The TSA methodology involves a series of simple steps that make it suitable for routine laboratory use. The following is a brief outline of the key steps involved in performing a TSA experiment:

• Protein sample preparation: purify the protein of interest ≥ 90% using established protocols, ensuring its quality and concentration.
• Fluorescent dye binding: add a fluorescent dye, such as SYPRO^TM Orange (a gold standard dye), to the protein solution. The dye selectively binds to hydrophobic regions exposed during protein unfolding.
• Thermal gradient generation: set up a thermal gradient by using a thermocycler. Typically, the temperature is increased in small increments, allowing for gradual protein denaturation.
• Fluorescence detection: monitor the fluorescence intensity of the dye at each temperature increment. The increase in fluorescence corresponds to protein denaturation.
• Data analysis: analyze the thermal-shift data to determine the protein melting temperature (T_m), which represents the temperature at which 50% of the protein is unfolded. T_m provides insights into protein stability and ligand interactions.

Overall, TSA offers numerous applications in various fields, including:

• Drug discovery: TSA enables screening of compound libraries for potential drug candidates. By measuring changes in the protein’s thermal stability in the presence of small molecules, compounds that stabilize or destabilize the target protein can be identified.
• Protein engineering: by assessing the thermal stability of protein mutants, TSA aids in the optimization of protein engineering strategies. It provides insights into how specific mutations or modifications affect the protein's stability, aiding in the design of more robust variants.
• Ligand interaction studies: TSA can evaluate the binding affinity and thermodynamics of protein-ligand interactions. By monitoring the shift in protein melting temperature upon ligand binding, the binding strength can be quantified and the impact of different ligands on protein stability can be assessed.
• Affinity and inhibition parameters: affinity parameters (K_d) can be directly extrapolated varying ligand concentrations. We demonstrated that these affinity parameters directly correlate with, for example, inhibitor parameters (IC₅₀, K_i).
• Quality control: in biopharmaceutical production, TSA plays a vital role in assessing the stability and quality of protein-based drugs. It ensures the consistency and integrity of protein formulations, preventing potential degradation during storage and transportation.

Additionally, TSA enables the ranking of compounds based on their affinity to the target protein, providing valuable insights into their binding properties. By using TSA, researchers can confidently evaluate compounds and identify those with a higher or lower affinity to the target protein, aiding in drug discovery and optimization processes.

When considering the use of TSA in early-stage drug discovery, we offer the following recommendations:

• TSA is highly effective in primary screening scenarios that involve small, focused or unbiased compound libraries. By utilizing TSA during the initial screening process, promising compounds can be quickly identified for further investigation.
• TSA can serve as secondary assay for hit validation and target engagement. By employing TSA in this capacity, additional insights can be gained into the interactions between compounds and target proteins, further validating potential hits.
• TSA is particularly useful in characterizing lead compounds during the hit-to-lead phase of drug discovery. These assay provides crucial information on the stability and binding properties of lead compounds, aiding in the selection of the most viable candidates for further development.
• For the identification of proteolysis targeting chimera (PROTAC^TM) scaffold molecules, TSA can be a valuable tool. The interactions between PROTACs and target proteins can be assessed by utilizing TSA, facilitating the development of effective protein-degradation strategies.
• TSA can also be employed to characterize protein-mutant variants. By subjecting mutant proteins to TSA, the impact of specific mutations on protein stability and ligand interactions can be evaluated, providing crucial insights into protein engineering and optimization.
• TSA offers several advantages over traditional methods of protein stability analysis, such as its simplicity, speed, and cost-effectiveness. In general, it requires measurably less protein amount than other classical biophysical methods and it can be easily miniaturized to 384 well/plate format, making it potentially suitable for mid/high-throughput screening.

One of the notable advantages of TSA is its ability to create a cell-free, label-free, and immobilization-free simplified environment for studying protein-ligand binding and assessing the effects of protein mutations. Because target proteins do not require labelling in TSA, the experimental setup is straightforward. This feature allows for a streamlined and efficient workflow, saving time and resources.

The main limitation of TSA is that it relies on the assumption that protein unfolding is accompanied by an increase in exposed hydrophobic regions, which may not always hold true. Certain protein classes may have complex unfolding profiles that cannot be accurately measured using TSA alone. In such cases, combining TSA with complementary techniques can provide a more comprehensive understanding of protein stability.

Moreover, although TSA is suitable for most target protein classes, it is important to note that it may not be ideal for studying membrane-bound proteins, such as G-protein coupled receptors (GPCRs). GPCRs, for example, have naturally exposed hydrophobic regions which allow the immediate binding of the fluorescent dye used in TSA, thus creating an unusually high fluorescence signal and preventing normal fluorescence curve readouts upon increasing temperature. Alternative techniques and/or fluorogenic dyes should be considered for such protein classes.

While TSA has its limitations, it remains a versatile technique that continues to contribute significantly to the advancement of biopharmaceutical research. By harnessing the power of TSA, new therapeutic targets can be uncovered, and more effective protein-binding drugs can be developed, bringing us closer to improved healthcare outcomes.