As a company dedicated to providing bioactive natural products target identification, we focus on label-free approaches that preserve native biological contexts. Among these, thermal proteome profiling (TPP) has emerged as a powerful, reliable, and scalable technology to identify drug–protein interactions directly in cells, tissues, or lysates.
What is TPP and Its Principle
TPP is a label-free, mass spectrometry–based technology for unbiased, proteome-wide identification of protein targets and affected pathways of bioactive compounds, including natural products. By enabling target discovery directly in cells, tissues, or lysates without chemical labeling or prior target assumptions, TPP is particularly well suited for compounds with complex structures and polypharmacological effects.
The scientific principle underlying TPP is based on the intrinsic relationship between protein thermal stability and ligand binding. Proteins possess defined three-dimensional structures that begin to unfold and aggregate when exposed to increasing temperatures, each exhibiting a characteristic thermal melting behavior. When a compound binds to a protein, this interaction can alter the protein's conformational stability, resulting in a measurable shift in its thermal melting profile. By quantitatively comparing protein stability across a temperature gradient in compound-treated versus control samples, TPP detects proteins whose thermal behavior is significantly altered upon compound exposure. Using high-resolution quantitative mass spectrometry, these thermal stability changes are measured simultaneously across thousands of proteins, enabling proteome-wide detection of target engagement. Proteins exhibiting significant thermal shifts may represent direct binding targets, while additional affected proteins can reflect stabilized protein complexes or downstream pathway modulation.
Fig. 1. Principle of TPP.
Our TPP-based Technology Platform
Our TPP-based technology platform has been specifically optimized for bioactive natural products and complex extracts. We combine advanced sample preparation, state-of-the-art mass spectrometry, and proprietary bioinformatics pipelines to deliver high-confidence target identification.
Key Features of Our Platform
- Compatibility with diverse natural products, including plant extracts, microbial metabolites, and marine compounds
- Label-free and native-condition analysis, preserving physiological relevance
- Flexible experimental formats, including cell-based TPP, lysate-based TPP, and tissue TPP
- Integration with orthogonal validation assays, such as DARTS and functional proteomics
Typical Workflow
Step 1
Sample Treatment
Living cells or cell lysates are incubated with the compound of interest and a vehicle control (DMSO). We offer various concentrations to determine dose-response relationships.
Step 2
Thermal Challenge
The samples are divided into several aliquots and heated to a range of temperatures (typically 37 °C to 67 °C). Following heating, the samples are cooled, and the precipitated (denatured) proteins are removed via ultracentrifugation.
Step 3
Protein Digestion & TMT Labeling
The remaining soluble proteins are reduced, alkylated, and digested into peptides. Each temperature point is labeled with a specific Tandem Mass Tag (TMT). This allows us to multiplex the samples, ensuring that all temperature points for a specific treatment are analyzed in a single MS run to minimize technical variation.
Step 4
LC-MS/MS Analysis
Peptides are separated by liquid chromatography and analyzed by high-resolution mass spectrometry.
Step 5
Data Processing & Bioinformatics
We utilize proprietary algorithms to fit melting curves for every identified protein. By comparing the Tm values between the treated and control groups, we calculate the ΔTm and identify statistically significant "hits."
Key Advantages of TPP
- Unbiased and Proteome-Wide: No prior knowledge of targets is required, enabling discovery-driven research.
- Label-Free: Eliminates the need for chemical modification of natural products, preserving activity and structure.
- Physiologically Relevant: Can be performed in intact cells or tissues, reflecting native biological environments.
- High Specificity and Sensitivity: Thermal shifts provide direct evidence of target engagement.
- Scalable and Reproducible: Suitable for both early discovery and advanced lead optimization stages.
- Multi-Target Detection: Particularly valuable for natural products with polypharmacological effects.
TPP represents a transformative, label-free approach for target identification, particularly in the field of bioactive natural products. By integrating robust experimental workflows with advanced proteomics and data analysis, our TPP-based technology platform enables researchers to uncover true molecular targets and accelerate drug discovery with confidence.
Online Inquiry
Frequently Asked Questions (FAQ)
Q1: Can TPP distinguish direct from indirect targets?
Q2: Is TPP compatible with complex natural product extracts?
A: Yes. Our platform has been optimized to handle complex mixtures and can identify multiple targets simultaneously.
Q3: What sample types can be used? What amount of compound is required?
A: TPP can be performed in cell lines, primary cells, tissues, or cell lysates, depending on the research question. Typically, we require only a few milligrams of the purified compound, depending on the potency and the number of replicates requested.
Q4: How does TPP differ from DARTS or SPROX?
A: While all are label-free, TPP is generally considered more robust for cellular applications and provides better proteomic depth due to its compatibility with TMT multiplexing and thermal shift principles.