NMR titration experiments with labeled human ubiquitin were employed in concert with chromatographic data obtained with a library of ubiquitin mutants to study the nature of protein adsorption in multimodal (MM) chromatography. The elution order of the mutants on the MM resin was significantly different from that obtained by ion-exchange chromatography. Further, the chromatographic results with the protein library indicated that mutations in a defined region induced greater changes in protein affinity to the solid support. Chemical shift mapping and determination of dissociation constants from NMR titration experiments with the MM ligand and isotopically enriched ubiquitin were used to determine and rank the relative binding affinities of interaction sites on the protein surface. The results with NMR confirmed that the protein possessed a distinct preferred binding region for the MM ligand in agreement with the chromatographic results. Finally, coarsegrained ligand docking simulations were employed to study the modes of interaction between the MM ligand and ubiquitin. The use of NMR titration experiments in concert with chromatographic data obtained with protein libraries represents a previously undescribed approach for elucidating the structural basis of protein binding affinity in MM chromatographic systems.ligand binding site | mixed mode chromatography | protein-ligand interactions | binding site mapping | pseudoaffinity T he development of efficient bioseparation processes for the production of high-purity biopharmaceuticals is one of the most pressing challenges facing the pharmaceutical and biotechnology industries today. In addition, high-resolution separations for complex bioanalytical applications are becoming increasingly important. Although it is generally accepted that nonspecific interactions can often complicate single mode chromatographic separations (e.g., ion-exchange, reversed-phase), these additional interactions can also result in unexpected selectivities (1, 2).Recent advances in the design of multimodal (MM) chromatographic systems have produced previously undescribed classes of chromatographic materials that can provide alternative and improved selectivities as compared to traditional single mode chromatographic materials (3-9). Johansson et al. have developed a library of MM ligands that can be employed for the capture of charged proteins under high salt conditions (4, 5). Liu et al. have developed a silica-based MM resin capable of weak anion-exchange and reversed-phase interactions for the simultaneous separation of acidic, basic, and neutral pharmaceutical compounds (9). Small ligand pseudoaffinity chromatographic materials such as those used for hydrophobic charge induction chromatography have resulted in previously undescribed classes of MM ligands that offer unique selectivities due more to multiple low affinity MM interactions than to specific binding to certain classes of proteins (10). In addition, several libraries of MM ligands have been recently developed and employed on chro...