate MS-TSA. Expansion to diverse cell types, tissues, and in vivo models will broaden the applicability of thermal profiling in drug discovery and chemical biology.

Conclusion

The combined application of ΦSDM, FAIMS, and an isobaric carrier channel (iMAATSA) significantly improves the depth, accuracy, and throughput of mass spectrometry–based thermal stability assays. This approach provides a powerful platform for comprehensive drug–target identification and quantitative proteome stability studies.

Instrumentation

• Thermo Fisher Scientific Easy Spray ES803 nano-LC system with 90 min gradient
• Orbitrap mass spectrometer with FAIMS Pro interface (CV −35, −50, −65)
• ΦSDM processing: 15 K resolution at m/z 200, 22 ms max ion time

References

1. Seashore-Ludlow B, Axelsson H, Lundbäck T. Perspective on CETSA literature: toward more quantitative data interpretation. SLAS Discov. 2020;25:118-126.
2. Savitski MM, Reinhard FB, Franken H, et al. Tracking cancer drugs in living cells by thermal profiling of the proteome. Science. 2014;346:1255784.
3. Molina DM, Jafari R, Ignatushchenko M, et al. Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay. Science. 2013;341:84.
4. Kelstrup C, Aizikov K, Batth TK, Kreutzmann A, Grinfeld D, Lange O, Mourad D, Makarov A, Olsen JV. Limits for resolving isobaric tandem mass tag reporter ions using phase-constrained spectrum deconvolution. J Proteome Res. 2018;17:1535-3893.
5. Schweppe DK, Eng JK, Yu Q, et al. Characterization and optimization of multiplexed quantitative analyses using high-field asymmetric-waveform ion mobility mass spectrometry. Anal Chem. 2019;91:4010-4016.
6. Yi L, Pigoń L, Haslam NJ, et al. Boosting to amplify signal with isobaric labeling (BASIL) strategy for comprehensive quantitative phosphoproteomic characterization of small populations of cells. Anal Chem. 2019;91:5794-5801.
7. Grinfeld D, Aizikov K, Kreutzmann A, Damoc E, Makarov A. Phase-constrained spectrum deconvolution for Fourier transform mass spectrometry. Anal Chem. 2017;89:1202-1211.
8. Jarzab A, Kurzawa N, Hopf T, et al. Meltome atlas—thermal proteome stability across the tree of life. Nat Methods. 2020;17:495-503.