Mapping protein receptor–ligand interactions via in vivo chemical crosslinking, affinity purification, and differential mass spectrometry

Protein receptor–ligand interactions play important roles in mediating enzyme catalysis, signal transduction, and other protein functions. Immunoaffinity purification followed by mass spectrometry analysis is a common method for identifying protein receptor–ligand complexes. However, it is difficult to distinguish between specific protein binding partners and non-specifically bound proteins that co-purify with the complex. In addition, weakly interacting binding partners may dissociate from the protein receptor–ligand complexes during immunoaffinity purification. The combination of chemical crosslinking, affinity purification, and differential mass spectrometry analysis provides a direct method for capturing stable, weak, and transient protein interactions that occur in vivo and in vitro. This approach enables the identification of functional receptor–ligand binding partners with high confidence. Herein, we describe a differential mass spectrometry approach coupled with in situ chemical crosslinking and immunoaffinity purification for identifying receptor–ligand binding partners. In particular, we identified a functional, counter-ligand structure of the natural killer cell p30-related protein.