Dissecting the binding mode of low affinity phage display peptide ligands to protein targets by hydrogen/deuterium exchange coupled to mass spectrometry

Ulrike Leurs, Brian Lohse, Shonoi Ming, Philip A. Cole, Rasmus P. Clausen, Jesper L. Kristensen, Kasper D. Rand

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)11734-11741
Number of pages8
JournalAnalytical Chemistry
Volume86
Issue number23
DOIs
StatePublished - Dec 2 2014

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Bacteriophages
Deuterium
Mass spectrometry
Hydrogen
Display devices
Ligands
Peptides
Proteins
Electron mobility
Scaffolds
Statistical methods
Ions

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

Dissecting the binding mode of low affinity phage display peptide ligands to protein targets by hydrogen/deuterium exchange coupled to mass spectrometry. / Leurs, Ulrike; Lohse, Brian; Ming, Shonoi; Cole, Philip A.; Clausen, Rasmus P.; Kristensen, Jesper L.; Rand, Kasper D.

In: Analytical Chemistry, Vol. 86, No. 23, 02.12.2014, p. 11734-11741.

Research output: Contribution to journalArticle

Leurs, Ulrike ; Lohse, Brian ; Ming, Shonoi ; Cole, Philip A. ; Clausen, Rasmus P. ; Kristensen, Jesper L. ; Rand, Kasper D. / Dissecting the binding mode of low affinity phage display peptide ligands to protein targets by hydrogen/deuterium exchange coupled to mass spectrometry. In: Analytical Chemistry. 2014 ; Vol. 86, No. 23. pp. 11734-11741.
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