Natural compounds exhibit remarkable structural diversity and biological activity, yet their mechanisms of action often remain elusive due to complex target interactions, low abundance of binding proteins, and transient ligand–protein interactions. To address these challenges, probe-based approaches have emerged as powerful and reliable technologies for target identification and mechanism-of-action studies.
As a company specializing in bioactive natural products target identification, we have developed and optimized a comprehensive suite of probe-based approaches. These technologies enable systematic, unbiased, and high-confidence identification of direct and functional protein targets in physiologically relevant systems. Our platform integrates chemical biology, proteomics, and advanced mass spectrometry to accelerate drug discovery from natural products.
What are Probe-based Approaches?
Probe-based approaches are chemical biology strategies that use small-molecule probes derived from bioactive compounds to directly interrogate protein targets in complex biological systems. These probes are rationally designed by modifying parent natural products with functional groups such as affinity tags, photo-reactive moieties, or reporter handles, while retaining biological activity. Once introduced into cells, tissues, or lysates, probes interact with their target proteins. The probe–protein complexes can then be selectively enriched, visualized, and identified using proteomic techniques. Compared with traditional genetic or phenotypic screening methods, Probe-based Approaches offer several key advantages:
- Direct identification of binding proteins
- Compatibility with native biological environments
- Applicability to endogenous proteins
- Ability to capture weak or transient interactions
These characteristics make probe-based approaches particularly well suited for studying complex natural products with unknown or multiple targets.
Explore Our Technology Platform
Our technology platform integrates multiple complementary probe-based approaches to ensure robust and comprehensive target identification. Each method is selected and optimized based on compound properties, biological context, and research objectives.
In this strategy, we use immobilized probe molecules in affinity chromatography to selectively enrich direct binding proteins from complex biological samples, enabling robust identification of high-affinity targets and associated protein complexes.
Activity-based Protein Profiling (ABPP)
For ABPP, we use probes designed to react with the active sites of specific enzyme families. By comparing probe labeling in the presence and absence of the natural product, we can identify which enzymes are functionally targeted.
For natural products that bind reversibly, PAL is indispensable. We incorporate a photoreactive group (e.g., diazirine) into the probe. Upon UV exposure, it forms a permanent covalent bond with the protein, "freezing" the interaction for rigorous analysis.
ABPP Coupled with SILAC Technique
We combine ABPP with stable isotope labeling by amino acids in cell culture (SILAC). By growing cells in "heavy" and "light" media, we use mass spectrometry to distinguish true targets from background noise with quantitative precision.
Instruments and Equipment at Our Platform
The successful implementation of Probe-based Approaches relies not only on sophisticated chemical probe design and experimental strategies, but also on advanced instrumentation and well-integrated laboratory infrastructure. At our platform, we have established a comprehensive set of state-of-the-art instruments and equipment that support the entire workflow of bioactive natural products target identification, from probe synthesis and biological evaluation to protein enrichment and high-resolution proteomic analysis. They include:
- High-performance liquid chromatography (HPLC) systems
- Liquid chromatography–mass spectrometry (LC-MS) systems
- Nuclear magnetic resonance (NMR) spectrometers
- Cell culture facilities and CO2 incubators
- Biosafety cabinets and automated cell counters
- Fluorescence and luminescence microplate readers
- SDS-PAGE and Western blotting systems
- Magnetic and agarose bead-based affinity purification systems
- High-speed and ultracentrifuges
- UV irradiation systems for Photoaffinity-Labelling (PAL)
- Automated and manual liquid handling systems
- Nano-flow liquid chromatography systems
- High-resolution LC–MS/MS platforms (Orbitrap / Q-TOF)
- Quantitative proteomics systems for SILAC and label-free analysis
- Bioinformatics and proteomics data analysis software platforms
Our probe-based approaches technology platform combines affinity chromatography, ABPP, PAL, and ABPP coupled with SILAC to address diverse target interaction scenarios, ranging from high-affinity binding to transient and low-abundance interactions. Supported by state-of-the-art instruments and comprehensive proteomics infrastructure, our platform delivers high-confidence, reproducible, and mechanistically informative results.
Online Inquiry
Frequently Asked Questions (FAQ)
Q1: Does modifying my natural product into a probe change its activity?
Q2: Can these approaches be used in living cells?
A: Yes. Techniques such as PAL and ABPP are fully compatible with live-cell experiments, preserving physiological relevance.
Q3: How do you reduce non-specific binding?
A: We employ competitive binding assays, quantitative proteomics (e.g., SILAC), and stringent washing conditions to ensure specificity.
Q4: Are these technologies suitable for low-abundance targets?
A: Yes. Probe enrichment and sensitive mass spectrometry allow detection of low-abundance proteins.
Q5: Can multiple targets be identified simultaneously?
A: Absolutely. Probe-based approaches are well suited for identifying multi-target profiles of complex natural products.