Dissecting the Binding Mode of Low Affinity Phage Display Peptide Ligands to Protein Targets by Hydrogen/Deuterium Exchange Coupled to Mass Spectrometry
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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 A; Cole, Philip A; Clausen, Rasmus P; Kristensen, Jesper L; Rand, Kasper D.
In: Analytical Chemistry, Vol. 86, No. 23, 17.10.2014, p. 11734-11741.Research output: Contribution to journal › Journal article › peer-review
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TY - JOUR
T1 - Dissecting the Binding Mode of Low Affinity Phage Display Peptide Ligands to Protein Targets by Hydrogen/Deuterium Exchange Coupled to Mass Spectrometry
AU - Leurs, Ulrike
AU - Lohse, Brian
AU - Ming, Shonoi A
AU - Cole, Philip A
AU - Clausen, Rasmus P
AU - Kristensen, Jesper L
AU - Rand, Kasper D
PY - 2014/10/17
Y1 - 2014/10/17
N2 - 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, non-binding 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.
AB - 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, non-binding 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.
U2 - 10.1021/ac503137u
DO - 10.1021/ac503137u
M3 - Journal article
C2 - 25325890
VL - 86
SP - 11734
EP - 11741
JO - Industrial And Engineering Chemistry Analytical Edition
JF - Industrial And Engineering Chemistry Analytical Edition
SN - 0003-2700
IS - 23
ER -
ID: 126111855