title: Structural Characterization of Native Proteins and Protein Complexes by Electron Ionization Dissociation-Mass Spectrometry.
abstract: Mass spectrometry (MS) has played an increasingly important role in the identification and structural and functional characterization of proteins. In particular, the use of tandem mass spectrometry has afforded one of the most versatile methods to acquire structural information for proteins and protein complexes. The unique nature of electron capture dissociation (ECD) for cleaving protein backbone bonds while preserving noncovalent interactions has made it especially suitable for the study of native protein structures. However, the intra- and intermolecular interactions stabilized by hydrogen bonds and salt bridges can hinder the separation of fragments even with preactivation, which has become particularly problematic for the study of large macromolecular proteins and protein complexes. Here, we describe the capabilities of another activation method, 30 eV electron ionization dissociation (EID), for the top-down MS characterization of native protein-ligand and protein-protein complexes. Rich structural information that cannot be delivered by ECD can be generated by EID. EID allowed for the comparison of the gas-phase and the solution-phase structural stability and unfolding process of human carbonic anhydrase I (HCA-I). In addition, the EID fragmentation patterns reflect the structural similarities and differences among apo-, Zn-, and Cu,Zn-superoxide dismutase (SOD1) dimers. In particular, the structural changes due to Cu-binding and a point mutation (G41D) were revealed by EID-MS. The performance of EID was also compared to that of 193 nm ultraviolet photodissociation (UVPD), which allowed us to explore their qualitative similarities and differences as potential valuable tools for the MS study of native proteins and protein complexes.
authors.affiliation: Department of Biological Chemistry, David Geffen School of Medicine, University of California , Los Angeles, California 90095, United States.
authors.forename: Huilin
authors.initials: H
authors.lastname: Li
authors.affiliation: Department of Chemistry and Biochemistry, UCLA/DOE Institute of Genomics and Proteomics, and UCLA Molecular Biology Institute, University of California , Los Angeles, California 90095, United States.
authors.forename: Yuewei
authors.initials: Y
authors.lastname: Sheng
authors.affiliation: Department of Chemistry, The University of Texas at Austin , Austin, Texas 78712, United States.
authors.forename: William
authors.initials: W
authors.lastname: McGee
authors.affiliation: Department of Chemistry, The University of Texas at Austin , Austin, Texas 78712, United States.
authors.forename: Michael
authors.initials: M
authors.lastname: Cammarata
authors.affiliation: Department of Chemistry, The University of Texas at Austin , Austin, Texas 78712, United States.
authors.forename: Dustin
authors.initials: D
authors.lastname: Holden
authors.affiliation: Department of Biological Chemistry, David Geffen School of Medicine, University of California , Los Angeles, California 90095, United States.|Department of Chemistry and Biochemistry, UCLA/DOE Institute of Genomics and Proteomics, and UCLA Molecular Biology Institute, University of California , Los Angeles, California 90095, United States.
authors.forename: Joseph A
authors.identifier: 0000-0001-9989-1437
authors.initials: JA
authors.lastname: Loo
mesh_terms: D024401:Carbonic Anhydrase I / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D006801:Humans; D008024:Ligands; D010782:Photolysis / Q000528:radiation effects; D017354:Point Mutation; D058767:Protein Unfolding; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D017550:Spectroscopy, Fourier Transform Infrared; D000072105:Superoxide Dismutase-1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014466:Ultraviolet Rays
chemical_list: D008024:Ligands; D000072105:Superoxide Dismutase-1; D024401:Carbonic Anhydrase I