Activity-Based Protein Profiling Principles and Applications Comprehensive Analysis
Proteomics methods such as DDA (Data-Dependent Acquisition) or TMT-labeled quantification focus on protein expression levels but often overlook the true functional state of proteins. Activity-Based Protein Profiling (ABPP), as a function-oriented proteomics technique, uses specific probes to label active proteins, providing a new perspective on 'who is working'. Biotai PegBio integrates ABPP strategies with high-resolution mass spectrometry platforms, offering clients one-stop technical services from active probe screening to target identification, especially suitable for enzyme target discovery, drug mechanism research, and disease biomarker mining.
1. Principle of ABPP: Using chemical probes to lock active proteins
The core of ABPP lies inactivity-based probes (ABPs). These small molecule probes consist of three parts:
1. Warhead
A chemically reactive structural unit that can covalently bind to the active sites of specific enzyme families. For example, FP (fluorophosphonate) class Warhead for serine hydrolases and AOMK class Warhead for cysteine cathepsins.
2. Linker
Connects the warhead and the tag part, usually a flexible structure to avoid steric hindrance affecting binding efficiency, and can adjust probe selectivity and stability.
3. Tag
Used for subsequent protein enrichment and detection, common tags include biotin (for streptavidin magnetic bead capture), fluorescein (for gel detection), or alkyne/azide groups (for click chemistry labeling).
2. Key Advantages
1、Targeting active proteins: Only binds to enzymes with catalytic activity, avoiding interference from 'dead' enzymes;
2、Suitable for complex samples: Such as cell lysates, tissue homogenates, and even live cells;
3、Strong scalability: Supports high-throughput screening and diverse tag applications.
3. Technical Process: From Sample Processing to Target Identification
Step 1: Active Probe Incubation
Add ABP to the sample to be analyzed (such as cell lysates, tissue homogenates, or live cells) under suitable conditions to selectively label active proteins with the probe.
Step 2: Tag Enrichment and Elution
Use tags (such as biotin) combined with streptavidin magnetic beads to enrich binding proteins and wash away unbound impurities. This step ensures that only enzyme targets with catalytic activity are retained.
Step 3: Digestion and Mass Spectrometry Analysis
Enriched proteins are digested with trypsin or LysC and analyzed by LC-MS/MS (liquid chromatography-tandem mass spectrometry) to identify peptide sequences and relative abundance.
Step 4: Data Analysis and Functional Annotation
Combine databases like UniProt, GO/KEGG for functional annotation, and clustering analysis to identify differentially active proteins and construct regulatory networks. Biotai PegBio applies its self-developed data analysis pipeline in this step to significantly improve target discovery efficiency and accuracy.
Biotai PegBio adoptsthe high-resolution Orbitrap Exploris 480 platform, combined with self-optimized data analysis processes, to achieve high-sensitivity probe protein detection in sub-microgram samples.
4. Typical Application Scenarios
1. Enzyme Target Screening and Drug Mechanism Research
ABPP can be used for target validation of small molecule inhibitors. By comparing the active protein profiles before and after drug treatment, it can be determined whether the inhibitor effectively binds to the target enzyme. For example, some anticancer drugs target the serine hydrolase family, and ABPP can visually verify their specific inhibitory effects.
2. Disease-Related Enzyme Biomarker Mining
In studies of tumors, neurodegenerative diseases, metabolic disorders, etc., ABPP can help discover key active enzymes upregulated or downregulated in pathological states, indicating potential biomarkers or therapeutic targets.
3. Complementary Metabolomics Analysis
Combining ABPP with metabolomics can reveal the relationship between changes in enzyme activity and disruptions in metabolic pathways, especially suitable for analyzing metabolic enzyme regulation mechanisms.
4. Development and Validation of New Probes
The ABPP platform is not only a tool for applications but also avalidation platform for chemical probe development, assessing the targeting and selectivity of new probes.
5. Advantages and Limitations of ABPP Technology
| Advantages | Challenges |
| Visualizing enzyme activity states | High threshold for probe design |
| High specificity in screening active proteins | Requirements for probe permeability (cell/in vivo experiments) |
| Supports in situ or in vivo analysis | High dependency on MS platforms and data analysis |
| Support for target validation and mechanism research | Specific enzyme family probes still need to be developed |
Bontai Parker BiotechnologyBy building a high-throughput probe screening library, optimizing enrichment and washing processes, and utilizing advanced mass spectrometry platforms such as Orbitrap Exploris 480, we have significantly enhanced the throughput and data depth of ABPP projects, providing customers with one-on-one technical support throughout the entire process.
Advantages of Bontai Parker ABPP Services
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One-stop service: from probe screening and protein enrichment to MS identification and data analysis;
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High-throughput platform: supports parallel analysis of multiple samples;
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Multi-domain coverage: oncology, metabolism, immunity, neurology, infection, etc.;
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Professional team support: a cross-disciplinary team composed of experts in organic synthesis, protein chemistry, and mass spectrometry analysis.
Activity-Based Protein Profiling has overcome the traditional proteomics dilemma of 'visible but intangible', precisely capturing active proteins from a functional perspective. It has become one of the core technologies in functional proteomics research. With the emergence of more highly selective probes and data analysis tools, ABPP willshow broader prospects in directions such as precision medicine, target discovery, and new drug development. Bontai Parker Biotechnology will continue to deepen the ABPP technology platform, assisting researchers in more quickly, accurately, and comprehensively uncovering protein function networks.
Bontai Parker Biotechnology--Characterization of biological products, high-quality multi-omics mass spectrometry detection service provider
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