Activity-based protein profiling: The serine hydrolases

Abstract

With the postgenome era rapidly approaching, new strategies for the functional analysis of proteins are needed. To date, proteomics efforts have primarily been confined to recording variations in protein level rather than activity. The ability to profile classes of proteins on the basis of changes in their activity would greatly accelerate both the assignment of protein function and the identification of potential pharmaceutical targets. Here, we describe the chemical synthesis and utility of an active-site directed probe for visualizing dynamics in the expression and function of an entire enzyme family, the serine hydrolases. By reacting this probe, a biotinylated fluorophosphonate referred to as FP-biotin, with crude tissue extracts, we quickly and with high sensitivity detect numerous serine hydrolases, many of which display tissue-restricted patterns of expression. Additionally, we show that FPbiotin labels these proteins in an activity-dependent manner that can be followed kinetically, offering a powerful means to monitor dynamics simultaneously in both protein function and expression.

Aryl Vinyl Sulfonates and Sulfones as Active Site-Directed and Mechanism-Based Probes for Protein Tyrosine Phosphatases

“…9 In particular, mechanism-based probes are available for covalent labeling of the cysteine and serine hydrolases, providing new insights into our understanding of these two families of proteases in cell biology and in diseases. 10,11 In addition to profiling the functional state of enzymes, mechanism-based probes are also seeing application in drug discovery, target identification, and discovery of previously uncharacterized enzyme activity. 9 To develop class-selective PTP probes, 4-fluromethylaryl phosphate was initially examined 12 as its hydrolysis generates a highly reactive quinone methide intermediate, which alkylate nucleophiles at, or near, the phosphatase active site.…”

How Shall We Use the Proteomics Toolbox for Biomarker Discovery?

“…It can be forecast that other positive selection strategies could be of interest, e.g. those based on active site classes 35 . However, due to the tremendous complexity of plasma, efficient strategies will be limited to those showing very high selectivity on the chemical sense of the word.…”

Activity Profiling of Papain-Like Cysteine Proteases in Plants

“…Activities of most Ser proteases can be profiled using FP-biotin, a biotinylated derivative of (DFP) (Liu et al, 1999), whereas papain-like Cys proteases can be profiled with DCG-04, a biotinylated derivative of E-64 (Greenbaum et al, 2000;Fig. 1A).…”