title: Robotically assisted titration coupled to ion mobility-mass spectrometry reveals the interface structures and analysis parameters critical for multiprotein topology mapping.
abstract: Multiprotein complexes have three-dimensional shapes and dynamic functions that impact almost every aspect of biochemistry. Despite this, our ability to rapidly assess the structures of such macromolecules lags significantly behind high-throughput efforts to identify their function, especially in the context of human disease. Here, we describe results obtained by coupling ion mobility-mass spectrometry with automated robotic sampling of different solvent compositions. This combination of technologies has allowed us to explore an extensive set of solution conditions for a group of eight protein homotetramers, representing a broad sample of protein structure and stability space. We find that altering solution ionic strength in concert with dimethylsulfoxide content is sufficient to disrupt the protein-protein interfaces of all of the complexes studied here. Ion mobility measurements captured for both intact assemblies and subcomplexes match expected values from available X-ray structures in all cases save two. For these exceptions, we find that distorted subcomplexes result from extreme disruption conditions, and are accompanied by small shifts in intact tetramers size, thus enabling the removal of distorted subcomplex data in downstream models. Furthermore, we find strong correlations between the relative intensities of disrupted protein tetramers and the relative number and type of interactions present at interfaces as a function of disrupting agent added. In most cases, this correlation appears strong enough to quantify various types of protein interfacial interactions within unknown proteins following appropriate calibration.
authors.affiliation: Department of Chemistry, University of Michigan , 930 North University Avenue, Ann Arbor, Michigan 48109, United States.
authors.forename: Yueyang
authors.initials: Y
authors.lastname: Zhong
authors.forename: Jun
authors.initials: J
authors.lastname: Feng
authors.forename: Brandon T
authors.initials: BT
authors.lastname: Ruotolo
mesh_terms: D000818:Animals; D002417:Cattle; D006801:Humans; D007474:Ion Exchange; D013058:Mass Spectrometry / Q000379:methods*; D046912:Multiprotein Complexes / Q000032:analysis / Q000737:chemistry*; D011487:Protein Conformation; D025941:Protein Interaction Mapping / Q000379:methods*; D017433:Protein Structure, Secondary; D011817:Rabbits; D012371:Robotics / Q000379:methods*; D014961:X-Ray Diffraction
chemical_list: D046912:Multiprotein Complexes