What Is ABPP and Why It Is Transforming Functional Proteomics?
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Warhead (reactive moiety): Covalently reacts with the active site of the enzyme.
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Linker: Provides structural flexibility and minimizes steric hindrance.
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Reporter group: Facilitates fluorescence imaging, affinity purification, or mass spectrometric identification.
- Protein presence does not necessarily imply protein functionality.
What Is ABPP?
Activity-Based Protein Profiling (ABPP) is a chemical probe–based proteomic strategy specifically designed to detect the catalytically active states of enzymes, rather than merely measuring their expression levels. ABPP employs Activity-Based Probes (ABPs) that selectively and covalently label the catalytic sites of active enzymes, thereby enabling the identification of enzymes with confirmed functional activity in complex biological samples. This technique represents a close integration of chemical biology, proteomics, and enzymology, and is particularly valuable for investigating enzyme function, screening drug targets, and comparing enzymatic activity under distinct physiological or pathological conditions.
The underlying principle is to design chemical probes capable of forming covalent bonds with enzyme active sites, such that probe labeling occurs exclusively when the enzyme is in a catalytically competent state. An ABPP probe typically comprises three core components:
Why Is ABPP Transforming Functional Proteomics?
Conventional proteomics approaches (e.g., label-free quantification, iTRAQ, TMT) primarily focus on protein abundance or expression levels. However, a fundamental limitation exists:
Many enzymes, even when highly expressed in cells, can be catalytically inactive due to inhibitory phosphorylation, conformational locking, or binding to endogenous inhibitors. Such distinctions are often beyond the resolution of conventional proteomics. ABPP overcomes this limitation, offering disruptive advantages in several key aspects:
1. Prioritizing Activity Over Abundance
By covalently labeling only catalytically active enzymes, ABPP avoids misinterpretation caused by inactive yet abundant proteins. For example, a cancer-associated protease might be highly expressed but rendered completely inactive due to conformational constraints, proteomics might incorrectly classify it as active, whereas ABPP accurately differentiates it.
2. High Specificity and Functional Targeting
Through the design of warheads tailored to distinct enzyme families, ABPP can selectively target specific classes of enzymes, including:
(1) Serine Hydrolases
(2) Cysteine Proteases
(3) Metalloproteases
(4) Deacetylases
(5) Esterases, Lipases, Sulfatases, and others
3. Compatibility with Complex Samples and in Situ Imaging
ABPP can be applied to cell lysates, tissue samples, living cells, and even animal models. Fluorescently tagged probes enable spatial imaging of active enzymes within cells, while biotinylated probes allow affinity-based isolation of active enzymes followed by identification through mass spectrometry.
4. Intrinsic Suitability for Drug Discovery
ABPP is highly compatible with drug target validation and high-throughput screening. Competitive ABPP assays can determine whether candidate small molecules effectively inhibit a target enzyme’s activity. A substantial reduction in activity upon compound treatment indicates successful binding to the enzyme’s active site.
Core Workflow of ABPP Technology
1. Selection or Synthesis of Activity-Based Probes
Warheads are designed according to the target enzyme family. Reporter groups may be fluorescent, biotin, or alkyne moieties.
2. Sample Preparation
Cells or tissues are lysed, or alternatively, live-cell or in situ tissue labeling can be performed.
3. Probe Incubation
Temperature, incubation time, and probe concentration are carefully controlled; competitive assays may be conducted by adding specific inhibitors.
4. Purification and Detection
Click chemistry–based enrichment is employed. Detection methods include SDS-PAGE, Western blotting, and LC–MS/MS.
5. Data Analysis
Active enzymes are qualitatively identified, and quantitative comparisons of activity levels can be made across experimental conditions (e.g., before and after drug treatment).
Technological Integration Driving ABPP Toward Multidimensional Functional Omics
In recent years, ABPP has been progressively integrated into multidimensional omics platforms. For instance, coupling ABPP with quantitative mass spectrometry (e.g., TMT, DIA) enables high-throughput quantification of enzyme activity, while integration with transcriptomics, metabolomics, or spatial omics reveals the temporal and spatial dynamics of enzymatic function. Moreover, advances in click chemistry–based probe design have extended ABPP applications to in situ imaging and in vivo studies, marking a transition from purely in vitro assays toward the forefront of precision biomedical research.
The transformative potential of ABPP lies in its ability to overcome the conceptual limitation that expression equals function, redefining protein functional states from the perspective of catalytic activity. This approach not only enhances our capacity to elucidate the regulatory mechanisms of protein function but also accelerates drug discovery, biomarker identification, and the advancement of precision medicine. As a leading functional proteomics service provider, MtoZ Biolabs has developed a comprehensive ABPP platform encompassing probe synthesis, sample preparation, click chemistry reactions, high-throughput mass spectrometry, and bioinformatic interpretation. Leveraging a proprietary library of high-activity chemical probes and state-of-the-art mass spectrometry capabilities, MtoZ Biolabs delivers high-quality, customized enzyme activity profiling solutions to advance life science research.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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