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Microfluidic Mobility Shift Assay for Real-Time Analysis of Peptide N-Palmitoylation.

Thomas Lanyon-Hogg1, Neki V Patel1, Markus Ritzefeld1

  • 11 Department of Chemistry, Imperial College London, London, UK.

SLAS Discovery : Advancing Life Sciences R & D
|March 16, 2017
PubMed
Summary

Researchers developed a novel microfluidic mobility shift assay (MSA) for direct, real-time measurement of Sonic hedgehog (Shh) palmitoylation. This assay overcomes limitations of existing methods, enabling efficient study of Hedgehog acyltransferase (Hhat) activity and inhibitor screening.

Keywords:
enzyme assays or enzyme kineticsfluorescence methodslipids or lipid metabolismmedicinal chemistry

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Area of Science:

  • Biochemistry and Molecular Biology
  • Chemical Biology
  • Microfluidics and Assay Development

Background:

  • The Hedgehog signaling pathway is crucial for development and implicated in various cancers.
  • Sonic hedgehog (Shh) protein requires dual lipidation, including N-terminal palmitoylation by Hedgehog acyltransferase (Hhat), for its function.
  • Existing Hhat activity assays have significant limitations including safety, cost, manual handling, and indirect measurements.

Purpose of the Study:

  • To develop a novel, direct, and real-time assay for quantifying Shh palmitoylation.
  • To overcome the limitations of existing peptide lipidation assays.
  • To enable efficient study of Hhat enzyme kinetics and small-molecule inhibitor screening.

Main Methods:

  • Development of a microfluidic mobility shift assay (MSA) utilizing fluorescently labeled Shh peptides.
  • Separation of Shh amine-substrate and palmitoylated Shh amide-product based on charge and hydrodynamic radius.
  • Online fluorescence intensity measurements for real-time quantification of Hhat activity and inhibitor IC50 determination.

Main Results:

  • The MSA successfully separated and quantified fluorescently labeled Shh substrate and product peptides.
  • The assay enabled real-time monitoring of Hhat-catalyzed palmitoylation reactions.
  • The MSA was used to determine kinetic parameters and IC50 values for small-molecule inhibitors.

Conclusions:

  • The microfluidic mobility shift assay (MSA) provides a direct, real-time, and sensitive method for measuring N-lipidation.
  • MSA offers a powerful alternative to existing assays for studying Hhat activity and developing inhibitors.
  • This assay facilitates a deeper understanding of Hhat function in biological processes and disease.