2014
Leurs, Ulrike; Lohse, Brian; Ming, Shonoi; Cole, Philip A; Clausen, Rasmus P; Kristensen, Jesper L; Rand, Kasper D
In: Anal Chem, vol. 86, no. 23, pp. 11734-41, 2014, ISSN: 1520-6882.
Abstract | Links | BibTeX | Tags: Binding Sites, Deuterium Exchange Measurement, Histone Demethylases, Humans, Jumonji Domain-Containing Histone Demethylases, Ligands, Mass Spectrometry, Models, Molecular, Molecular Structure, Peptides
@article{1299975,
title = {Dissecting the binding mode of low affinity phage display peptide ligands to protein targets by hydrogen/deuterium exchange coupled to mass spectrometry},
author = {Ulrike Leurs and Brian Lohse and Shonoi Ming and Philip A Cole and Rasmus P Clausen and Jesper L Kristensen and Kasper D Rand},
doi = {10.1021/ac503137u},
issn = {1520-6882},
year = {2014},
date = {2014-12-01},
journal = {Anal Chem},
volume = {86},
number = {23},
pages = {11734-41},
abstract = {Phage display (PD) is frequently used to discover peptides capable of binding to biological protein targets. The structural characterization of peptide-protein complexes is often challenging due to their low binding affinities and high structural flexibility. Here, we investigate the use of hydrogen/deuterium exchange mass spectrometry (HDX-MS) to characterize interactions of low affinity peptides with their cognate protein targets. The HDX-MS workflow was optimized to accurately detect low-affinity peptide-protein interactions by use of ion mobility, electron transfer dissociation, nonbinding control peptides, and statistical analysis of replicate data. We show that HDX-MS can identify regions in the two epigenetic regulator proteins KDM4C and KDM1A that are perturbed through weak interactions with PD-identified peptides. Two peptides cause reduced HDX on opposite sides of the active site of KDM4C, indicating distinct binding modes. In contrast, the perturbation site of another PD-selected peptide inhibiting the function of KDM1A maps to a GST-tag. Our results demonstrate that HDX-MS can validate and map weak peptide-protein interactions and pave the way for understanding and optimizing the binding of peptide scaffolds identified through PD and similar ligand discovery approaches.},
keywords = {Binding Sites, Deuterium Exchange Measurement, Histone Demethylases, Humans, Jumonji Domain-Containing Histone Demethylases, Ligands, Mass Spectrometry, Models, Molecular, Molecular Structure, Peptides},
pubstate = {published},
tppubtype = {article}
}
Phage display (PD) is frequently used to discover peptides capable of binding to biological protein targets. The structural characterization of peptide-protein complexes is often challenging due to their low binding affinities and high structural flexibility. Here, we investigate the use of hydrogen/deuterium exchange mass spectrometry (HDX-MS) to characterize interactions of low affinity peptides with their cognate protein targets. The HDX-MS workflow was optimized to accurately detect low-affinity peptide-protein interactions by use of ion mobility, electron transfer dissociation, nonbinding control peptides, and statistical analysis of replicate data. We show that HDX-MS can identify regions in the two epigenetic regulator proteins KDM4C and KDM1A that are perturbed through weak interactions with PD-identified peptides. Two peptides cause reduced HDX on opposite sides of the active site of KDM4C, indicating distinct binding modes. In contrast, the perturbation site of another PD-selected peptide inhibiting the function of KDM1A maps to a GST-tag. Our results demonstrate that HDX-MS can validate and map weak peptide-protein interactions and pave the way for understanding and optimizing the binding of peptide scaffolds identified through PD and similar ligand discovery approaches.