Time-Resolved Acetaldehyde-Based Accessibility Profiling Maps Ligand-Target Interactions

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.

Automated summary

The study introduces an acetaldehyde labeling approach to complement formaldehyde labeling for probing ligand-protein interactions. The use of acetaldehyde allows for cleaner and more moderate reaction profiles compared to formaldehyde, enabling the identification of lysines involved in ligand-protein binding. Time-dependent changes in lysine accessibility profiles were detected using acetaldehyde labeling, further quantified by multiple reaction monitoring to sensitively determine ligand-binding sites and differentiate binding affinities among various ligands, which was confirmed by native mass spectrometry and molecular docking. Monitoring the chemical accessibility of these responsive peptides in cell lysates demonstrates the potential of these methods in characterizing ligand-protein interactions in complex cellular environments.