Top-down mass spectrometry of intact membrane protein complexes reveals oligomeric state and sequence information in a single experiment

Albert Konijnenberg, Ludovic Bannwarth, Duygu Yilmaz, Armagan Kocer, Catherine Venien-Bryan, Frank Sobott*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

26 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)1292-1300
Number of pages9
JournalProtein Science
Volume24
Issue number8
DOIs
Publication statusPublished - Aug-2015

Keywords

  • mass spectrometry
  • ion channels
  • top-down sequencing
  • integral membrane proteins
  • collision-induced dissociation
  • HEPATITIS-C VIRUS
  • ELECTROSPRAY-IONIZATION
  • GAS-PHASE
  • ION-CHANNEL
  • DISSOCIATION
  • ASSEMBLIES
  • PROTEOMICS
  • RELEASE
  • LIPIDS
  • P7

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