Activity-Based Protein Profiling of the Serine Hydrolases
Activity-based protein profiling of the serine hydrolases is an analytical approach that integrates enzyme-reactive probe chemistry with high-resolution mass spectrometry to investigate the expression patterns and functional states of serine hydrolase family members. Serine hydrolases are ubiquitous in both eukaryotic and prokaryotic systems, playing vital roles in digestion, immune regulation, development, and apoptosis. Their catalytic mechanism involves a conserved serine residue at the active site, which mediates substrate hydrolysis through nucleophilic attack. Unlike conventional proteomics that focuses primarily on protein abundance, activity-based protein profiling of the serine hydrolases emphasizes the functional activity of enzymes. Notably, serine hydrolase activity is frequently dysregulated in various diseases without corresponding changes in expression levels. Such functional alterations can only be uncovered through activity-based labeling, underscoring the unique value of this approach in elucidating disease-relevant enzyme behavior. This makes activity-based protein profiling of the serine hydrolases especially powerful in studies of enzymatic regulation, signaling pathway modulation, and drug response profiling. In practice, this strategy has found extensive application in oncology, infectious disease, and neurodegenerative disorder research. Additionally, it serves as a key technique for drug mechanism elucidation, including the evaluation of candidate inhibitors targeting serine hydrolases and the validation of their potential as therapeutic targets.
The core principle of activity-based protein profiling of the serine hydrolases relies on the use of active-site-specific chemical probes—such as fluorophosphonate or phosphotriester derivatives—that covalently bind only to catalytically active serine hydrolases. These probes are typically conjugated with affinity tags (e.g., biotin or fluorescein) or isotopic labels to enable subsequent enrichment and quantification. Probe-labeled samples are then subjected to high-resolution mass spectrometric analysis, allowing for the identification and relative quantification of active enzyme populations. This technique offers high specificity, excellent throughput, and robust reproducibility, enabling the simultaneous profiling of hundreds of serine hydrolases and facilitating in-depth exploration of enzymatic regulatory networks.
The technical workflow of activity-based protein profiling of the serine hydrolases typically comprises three key stages. First, sample preparation and probe labeling must be conducted under non-denaturing conditions to preserve the enzymes' native tertiary structure. Second, labeled enzymes are enriched—commonly via streptavidin magnetic beads targeting biotin-tagged probes. Finally, the enriched samples undergo LC-MS/MS analysis coupled with database searching to achieve confident protein identification and quantification. To enhance biological interpretation, results are integrated with bioinformatic analyses such as Gene Ontology (GO) annotation, family-based enrichment, and hierarchical clustering heatmaps, providing a comprehensive view of the active serine hydrolase landscape.
MtoZ Biolabs specializes in functional proteomics research, offering high-quality chemical proteomics services. Powered by a skilled experimental team and advanced analytical platforms, we deliver detailed activity maps of the proteome to support clients in enzymatic target discovery and mechanistic disease studies, thereby facilitating the translation of activity-based protein profiling of the serine hydrolases into impactful drug development and clinical applications.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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