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Microfluidic mobility shift assay for real-time analysis of peptide n-palmitoylation

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Title: Microfluidic mobility shift assay for real-time analysis of peptide n-palmitoylation
Authors: Lanyon-Hogg, T
Patel, NV
Ritzefeld, M
Boxall, KJ
Burke, R
Blagg, J
Magee, AI
Tate, EW
Item Type: Journal Article
Abstract: The Hedgehog pathway is a key developmental signaling pathway but is also implicated in many types of cancer. The extracellular signaling protein Sonic hedgehog (Shh) requires dual lipidation for functional signaling, whereby N-terminal palmitoylation is performed by the enzyme Hedgehog acyltransferase (Hhat). Hhat is an attractive target for small-molecule inhibition to arrest Hedgehog signaling, and methods for assaying Hhat activity are central to understanding its function. However, all existing assays to quantify lipidation of peptides suffer limitations, such as safety hazards, high costs, extensive manual handling, restriction to stopped-assay measurements, or indirect assessment of lipidation. To address these limitations, we developed a microfluidic mobility shift assay (MSA) to analyze Shh palmitoylation. MSA allowed separation of fluorescently labeled Shh amine-substrate and palmitoylated Shh amide-product peptides based on differences in charge and hydrodynamic radius, coupled with online fluorescence intensity measurements for quantification. The MSA format was employed to study Hhat-catalyzed reactions, investigate Hhat kinetics, and determine small-molecule inhibitor IC50 values. Both real-time and stopped assays were performed, with the latter achieved via addition of excess unlabeled Shh peptide. The MSA format therefore allows direct and real-time fluorescence-based measurement of acylation and represents a powerful alternative technique in the study of N-lipidation.
Issue Date: 31-Jan-2017
Date of Acceptance: 27-Dec-2016
URI: http://hdl.handle.net/10044/1/46069
DOI: https://dx.doi.org/10.1177/2472555216689529
ISSN: 2472-5552
Publisher: SAGE
Start Page: 418
End Page: 424
Journal / Book Title: SLAS Discovery
Volume: 22
Issue: 4
Copyright Statement: © 2017 Society for Laboratory Automation and Screening. SAGE publications The final, definitive version of this paper has been published in SLAS Discovery, Vol 22, Issue 4 by Sage Publications Ltd. It is available at: http://journals.sagepub.com/home/jbx
Sponsor/Funder: Cancer Research UK
European Commission
Funder's Grant Number: C6433/A16402
FP7-PEOPLE-2013-IEF
Keywords: Science & Technology
Life Sciences & Biomedicine
Physical Sciences
Biochemical Research Methods
Biotechnology & Applied Microbiology
Chemistry, Analytical
Biochemistry & Molecular Biology
Chemistry
enzyme assays or enzyme kinetics
lipids or lipid metabolism
fluorescence methods
medicinal chemistry
FLUORESCENCE-BASED ASSAY
HEDGEHOG ACYLTRANSFERASE
SONIC-HEDGEHOG
CANCER-CELLS
PROTEIN
INHIBITORS
ACTIVATION
MECHANISMS
MONITOR
TARGET
Appears in Collections:Chemistry
Biological and Biophysical Chemistry
National Heart and Lung Institute
Faculty of Natural Sciences