title: Native top-down mass spectrometry for the structural characterization of human hemoglobin.
abstract: Native mass spectrometry (MS) has become an invaluable tool for the characterization of proteins and noncovalent protein complexes under near physiological solution conditions. Here we report the structural characterization of human hemoglobin (Hb), a 64 kDa oxygen-transporting protein complex, by high resolution native top-down MS using electrospray ionization and a 15-Tesla Fourier transform ion cyclotron resonance mass spectrometer. Native MS preserves the noncovalent interactions between the globin subunits, and electron capture dissociation (ECD) produces fragments directly from the intact Hb complex without dissociating the subunits. Using activated ion ECD, we observe the gradual unfolding process of the Hb complex in the gas phase. Without protein ion activation, the native Hb shows very limited ECD fragmentation from the N-termini, suggesting a tightly packed structure of the native complex and therefore a low fragmentation efficiency. Precursor ion activation allows a steady increase in N-terminal fragment ions, while the C-terminal fragments remain limited (38 c ions and four z ions on the α chain; 36 c ions and two z ions on the β chain). This ECD fragmentation pattern suggests that upon activation, the Hb complex starts to unfold from the N-termini of both subunits, whereas the C-terminal regions and therefore the potential regions involved in the subunit binding interactions remain intact. ECD-MS of the Hb dimer shows similar fragmentation patterns as the Hb tetramer, providing further evidence for the hypothesized unfolding process of the Hb complex in the gas phase. Native top-down ECD-MS allows efficient probing of the Hb complex structure and the subunit binding interactions in the gas phase. It may provide a fast and effective means to probe the structure of novel protein complexes that are intractable to traditional structural characterization tools.
authors: Department of Chemistry and Biochemistry. jzhang@uwalumni.com.
authors: Jiang
authors: J
authors: Zhang
authors: Department of Chemistry and Biochemistry. rezak1@gmail.com.
authors: G
authors: G
authors: Reza Malmirchegini
authors: Department of Chemistry and Biochemistry, UCLA/DOE Institute of Genomics and Proteomics, University of California, Los Angeles, California, 90095, United States. rclubb@mbi.ucla.edu.
authors: Robert T Clubb T
authors: RT
authors: Clubb
authors: De partment of Chemistry and Biochemistry, Department of Biological Chemistry, David Geffen School of Medicine, UCLA/DOE Institute of Genomics and Proteomics, University of California, Los Angeles, California, 90095, United States. Jloo@chem.ucla.edu.
authors: Joseph A
authors: JA
authors: Loo
mesh_terms: D017357:Cyclotrons; D006454:Hemoglobins / Q000737:chemistry* / Q000648:ultrastructure*; D006801:Humans; D008956:Models, Chemical; D008958:Models, Molecular; D021122:Protein Subunits; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*
chemical_list: D006454:Hemoglobins; D021122:Protein Subunits