Collision-Induced Affinity Selection Mass Spectrometry for Identification of Ligands

[Image: see text] Hyphenated mass spectrometry has been used to identify ligands binding to proteins. It involves mixing protein and compounds, separation of protein–ligand complexes from unbound compounds, dissociation of the protein–ligand complex, separation to remove protein, and injection of the supernatant into a mass spectrometer to observe the ligand. Here we report collision-induced affinity selection mass spectrometry (CIAS-MS), which allows separation and dissociation inside the instrument. The quadrupole was used to select the ligand–protein complex and allow unbound molecules to be exhausted to vacuum. Collision-induced dissociation (CID) dissociated the protein–ligand complex, and the ion guide and resonance frequency were used to selectively detect the ligand. A known SARS-CoV-2 Nsp9 ligand, oridonin, was successfully detected when it was mixed with Nsp9. We provide proof-of-concept data that the CIAS-MS method can be used to identify binding ligands for any purified protein.