An improved synthesis of a fluorophosphonate-polyethylene glycol-biotin probe and its use against competitive substrates.

ABSTRACT: The fluorophosphonate (FP) moiety attached to a biotin tag is a prototype chemical probe used to quantitatively analyze and enrich active serine hydrolases in complex proteomes in an approach called activity-based protein profiling (ABPP). In this study we have designed a novel synthetic route to a known FP probe linked by polyethylene glycol to a biotin tag (FP-PEG-biotin). Our route markedly increases the efficiency of the probe synthesis and overcomes several problems of a prior synthesis. As a proof of principle, FP-PEG-biotin was evaluated against isolated protein mixtures and different rat-tissue homogenates, showing its ability to specifically target serine hydrolases. We also assessed the ability of FP-PEG-biotin to compete with substrates that have high enzyme turnover rates. The reduced protein-band intensities resulting in these competition studies demonstrate a new application of FP-based probes seldom explored before.

SUBMITTER: Xu H 

PROVIDER: S-EPMC3566859 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature