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Micro-Immobilized-Enzyme Reactor Based Limited Proteolysis Coupled to Native Mass Spectrometry (IMER-based LiP-nMS) for the Structural Characterization of Protein Complexes

ABSTRACT: Native mass spectrometry (nMS) has emerged as a powerful analytical technique for structural biology; however, nMS alone can only provide limited structural information regarding tertiary and quaternary structure, subunit connectivity, and ligand binding location within the complexes. Here, we developed a micro-immobilized-enzyme reactor-based limited proteolysis coupled to native Mass Spectrometry (IMER-based LiP-nMS) for the structural characterization of protein complexes. Take a pair of allosteric protein complexes, glycogen phosphorylase a and b (GPa and GPb), for example, we demonstrate that IMER-based LiP-nMS approach allows us to rapidly pinpoint the surface accessible sites, stable domains, and regions involved in subunit interaction interface. The structural difference induced by phosphorylation at Ser14 between GPa and GPb were also observed and consistent with known structural evidence. This new approach can greatly enhance the structural output of nMS technique and should be applicable to examining protein complexes whose topology or crystal structures are not fully known.

INSTRUMENT(S): SYNAPT G2-Si

ORGANISM(S): Oryctolagus Cuniculus (ncbitaxon:9986)

SUBMITTER: Huilin Li

PROVIDER: MSV000087466 | MassIVE | Thu May 20 09:18:00 BST 2021

REPOSITORIES: MassIVE

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