Thermal Proteome Profiling (TPP) is a key method within Cellular Thermal Shift Assay (CETSA), designed to detect the binding efficiency between drugs and target proteins within cells. The principle is that when a protein binds to a drug molecule, it typically becomes more stable. As temperature increases, proteins generally denature; however, drug-bound proteins exhibit increased stability, resulting in a rightward shift of their thermal melting curves. By combining this principle with mass spectrometry, it's possible to assess the thermal denaturation states of the entire proteome across multiple temperatures, thereby determining the thermal shift of each protein.

Keywords: Thermal Proteome Profiling Technology

Technical Principle

In Thermal Proteome Profiling (TPP), compounds and control samples are incubated separately with the biological sample. These mixtures are then subjected to a range of temperatures. Upon heating, ligand-bound proteins remain relatively stable and folded, whereas unbound proteins denature and precipitate. The soluble proteins are then analyzed using immunoblotting or mass spectrometry to assess their thermal stability, confirming compound-protein interactions within cells.

Applications

- Small molecules with complex structure-activity relationships or difficult to modify

- One-to-one validation of small molecule binding to target proteins

Experimental Workflow

TPP encompasses several approaches:

- TPP-TR (Temperature Range): Analyzes protein stability across a range of temperatures.

- TPP-CCR (Compound Concentration Range): Assesses protein stability across varying compound concentrations.

- 2D-TPP (Two-Dimensional TPP): Combines both temperature and concentration variations.

General steps include:

1. Sample Preparation

Involves preparing cell materials under various conditions, which can include cell extracts, intact cells, tissues, body fluids (like blood), or microorganisms (such as archaea, bacteria, protozoa, fungi). Conditions may vary chemically (e.g., drugs or metabolites), genetically (e.g., gene knockouts or overexpression), environmentally, or by cell state (e.g., different cell cycle stages).

2. Thermal Treatment

Samples are briefly heated (typically for 3 minutes) to induce protein denaturation and aggregation.

3. Collection of Soluble Proteins

Soluble proteins are collected via ultracentrifugation or low-speed centrifugation using porous filter plates.

4. Proteomic Analysis

Soluble proteins are digested into peptides using proteases. Samples are labeled with tandem mass tags (TMT) for quantification and analyzed via mass spectrometry.

5. Data Analysis

Mass spectrometry data is processed using proteomics databases to identify and quantify proteins. It aims to identify proteins whose thermal stability changes under different conditions, depending on the TPP approach used.

Advantages

- No need for small molecule modification

- Identifies direct binding targets, reflecting natural binding states

- Rapid one-to-one validation of small molecule and target protein interactions