Activity Based Protein Profiling Service
- The tag facilitates detection and enrichment of labeled proteins.
- Fluorescent tags (e.g., FITC, Cy5) enable direct visualization.
- Biotin tags allow for affinity purification via streptavidin-based systems.
- Click chemistry tags (e.g., alkyne groups) offer modularity for post-labeling modifications.
Activity Based Protein Profiling is an innovative approach in functional proteomics, designed to investigate the active state of proteins in complex biological systems. Unlike traditional proteomics, which focuses on protein abundance, Activity Based Protein Profiling uses activity-based probes (ABPs) to covalently label the active sites of target proteins. These labeled proteins are then identified and quantified using advanced techniques such as mass spectrometry, revealing functional insights beyond static expression levels. By bridging the gap between protein expression and function, Activity Based Protein Profiling enables researchers to explore dynamic biological processes, identify enzyme activities, and uncover potential drug targets. MtoZ Biolabs provides Activity Based Protein Profiling Service to empower your functional proteomics research. Combining state-of-the-art mass spectrometry platforms such as the Orbitrap Fusion Lumos with expertise in chemical probe design, our service ensures precise identification and quantification of active proteins.
The success of Activity Based Protein Profiling (ABPP) heavily relies on the design and synthesis of high-quality activity-based probes (ABPs). An effective ABP typically comprises three essential components:
1. Reactive Group (RG)
The reactive group is the functional core of the probe, designed to covalently bind to the active site of the target protein. Commonly used reactive groups include fluorophosphonates (FPs), chloromethyl ketones (CMKs), sulfonyl fluorides (SFs), and vinyl sulfones (VSs). These groups interact specifically with catalytic residues in active proteins.
2. Linker Group
The linker connects the reactive group with the tag group, optimizing the probe's physical and chemical properties, such as solubility and stability. The length and flexibility of the linker are carefully tuned to enhance the probe's specificity and reactivity.
3. Tag Group
Figure 1. Schematics of a Typical Activity-Based Probe
In Activity Based Protein Profiling, labeling methods are crucial for selective detection of active proteins, with two primary strategies: one-step and two-step labeling. One-step labeling directly combines the reactive group and detectable tag within the probe. During incubation, the probe binds to active proteins and enables immediate detection or enrichment, offering simplicity and speed. However, it has limited flexibility in tag selection. Two-step labeling separates binding and tagging processes. Probes carry a small bioorthogonal group (e.g., alkyne), allowing versatile tags to be attached later via click chemistry. This method minimizes steric hindrance and enhances sensitivity but requires additional steps. At MtoZ Biolabs, we offer both methods, ensuring tailored solutions for diverse research needs.
Figure 2. Two Primary Labeling Strategies in Activity Based Protein Profiling
Analysis Workflow
Wang, S. et al. Front Pharmacol. 2018.
Figure 3. A General Representation of the Activity Based Protein Profiling Workflow
Service Advantages
1. Advanced Analysis Platform: MtoZ Biolabs established an advanced Activity Based Protein Profiling Service platform, guaranteeing reliable, fast, and highly accurate analysis service.
2. One-stop service: We provide a comprehensive workflow covering from probe design to detection techniques.
3. Customized Strategic Planning: Tailors specific strategies to analyze dynamic protein changes in complex samples.
4. High-Data-Quality: Deep data coverage with strict data quality control. AI-powered bioinformatics platform integrates all activity based protein profiling data, providing clients with a comprehensive data report.
5. One-Time-Charge: Our pricing is transparent, no hidden fees or additional costs.
Applications
1. Activity Based Protein Profiling for the Functional Annotation of Enzymes
Activity Based Protein Profiling has emerged as a transformative tool in cancer research and enzyme characterization. For example, using fluorophosphonate probes, ABPP identified KIAA1363 as a highly active enzyme elevated in aggressive cancer cells. Competitive ABPP was then employed to develop a selective inhibitor, AS115, which enabled precise disruption of KIAA1363 activity. Metabolomic analysis revealed KIAA1363's critical role as a 2-acetyl MAGE hydrolase, regulating MAGE lipid levels and connecting key lipid signaling pathways involved in cancer progression. This multidimensional profiling strategy showcases Activity Based Protein Profiling's ability to uncover disease-related enzyme functions, design targeted inhibitors, and provide insights into complex metabolic networks, offering valuable applications in biomarker discovery, drug development, and cancer biology.
Barglow, K. T. et al. Nat Methods. 2007.
Figure 4. Multidimensional Profiling Strategy for the Annotation of the Cancer-Related Enzyme KIAA1363
2. Activity Based Protein Profiling for the Drug Target Identification and Mechanism Elucidation
Activity Based Protein Profiling serves as a robust tool for identifying drug targets and elucidating molecular mechanisms. For instance, the mechanism of action of resveratrol, a natural anticancer compound, was explored using a biotinylated activity-based probe (ABP). This probe, designed through structure-activity relationship (SAR) studies to preserve resveratrol's bioactivity, was incubated with melanoma cell lysates and enriched with streptavidin beads. Subsequent analysis identified histone deacetylase I (HDAC1) as the target protein, uncovering an epigenetic regulatory pathway involving focal adhesion kinase. ABPs operate in physiological conditions, interacting with active proteomes and living cells to reveal authentic drug-protein interactions. Biotin, frequently incorporated as a reporter group, enables effective enrichment and detection of probe-protein complexes, facilitating target identification. These capabilities make Activity Based Protein Profiling an invaluable tool for advancing drug discovery, understanding epigenetic mechanisms, and exploring therapeutic targets in complex biological systems.
Chen, X. et al. Signal Transduct Target Ther. 2020.
Figure 5. Target Identification of Resveratrol with Its Activity-Based Probe
Deliverables
1. Comprehensive Experimental Details
2. Materials, Instruments, and Methods
3. Total Ion Chromatogram & Quality Control Assessment (project-dependent)
4. Data Analysis, Preprocessing, and Estimation (project-dependent)
5. Bioinformatics Analysis
6. Raw Data Files
MtoZ Biolabs, an integrated Chromatography and Mass Spectrometry (MS) Services Provider, provides advanced proteomics, metabolomics, and biopharmaceutical analysis services to researchers in biochemistry, biotechnology, and biopharmaceutical fields. Our ultimate aim is to provide more rapid, high-throughput, and cost-effective analysis, with exceptional data quality and minimal sample consumption. If you are interested in our Activity Based Protein Profiling Service, please feel free to contact us.
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