Principles
Activity-Based Protein Profiling (ABPP) is a core technology in chemical proteomics that uses activity-site-specific covalent probes to achieve in situ labeling, enrichment, and identification of functional active proteins in complex biological samples.
The probe consists of three parts: a reaction group (targeting catalytic centers of serine hydrolases, cysteine proteases, oxidoreductases, etc.), a linker arm, and a reporter group (biotin, fluorophore, alkyne/azide).
Core Advantages
- Function-oriented: Labels only activated proteins, not inactive or precursor forms
- In situ physiological environment: Compatible with live cells, tissues, and body fluids
- High-precision identification: Combines LC-MS/MS for high-specificity enrichment and precise identification
- Wide dynamic range: Detects low-abundance active proteins with 8–10× sensitivity improvement
Standardized Workflow
- Probe design & activity validation (IC₅₀ within 3× of parent molecule)
- In situ labeling & competition control (37°C, 1–4 h incubation)
- Click chemistry enrichment (Cu(I)-catalyzed + streptavidin magnetic beads)
- Tryptic digestion + high-resolution LC-MS/MS detection (DDA/PRM mode)
- Bioinformatics analysis & target validation (Western Blot / CETSA / molecular docking)