Native mass spectrometry (MS) is an approach that enables analysis of large biomolecules and complexes in a ‘native-like’ state. Increasing applications of native MS in recent years have exemplified its merit as a rapid, sensitive and automatable technique for insight into noncovalent protein-ligand interactions, binding stoichiometry, relative or absolute affinity, and target selectivity. In particular, native MS has proven valuable in fragment-based drug discovery, providing data complementary to those obtained by traditional biophysical techniques such as nuclear magnetic resonance spectroscopy (NMR), X-ray crystallography and surface plasmon resonance (SPR). Importantly, native MS addresses several limitations of these traditional approaches, utilising only picomole quantities of protein and ligand, and having fewer restrictions on analyte size. Moreover, native MS has the unique capability to detect multiple species, enabling the affinity and specificity of multiple ligands to be evaluated simultaneously, making the technique well suited for ligand screening.
We have developed a native MS workflow using Orbitrap detection for the analysis of protein-ligand interactions and protein mixtures. Conditions were optimised using well-characterised proteins of ongoing interest, predominantly SPRY domain-containing SOCS box protein 2 (SPSB2) and fatty acid binding proteins (FABPs) 1-5. Using SPSB2 and its nanomolar affinity ligands as a model system, we demonstrated that native MS-derived absolute binding affinities are consistent with those generated by SPR. Furthermore, we exploited the unique features of native MS to simultaneously evaluate and rank the binding affinities of multiple ligands for FABP4, as well as the selectivity of a single ligand in a mixture containing FABPs 1-5. We found good agreement for both relative binding affinity and target selectivity with those derived from NMR, SPR and isothermal calorimetry. This study demonstrates the potential of native MS as a rapid and efficient orthogonal method for fragment elaboration and target identification.