Activity-Based Protein Profiling (ABPP) is a powerful chemical proteomics strategy that integrates synthetic chemistry, cell biology, and mass spectrometry. It employs specially designed activity-based probes (ABPs) to selectively bind active enzymes or proteins in complex biological systems such as cell lysates, live cells, and tissues. Unlike traditional proteomics, ABPP focuses on profiling protein activity rather than mere abundance, enabling the identification of functional targets of small molecules.

Keywords: ABPP, Activity-Based Protein Profiling, Activity-Based Probe

Technical Principle

Activity-based probes (ABPs) are derived from bioactive small molecules by appending functional tags such as photo-reactive groups or clickable handles, without compromising the molecule's biological activity. These probes are introduced into live cells, where they interact with active proteins. Upon UV irradiation, the photo-crosslinking group forms a covalent bond with nearby target proteins. Using click chemistry and biotin affinity tags, labeled proteins are enriched and then identified via proteomic mass spectrometry.

Experimental Workflow

1. Identification of Small Molecule Lead Compounds

Bioactive small molecules, usually under 1500 Da, may derive from chemical drugs, natural products, endogenous metabolites, or traditional Chinese medicine monomers. Lead compounds may be sourced from literature, metabolomics data, or in-house screening. They are acquired through commercial purchase, extraction, or synthesis.

2. Design and Synthesis of Activity-Based Probes (ABPs)

Reactive groups (e.g., hydroxyl, amino, halogen) on the small molecule are modified with a photoaffinity label (such as diazirine) and an alkyne moiety. Upon UV exposure, the photo-reactive diazirine forms a carbene that covalently cross-links to nearby proteins. The alkyne group is then used in a click chemistry reaction with azide-biotin to label the target with biotin. Biotin binds strongly to streptavidin, facilitating affinity purification of labeled proteins.

3. Cell Labeling and Enrichment

Cells are incubated with the ABP, followed by UV cross-linking, click chemistry tagging, and magnetic bead-based enrichment.

Three experimental groups are included: probe group, negative control, and competition group. The probe group identifies binding targets. The negative control (DMSO or parent molecule) helps eliminate background. The competition group, using excess unmodified molecule, distinguishes specific interactions.

Initial fluorescence gel scanning optimizes probe concentrations and competition conditions. Each group includes three biological replicates, totaling nine samples for proteomic mass spectrometry analysis.

4. Proteomic Mass Spectrometry Analysis

Mass spectrometry is conducted on all samples to identify proteins labeled by the ABP. Candidate targets are selected for further biological validation (validation strategy is handled separately).

Application Areas

- Functional elucidation of drug targets

- Target identification for TCM monomers and natural products

- Drug repurposing and mechanism studies

- Investigation of disease-associated protein activities

- Analysis of endogenous small molecule–protein interactions

Advantages

- Profiles active proteins in live-cell environments

- Identifies functionally relevant small molecule targets

- End-to-end service: probe synthesis, cell labeling, and target ID

- Interdisciplinary team of PhD-level chemists and biologists