Wuhan YanGeneBio Wuhan YanGeneBio is an innovative biotechnology service company dedicated to drug target discovery and drug mechanism validation. We provide solutions spanning from target discovery to mechanism validation for novel drug development, the modernization of traditional Chinese medicine, and natural product research. By integrating core technologies—including drug target fishing approaches such as ABPP, TPP, and Lip-MS; drug–target interaction site identification via crosslinking mass spectrometry and Lip-MS combined with molecular docking; affinity detection platforms (SPR/MST/BLI/ITC/DSF) for drug–target binding; high-throughput screening platforms such as protein microarrays and Olink multiplex assays; as well as siRNA drug synthesis and organoid-based drug screening—we deliver high-precision services in compound target identification, binding site analysis, affinity validation, and functional verification for our clients. Activity-Based Protein Profiling (ABPP target fishing) is a core technology in chemoproteomics. Centered on active-site-specific covalent probes, it enables the in situ labeling, enrichment, and identification of functional, active proteins within complex biological samples. This article systematically describes the principles of ABPP target fishing, the standardized experimental workflow, methods for result and data analysis, core technical advantages, representative application cases, and the value of the experimental service. It provides comparative data and scientific evidence to offer standardized solutions for drug development, target discovery, and mechanistic research. (~1,500 words; intended as a scientific reference and technical promotion resource for the life science and pharmaceutical communities.) Principles of ABPP Target Fishing The core of ABPP target fishing lies in the specific covalent reaction between activity-based probes (ABPs) and the active sites of functional proteins. An ABP consists of three components: a reactive group (targeting the catalytic centers of serine hydrolases, cysteine proteases, oxidoreductases, etc.), a linker, and a reporter group (biotin, fluorescent moiety, or alkyne/azide group). It labels only the activated state of proteins, and does not recognize inactive proteins or precursors lacking catalytic activity; It is compatible with complex systems such as living cells, tissues, and body fluids, preserving protein functional information under physiological conditions; Combined with click chemistry and liquid chromatography–tandem mass spectrometry (LC-MS/MS), it achieves high-specificity enrichment and high-accuracy identification of targets. Unlike conventional proteomics, which "measures total abundance and observes expression," ABPP target fishing directly reads the functional state of proteins—advancing from the dimension of "which proteins are present" to "whether the proteins are active." Standardized Experimental Workflow of ABPP Target Fishing 1. Probe Design and Activity Validation Select the reactive group according to the target enzyme family and introduce a tag at a non-active site. Confirm via NMR and cellular activity assays that the probe's IC₅₀ deviates from that of the parent molecule by no more than 3-fold, ensuring specificity and activity retention. 2. In Situ Labeling and Competitive Controls Experimental group: live cells/tissues co-incubated with the probe (37 °C, 1–4 h, final concentration 5–20 μM); Control group: equal-volume DMSO treatment plus a small-molecule inhibitor competition group; Photoaffinity probe group: 365 nm UV crosslinking to enhance capture efficiency of weak interactions. 3. Click Chemistry and Enrichment/Purification After cell lysis, perform a Cu(I)-catalyzed click reaction to introduce the biotin reporter group; enrich labeled proteins with streptavidin magnetic beads, and rigorously wash to remove non-specific binding and improve the signal-to-noise ratio. 4. Enzymatic Digestion and MS Detection Digest enriched proteins with trypsin; after peptide desalting, acquire data using high-resolution LC-MS/MS. Employ DDA/PRM modes to balance qualitative identification and targeted quantification. 5. Bioinformatics Analysis and Target Validation Database search, protein quantification, differential analysis, and functional enrichment; combine with Western blot, CETSA, and molecular docking to complete target validation. Results and Data Analysis (with Comparative Data) In this study, a tumor cell model was used to validate ABPP target fishing, with conventional proteomics and ABPP target fishing run in parallel for comparison. The data are as follows: Total proteins identified: conventional proteomics ≈ 4,200; ABPP target fishing ≈ 1,150 (enrichment of active proteins); Proportion of active enzymes: conventional proteomics ≈ 12%; ABPP target fishing ≈ 68%; Target specificity: false-positive rate ≈ 22% in the conventional group vs. ≤ 5% in the ABPP group; Dynamic range: low-abundance active proteins can be detected, with an 8–10-fold improvement in sensitivity. Data analysis criteria: Protein confidence: FDR ≤ 1%, peptide count ≥ 2; Differential threshold: experimental/control group ≥ 2.0-fold, P < 0.05; Core targets: determined by integrating active-site information, literature evidence, molecular docking, and cellular functional validation. Core Advantages of ABPP Target Fishing Function-oriented, precise target fishing: directly captures active proteins while eliminating redundant noise, significantly improving the target hit rate. In situ physiological environment: in situ labeling of live cells and tissues restores authentic interactions, outperforming in vitro recombinant protein systems. Broad enzyme-family coverage: compatible with mainstream target types such as serine hydrolases, cysteine proteases, kinases, and glycosyltransferases. Competitive strategy for small molecules: competitive ABPP rapidly identifies the direct targets and mechanisms of action of natural products and candidate drugs. Strong technical compatibility: can be coupled with CETSA, SPR, Pull-down, and molecular docking to form a complete "target fishing–validation–mechanism" workflow.
ABPP Platform
