Serine Hydrolase Active-site Probes for Activity-based Enzyme Profiling

New fluorophosphonate probes selectively label and enrich active serine hydrolases

by Ryan D. Bomgarden, Ph.D.; Chris L. Etienne, Ph.D.; Rosa I. Viner, Ph.D.1; Monica Noonan, M.S.; John C. Rogers, Ph.D. - 06/01/11

The Thermo Scientific ActivX Serine Hydrolase Probes consist of a tag linked to a fluorophosphonate (FP) group, which specifically and covalently labels serines of enzymatically active serine hydrolases (Figure 1).^1-3 In addition to activity assessment, FP probes can be used to screen small-molecule inhibitors against enzymes derived from cell lysates, subcellular fractions, tissues and recombinant proteins.

Figure 1. Structure and labeling mechanism of serine hydrolase probes. Panel A: Structures of azido, desthiobiotin and fluorescent fluorophosphonate (FP) probes for affinity enrichment or detection of labeled enzymes. Panel B: Fluorophosphonate probes covalently label the active-site serine of enzymatically active serine hydrolases.

Depending on the active-site probe tag group, FP probe-labeled enzymes can be detected and quantified by Western blot, fluorescent gel imaging or mass spectrometry (MS) (Figure 2). TAMRA-FP probes can be used to label and detect serine hydrolase activity in samples using fluorescent gel imaging, capillary electrophoresis or mass spectrometry.³ Azido-FP probes are used in combination with phosphine- or alkyne-derivatized tags for either detection or enrichment. Desthiobiotin-FP probes can be used for both enrichment and detection of active-site-labeled proteins by western blot.

For the MS workflow, labeled proteins are reduced, alkylated and enzymatically digested to peptides. Only the active site-labeled peptides are enriched for analysis by LC-MS/MS. Both Western blotting and MS can be used for determining inhibitor target binding, but only the MS workflow can identify global inhibitor targets and off-targets.

Inhibitor profiling with serine hydrolase probes

The serine hydrolase superfamily is one of the largest, most diverse enzyme families in eukaryotic proteomes.⁴ Serine hydrolases are generally grouped into two large 100+ member families: serine proteases (e.g., trypsin, elastase and thrombin) and metabolic serine hydrolases. Although many family members share a common catalytic active site, metabolic serine hydrolases are divided into multiple enzyme subclasses, including esterases, lipases, amidases and peptidases based on differences in structure, catalytic mechanism and substrate preference. Because many of the proteolytic enzymes in this family are expressed as inactive pro-enzymes (zymogens), active-site probes are advantageous for activity assessment, especially when compared with other expression profiling techniques that measure only abundance.

To demonstrate the utility and specificity of active-site probes for serine hydrolase activity profiling, mouse brain and liver tissue lysates were labeled with TAMRA-FP probe and analyzed by fluorescent gel scanning (Figure 3A). Using the fluorescent gel workflow revealed that lysates pretreated with protease (AEBSF) or hydrolase (URB597 and CAY10401) inhibitors had different inhibition patterns of TAMRA-labeled serine hydrolases compared to untreated control samples. Probe labeling was also specific for active serine hydrolases, as heat denatured samples had signal similar to unlabeled controls samples.

Figure 3. Screening different inhibitors in mouse tissue lysates using serine hydrolase probes. Panel A: Mouse brain or liver tissue lysates (50μg) were pretreated with either DMSO (0) or serine hydrolase inhibitors 100μM AEBSF (A), URB597 (U) or CAY10401 (C) for 1 hour before labeling with 2μM TAMRA-FP probe. Labeled proteins were separated by SDS-PAGE and analyzed by fluorescent gel scanning using a Typhoon* Imager (GE Healthcare Life Sciences). Unlabeled lysate (-) and heat-denatured (Δ) lysate were used as controls to show probe labeling specificity. FAAH is indicated by an arrow. Panel B: Mouse brain tissue lysate (500μg) was pretreated as in Panel A and labeled with 2μM of desthiobiotin-FP probe. Desthiobiotin-FP-labeled proteins were denatured and enriched using streptavidin agarose before SDS-PAGE and Western blotting with a specific FAAH antibody.

We also used the desthiobiotin-FP serine hydrolase probe to enrich labeled proteins before western blotting. Hydrolase inhibitors URB597 and CAY10401 inhibited fatty acid amide hydrolase (FAAH; Figure 3B); whereas, the protease inhibitor AEBSF had no effect on enzyme activity. In addition to profiling inhibitor target specificity, active-site probes can be used to determine drug-binding constants and relative potency using inhibitor dose-response curves (IC₅₀).

Identify serine hydrolase active sites using mass spectrometry

Although assessment of inhibitor targets by fluorescent gel scanning or western blotting are simple methods to evaluate inhibitors, this workflow is limited by SDS-PAGE separation of proteins and the specific antibodies used. A more global approach to inhibitor assessment is required to identify drug targets and off-target effects. The MS workflow can identify protein targets by analyzing enriched proteins or the desthiobiotin-FP-labeled, active-site peptides.

Using the mass spectrometry workflow (Figure 2), we were able to determine active-site labeling sites of over 30 serine hydrolase active-site peptides from mouse brain tissue, including FAAH (Figure 4). We were also able to conclusively map the active-site serine of this enzyme using the electron transfer dissociation (ETD) MS/MS fragmentation technique. Using this approach to generate proteomic target lists, inhibitors can be compared for multiple cell and tissue types.

1. Liu,Y., et al. (1999). Activity-based protein profiling: The serine hydrolases. Proc Natl Acad Sci USA96(26):14694-9. 
2. Patricelli, M.P., et al. (2001). Direct visualization of serine hydrolase activities in complex proteomes using fluorescent active site-directed probes. Proteomics1:1067-71. 
3. Okerberg, E.S., et al. (2005). High-resolution functional proteomics by active-site peptide profiling. Proc Natl Acad Sci USA 102(14):4996-5001. 
4. Simon, G.M. and Cravatt, B.F. (2010). Activity-based proteomics of enzyme superfamilies: Serine hydrolases as a case study. JBC285(15):11051-5. 

ActivX FP Serine Hydrolase Probes are exclusively licensed from ActivX Biosciences Inc. to Thermo Fisher Scientific for research use only.