Dissecting the Binding Mode of Low Affinity Phage Display Peptide Ligands to Protein Targets by Hydrogen/Deuterium Exchange Coupled to Mass Spectrometry

详细信息    查看全文

• 作者：Ulrike Leurs ; Brian Lohse ; Shonoi Ming ; Philip A. Cole ; Rasmus P. Clausen ; Jesper L. Kristensen ; Kasper D. Rand
• 刊名：Analytical Chemistry
• 出版年：2014
• 出版时间：December 2, 2014
• 年：2014
• 卷：86
• 期：23
• 页码：11734-11741
• 全文大小：525K
• ISSN：1520-6882

文摘

Phage display (PD) is frequently used to discover peptides capable of binding to biological protein targets. The structural characterization of peptide鈥損rotein 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鈥損rotein 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鈥損rotein interactions and pave the way for understanding and optimizing the binding of peptide scaffolds identified through PD and similar ligand discovery approaches.