title: Revealing ligand binding sites and quantifying subunit variants of noncovalent protein complexes in a single native top-down FTICR MS experiment.
abstract: "Native" mass spectrometry (MS) has been proven to be increasingly useful for structural biology studies of macromolecular assemblies. Using horse liver alcohol dehydrogenase (hADH) and yeast alcohol dehydrogenase (yADH) as examples, we demonstrate that rich information can be obtained in a single native top-down MS experiment using Fourier transform ion cyclotron mass spectrometry (FTICR MS). Beyond measuring the molecular weights of the protein complexes, isotopic mass resolution was achieved for yeast ADH tetramer (147 kDa) with an average resolving power of 412,700 at m/z 5466 in absorption mode, and the mass reflects that each subunit binds to two zinc atoms. The N-terminal 89 amino acid residues were sequenced in a top-down electron capture dissociation (ECD) experiment, along with the identifications of the zinc binding site at Cys46 and a point mutation (V58T). With the combination of various activation/dissociation techniques, including ECD, in-source dissociation (ISD), collisionally activated dissociation (CAD), and infrared multiphoton dissociation (IRMPD), 40% of the yADH sequence was derived directly from the native tetramer complex. For hADH, native top-down ECD-MS shows that both E and S subunits are present in the hADH sample, with a relative ratio of 4:1. Native top-down ISD of the hADH dimer shows that each subunit (E and S chains) binds not only to two zinc atoms, but also the NAD/NADH ligand, with a higher NAD/NADH binding preference for the S chain relative to the E chain. In total, 32% sequence coverage was achieved for both E and S chains.
authors.affiliation: Department of Biological Chemistry, David Geffen School of Medicine at UCLA, University of California, Los Angeles, CA, 90095, USA.
authors.forename: Huilin
authors.initials: H
authors.lastname: Li
authors.forename: Piriya
authors.initials: P
authors.lastname: Wongkongkathep
authors.forename: Steve L
authors.initials: SL
authors.lastname: Van Orden
authors.forename: Rachel R
authors.initials: RR
authors.lastname: Ogorzalek Loo
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Loo
mesh_terms: D000426:Alcohol Dehydrogenase / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D006736:Horses; D008024:Ligands; D008099:Liver / Q000201:enzymology; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D011485:Protein Binding; D011506:Proteins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*
chemical_list: D008024:Ligands; D011506:Proteins; D000426:Alcohol Dehydrogenase