LiF-MS: Mapping unstructured peptide-protein interactions using Ligand-Footprinting Mass Spectrometry

Unstructured peptides, or linear motifs, present a poorly understood molecular language within the context of cellular signaling. These modular regions are often short, unstructured and interact weakly and transiently with folded target proteins. Thus, they are difficult to study with conventional structural biology methods. We present Ligand-Footprinting Mass Spectrometry, or LiF-MS, as a method of mapping the binding sites and dynamic disorder of these peptides on folded protein domains. LiF-MS uses a cleavable crosslinker to mark regions of a protein contacted by a bound linear motif. We demonstrate this method can detect both conformation ensembles and binding orientations of a linear motif in its binding pocket to amino-acid-level detail. Furthermore, marked amino acids can be used as constraints in peptide-protein docking simulations to improve model quality. In conclusion, LiF-MS proves a simple and novel method of elucidating peptide docking structural data not accessible by other methods in the context of a purified system.