Application of activity-based protein profiling to study enzyme function in adipocytes

Abstract

Activity-based protein profiling (ABPP) is a chemical proteomics approach that utilizes small-molecule probes to determine the functional state of enzymes directly in native systems. ABPP probes selectively label active enzymes, but not their inactive forms, facilitating the characterization of changes in enzyme activity that occur without alterations in protein levels. ABPP can be a tool superior to conventional gene expression and proteomic profiling methods to discover new enzymes active in adipocytes and to detect differences in the activity of characterized enzymes that may be associated with disorders of adipose tissue function. ABPP probes have been developed that react selectively with most members of specific enzyme classes. Here, using as an example the serine hydrolase family that includes many enzymes with critical roles in adipocyte physiology, we describe methods to apply ABPP analysis to the study of adipocyte enzymatic pathways.

Keywords: ABPP probes; Activity-based protein profiling; Adipocyte enzymes; Chemoproteomics methods; Enzyme activity; Functional proteomics; Serine hydrolases.

Figure 1. Basics of Activity-Based Protein Profiling (ABPP)

(a) Schematic of ABPP probes showing their basic features: 1) a reactive head group that targets a specific enzyme class, and 2) a reporter tag. (b) Gel-based ABPP. A fluorophosphonate-reactive group can be coupled to a flurophore tag (e.g., rhodamine) to covalently label and detect active serine hydrolases by SDS-PAGE and in-gel fluorescence scanning. (c) ABPP-MudPIT. Biotin-conjugated ABPP probes can be used to label, enrich, and identify by mass spectrometry active serine hydrolases (SH).

Figure 2. ABPP probes are available for a variety of enzyme classes

Figure 3. Competitive ABPP for enzyme and inhibitor discovery

In a competitive Activity-Based Protein Profiling experiment, cells, animals, or prepared proteomes are first treated with either an inhibitor or vehicle and subsequently labeled with a broad ABPP probe. Gel-based ABPP or ABPP-MudPIT is then used to either visualize or identify active enzymes (i.e. labeled by the ABPP probe). An enzyme that is the target of an inhibitor will show reduced signals in the inhibitor-treated samples relative to vehicle controls.

Figure 4. Sample ABPP data obtained with FP-based probes

(a) Reporter-tagged fluorophosphonate ABPP probes that react covalently only with the active form of serine hydrolases can be used to (b) profile the pattern of serine hydrolase activity by gel-based ABPP during differentiation of 10T1/2 adipocytes or (c) identify active serine hydrolases in mouse white adipose tissue using ABPP-MudPIT.

Figure 5. Competitive ABPP-SILAC to identify the target of enzyme inhibitors

Cells are grown in different media with isotopically stable amino acids. Proteomes prepared from compound-treated “heavy” and vehicle-treated “light” cells are labeled with a biotin-conjugated ABPP probe, and mixed in a 1:1 ratio. Following avidin enrichment and on-bead digestion, samples are analyzed by mass spectrometry and representative peptides belonging to treated or control cells are identified by mass-to-charge shift.