How to Identify Enzyme Activity Targets Using ABPP Technology?

Activity-Based Protein Profiling (ABPP) technology, which is proteomics analysis based on active sites. By covalently labeling active proteins with specific small molecule probes, ABPP can efficiently and accurately identify enzyme molecules in functional states, providing a revolutionary tool for target discovery and drug mechanism research.

 

I. The Principle Basis of Enzyme Target Identification by ABPP Technology

1. What is an 'Enzyme Activity Target'?

Enzyme activity targets refer to enzyme molecules that exhibit functional state changes under specific physiological or pathological conditions. For example, a certain protease shows significantly increased activity in cancer tissues, suggesting that it may play a key role in tumorigenesis. Identifying these enzyme targets not only helps to understand disease mechanisms but also provides specific directions for drug design.

 

2. Basic Principle of ABPP

The core of ABPP technology ischemical probes selectively labeling 'active' enzyme proteins. Its basic components include:

(1) Reactive Group (Warhead): Recognizes enzyme active sites and covalently binds;

（2）Linker: Adjusts probe structural stability;

（3）Tag: Used for capture and detection, such as biotin, fluorescein, alkyne, etc.

By adding these probes to biological samples, ABPP can specifically capture enzymes with catalytic functions and, through enrichment, enzymatic digestion, and LC-MS/MS analysis, ultimately pinpoint specific 'active targets'.

 

II. Detailed Experiment Workflow: How to Screen Enzyme Targets via ABPP?

Step 1: Sample Preparation and Probe Incubation

Add the designed ABP into cell lysates, tissue homogenates, or live cell systems. The probe covalently binds to the catalytic centers of specific enzymes, only labeling enzymes in an active state. If researching drug mechanisms, set up 'drug-treated groups' and 'untreated control groups' to facilitate subsequent identification of drug-inhibited targets.

 

Step 2: Protein Enrichment and Elution

The tag carried by the probe (such as biotin) can achieve selective capture of target proteins through beads (such as streptavidin beads). After multiple rounds of washing to remove background noise, the enriched samples primarily retain active enzyme proteins.

 

Step 3: Enzymatic Digestion and Mass Spectrometry Identification

The captured protein samples are digested by trypsin (or LysC) and analyzed using a high-resolution LC-MS/MS platform, allowing precise identification of peptide segments and protein identities bound to the probes. BGI usesOrbitrap Exploris 480 mass spectrometry system, combined with optimized digestion and elution workflows, can detect thousands of functional enzyme targets in samples as low as μg level.

 

Step 4: Quantitative Analysis and Target Screening

By comparing enzyme activity profiles between different experimental groups (such as drug-treated group vs. control group), significantly downregulated enzymes (i.e., inhibited) can be identified, suggesting they may be direct or indirect drug targets.

 

III. Advantages and Applicable Scenarios of ABPP Target Identification

1. High Specificity: Only Identifies Enzymes Truly 'At Work'

Unlike traditional expression profiling methods, ABPP can exclude false positive signals from 'dead enzymes' or non-catalytic states.

 

2. Allows 'Pre-Post Treatment' Comparisons

Enzyme activity profiles under drug, pathological conditions, etc., can be comparatively analyzed, facilitating the screening of active targets with regulatory significance.

 

3. Adaptable to Various Enzymes and Tissues

By choosing different Warheads, ABPP can cover various enzymes such as serine hydrolases, caspases, metalloproteases, protein tyrosine phosphatases, and more.

 

IV. Highlights of BGI's ABPP Target Identification Services

 

Service Modules	BGI Advantages
Probe Screening and Customization	Customized Warhead/Linker/Tag structures according to client targets
Sample Processing Solutions	Adaptable to various sample types such as cells, tissues, serum, in vivo, etc.
High Sensitivity Mass Spectrometry Platform	Using Orbitrap series, high sensitivity, strong quantitative accuracy
Multi-omics Integrated Analysis	Provides ABPP+metabolomics/transcriptomics integration solutions
Data Analysis Support	Provides comprehensive reports including target screening, pathway analysis, PPI network visualization, etc.

In target discovery, covalent drug mechanism validation, and disease mechanism research, ABPP technology is not just about 'seeing' the presence of proteins but truly identifying 'active' functional units. By scientifically designing experimental systems, coupled with high-quality probes and advanced mass spectrometry platforms, researchers canquickly, accurately, and systematically identify potential enzyme activity targets, laying the foundation for downstream functional validation and drug development. As a leading functional proteomics service platform in China,BGI BiotechnologyEstablished a comprehensive service system in the ABPP fieldIncluding probe design, sample processing, high-resolution mass spectrometry detection, and target analysisThis system has been widely applied in cutting-edge fields such as oncology, drug development, and immunometabolism.

 

Biotake Pike Biotechnology – Characterization of Biologics, High-Quality Multi-Omics Mass Spectrometry Service Provider

 

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