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Profiling serine hydrolase activities in complex proteomes.

D Kidd1, Y Liu, B F Cravatt

  • 1The Skaggs Institute for Chemical Biology and Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037, USA.

Biochemistry
|April 13, 2001
PubMed
Summary
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Researchers developed new chemical probes, FP-biotin and FP-peg-biotin, to analyze serine hydrolase activities in complex biological samples. These probes enable faster functional characterization and identification of enzymes like proteases and lipases.

Area of Science:

  • Biochemistry
  • Proteomics
  • Chemical Biology

Background:

  • Serine hydrolases are a large enzyme family with diverse functions.
  • Posttranslational modifications complicate their functional characterization using standard methods.
  • Activity-based probes are needed for global analysis of enzyme activities.

Purpose of the Study:

  • To synthesize and evaluate a new activity-based probe, FP-peg-biotin, for serine hydrolases.
  • To compare the coverage and kinetics of FP-biotin and FP-peg-biotin.
  • To demonstrate the utility of these probes for inhibitor selectivity studies and protein identification.

Main Methods:

  • Synthesis of FP-peg-biotin, a poly(ethylene glycol)-linked variant of FP-biotin.
  • Incubation of probes with soluble and membrane proteomes.

Related Experiment Videos

  • Kinetic analysis of enzyme-probe interactions.
  • Avidin-based affinity isolation of biotinylated proteins.

    • Main Results:

    • FP-biotin and FP-peg-biotin achieved similar maximal coverage of serine hydrolase activity.
    • Different serine hydrolases exhibited distinct reaction rates with each probe.
    • The probes facilitated the assessment of inhibitor selectivity in complex proteomes.
    • A method for identifying multiple enzyme classes (peptidases, lipases, esterases) was established.

    Conclusions:

    • Biotinylated fluorophosphonates are effective tools for global serine hydrolase activity profiling.
    • FP-peg-biotin offers an alternative probe with potentially different kinetic properties.
    • These chemical probes accelerate enzyme functional characterization and molecular identification in proteomics.