title: Top-down mass spectrometry of intact membrane protein complexes reveals oligomeric state and sequence information in a single experiment.
abstract: Here we study the intact stoichiometry and top-down fragmentation behavior of three integral membrane proteins which were natively reconstituted into detergent micelles: the mechano-sensitive ion channel of large conductance (MscL), the Kirbac potassium channel and the p7 viroporin from the hepatitis C virus. By releasing the proteins under nondenaturing conditions inside the mass spectrometer, we obtained their oligomeric sizes. Increasing the ion activation (collision energy) causes unfolding and subsequent ejection of a highly charged monomer from the membrane protein complexes. Further increase of the ion activation then causes collision-induced dissociation (CID) of the ejected monomers, with fragments observed which were predominantly found to stem from membrane-embedded regions. These experiments show how in a single experiment, we can probe the relation between higher-order structure and protein sequence, by combining the native MS data with fragmentation obtained from top-down MS.
authors.affiliation: Department of Chemistry, Biomolecular & Analytical Mass Spectrometry group, University of Antwerp, Antwerp, Belgium.
authors.forename: Albert
authors.initials: A
authors.lastname: Konijnenberg
authors.affiliation: Bioinformatique et BioPhysique, Institut de Minéralogie, de Physique des Matériaux et de Cosmochimie (IMPMC), Université Pierre et Marie Curie, Paris, France.
authors.forename: Ludovic
authors.initials: L
authors.lastname: Bannwarth
authors.affiliation: Department of Neuroscience, University of Groningen, Groningen, The Netherlands.
authors.forename: Duygu
authors.initials: D
authors.lastname: Yilmaz
authors.affiliation: Department of Neuroscience, University of Groningen, Groningen, The Netherlands.
authors.forename: Armağan
authors.initials: A
authors.lastname: Koçer
authors.affiliation: Bioinformatique et BioPhysique, Institut de Minéralogie, de Physique des Matériaux et de Cosmochimie (IMPMC), Université Pierre et Marie Curie, Paris, France.
authors.forename: Catherine
authors.initials: C
authors.lastname: Venien-Bryan
authors.affiliation: Department of Chemistry, Biomolecular & Analytical Mass Spectrometry group, University of Antwerp, Antwerp, Belgium.|UA-VITO Centre for Proteomics, University of Antwerp, Antwerp, Belgium.
authors.forename: Frank
authors.initials: F
authors.lastname: Sobott
mesh_terms: D000595:Amino Acid Sequence; D016174:Hepacivirus / Q000737:chemistry*; D006526:Hepatitis C / Q000821:virology; D006801:Humans; D007473:Ion Channels / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015221:Potassium Channels / Q000737:chemistry; D055503:Protein Multimerization; D021241:Spectrometry, Mass, Electrospray Ionization; D014764:Viral Proteins / Q000737:chemistry*
chemical_list: D007473:Ion Channels; D015221:Potassium Channels; D014764:Viral Proteins
keywords: collision-induced dissociation; integral membrane proteins; ion channels; mass spectrometry; top-down sequencing