Limited proteolysis combined with mass spectrometry (LiP-MS) is an informative technique for studying changes in protein structure upon ligand binding. LiP-MS utilizes proteases to cleave proteins under native conditions, revealing a structure-specific proteolytic pattern. This method distinguishes between free and drug-bound states by the variations in susceptibility to proteolysis, where small molecule binding stabilizes the protein structure, resulting in fewer exposed cleavage sites.

Here, we have characterized the interaction of drug candidates with G12D mutant KRas protein by LiP-MS. We used intact protein mass spectrometry and top-down analysis to detect and identify KRas cleavage products, respectively, and therefore to locate the cleavage site in the protein sequence. Responsive to the drug binding, cleavage sites corresponded to the location of the drug binding site. Limited proteolysis yield can be estimated quantitatively using intact protein mass spectrometry and we found that the degree of cleavage depends on the binding affinity and presence of particular functional groups in the drug molecule structure. The approach was applicable to characterizing the drug binding with affinities in the micromolar range. This makes it potentially applicable for the screening of the drug leads libraries.

Molecular dynamics simulations showed that the responsive to the drug binding cleavage sites were located in the disordered regions of the protein structure, which were stabilized upon drug binding. It also sheds light on the contribution of the drug molecule functional groups to the stabilization of the protein structure and consequently to the degree of cleavage. The approach can be used for the identification of the drug binding sites, quantitative characterization of the binding, and drug design.