Activity-based protein profiling (ABPP) provides a functional proteomics solution by using reactive chemical probes to label active enzymes directly in complex biological systems. Competitive ABPP further extends this concept by allowing label-free target identification of unmodified compounds, making it ideally suited for natural product research. As a company dedicated to bioactive natural products target identification, we have built a comprehensive competitive ABPP platform that integrates advanced chemical probe design, state-of-the-art mass spectrometry, and robust bioinformatics to support drug discovery from hit identification to lead optimization.
Introduction to Competitive ABPP
One of the major limitations of conventional ABPP strategies is non-specific binding. Such non-specific interactions can complicate data interpretation and obscure the identification of true biological targets of the parent compound. To overcome this challenge, the competitive ABPP strategy has received increasing attention and has become a powerful refinement of traditional ABPP. In competitive ABPP, a biological proteome is first pre-incubated with the parent compound, which refers to the original bioactive molecule prior to any chemical transformation into a probe. The system is then treated with an activity-based probe that targets a specific class of functional proteins. If the parent compound interacts with a given protein, it competes with the probe for the same or overlapping binding site, thereby decreasing or completely preventing probe labeling of that protein. By quantitatively comparing probe labeling in the presence and absence of the parent compound, competitive ABPP enables the identification of proteins that are directly or functionally engaged by the compound of interest.
Fig. 1. Diagram of the competitive ABPP strategy [1].
Our Competitive ABPP Technology Platform
Our competitive ABPP technology platform is specifically designed to support target identification for bioactive natural products and small molecules.
Our Platform Integrates Four Core Components:
- Extensive Probe Library: We maintain a broad collection of validated activity-based probes covering major enzyme families, including serine hydrolases, cysteine proteases, kinases, deubiquitinases, oxidoreductases, and metabolic enzymes.
- Advanced Proteomics Infrastructure: High-resolution LC-MS/MS systems combined with quantitative proteomics strategies enable sensitive and reproducible detection of probe-labeled proteins.
- Bioinformatics and Data Analysis: Our proprietary data analysis pipelines provide statistically robust identification of competed targets, pathway enrichment analysis, and target prioritization based on biological relevance.
- Expert Scientific Support: Our multidisciplinary team of chemists, biologists, and proteomics experts collaborates closely with clients to design experiments, interpret data, and translate findings into actionable insights.
Our Competitive ABPP Workflow
We have refined our competitive ABPP workflow to ensure maximum sensitivity and reproducibility. The process typically follows these five main stages:
Step 1
Proteome Preparation
Extraction of proteins from relevant cell lines, tissues, or subcellular fractions while maintaining enzymatic activity. Where appropriate, competitive ABPP can also be performed in live cells to capture in situ target engagement.
Step 2
Competition Incubation
The proteome is pre-incubated with varying concentrations of the unmodified natural product (the "competitor").
Step 3
Probe Labeling
A specific ABP (containing a reactive group and a reporter tag like biotin) is added. The probe reacts with all available active sites not blocked by the natural product.
Step 4
Enrichment and Digestion
Biotinylated proteins are captured using streptavidin beads, followed by on-bead tryptic digestion.
Step 5
LC-MS/MS Analysis & Data Interpretation
Peptides are analyzed via quantitative mass spectrometry. A decrease in the MS signal for a specific protein in the treated sample identifies it as a potential target.
Advantages of Competitive ABPP Platform
- No chemical modification of parent compounds required
- Reduced non-specific binding and false positives
- Direct functional readout of target engagement
- Proteome-wide, unbiased target discovery
- Compatibility with complex and native biological systems
- Scalable for early discovery through lead optimization
Competitive ABPP has become a cornerstone technology for functional target identification in natural product-based drug discovery. Through our integrated platform and deep scientific expertise, we enable researchers to confidently uncover molecular targets, clarify mechanisms of action, and accelerate the translation of bioactive compounds into therapeutic opportunities.
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Reference
- Li G., et al. Currently available strategies for target identification of bioactive natural products[J]. Frontiers in Chemistry, 2021, 9: 761609.