Time-Resolved Acetaldehyde-Based Accessibility Profiling Maps Ligand–Target Interactions
- 31 December 2020
- journal article
- research article
- Published by American Chemical Society (ACS) in Journal of the American Society for Mass Spectrometry
- Vol. 32 (2), 519-530
- https://doi.org/10.1021/jasms.0c00382
Abstract
Elucidating ligand–protein interactions is important in understanding the biochemical machinery for given proteins. Previously, formaldehyde (FH)-based labeling has been employed to obtain such structural knowledge, since reactive residues that participate in ligand–target interactions display reduced accessibility to FH-labeling reagents, and thus can be identified by quantitative proteomics. Although being rapid and efficient for probing proteinaceous lysine accessibility, here, we report an acetaldehyde (AcH)-labeling approach that complements with FH for probing ligand–target interactions. AcH labeling examines lysine accessibility at a more moderate reaction speed and hence delivers a cleaner reaction when compared to that of FH. The subsequent application of AcH to label RNase A without and with ligands has assisted to assign lysines involved in ligand–RNase A binding by detecting the time-dependent changes in accessibility profiles. We further employed multiple reaction monitoring (MRM) to quantify these ligand-binding-responsive sites when a variety of potential ligands were queried. We noted that the time-resolved abundance changes of these peptides can sensitively determine the ligand-binding sites and differentiate binding affinities among these ligands, which was confirmed by native mass spectrometry (MS) and molecular docking. Lastly, we demonstrated that the binding sites can be recognized by monitoring the chemical accessibility of these responsive peptides in cell lysates. Together, we believe that the proposed combined use of AcH-based lysine accessibility profiling, native MS, and MRM screening is a powerful toolbox in characterizing ligand–target interactions, mapping topography, and interrogating affinities and holds promise for future applications in a complex cellular environment.Keywords
Funding Information
- Ministry of Science and Technology of the People's Republic of China (2018ZX09711001-002-003, 2018ZX09711002-001-004)
- China Pharmaceutical University (CPU2018GF09, CPU2018GY09)
- Natural Science Foundation of Jiangsu Province (BK20180079)
- National Natural Science Foundation of China (81720108032, 81872838, 81930109)
- Overseas Expertise Introduction Project for Discipline Innovation (G20582017001)
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