Mass Spectrometry Analysis of Chemically and Collisionally Dissociated Molecular Glue- and PROTAC-Mediated Protein Complexes Informs on Disassembly Pathways.

Abstract

Molecular glues (MGs) and proteolysis-targeting chimeras (PROTACs) are used to modulate protein-protein interactions (PPIs), via induced proximity between compounds that have little or no affinity for each other naturally. They promote either reversible inhibition or selective degradation of a target protein, including ones deemed undruggable by traditional therapeutics. Though native MS (nMS) is capable of analyzing multiprotein complexes, the behavior of these artificially induced compounds in the gas phase is still not fully understood, and the number of publications over the past few years is still rather limited. Here, we studied two MG-induced complexes between mTOR_FRB and FKBP12 as well as a PROTAC-induced complex between FKBP51_FK1 and the von Hippel-Lindau E3 ligase (VHL). Native MS combined with collision-induced dissociation (CID) provided a way of measuring not only the formation of these complexes but also their dissociation pathways. Both protein complexes seem to eject preferably the centrally located small (compared to the mass of the proteins) ligand upon CID, rather than dissociating a peripheral subunit, as is often observed for naturally occurring protein complexes. In contrast, chemically induced dissociation in solution generated complementary data to CID, by disrupting the PPI surface, which resulted in more diverse MS spectra that preserved the stronger interactions in solution.