Top-down mass spectrometry of intact membrane protein complexes reveals oligomeric state and sequence information in a single experiment |
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Authors: | Albert Konijnenberg Ludovic Bannwarth Duygu Yilmaz Arma?an Ko?er Catherine Venien-Bryan Frank Sobott |
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Affiliation: | 1.Department of Chemistry, Biomolecular & Analytical Mass Spectrometry group, University of Antwerp, Antwerp, Belgium;2.Bioinformatique et BioPhysique, Institut de Minéralogie, de Physique des Matériaux et de Cosmochimie (IMPMC), Université Pierre et Marie Curie, Paris, France;3.Department of Neuroscience, University of Groningen, Groningen, The Netherlands;4.UA-VITO Centre for Proteomics, University of Antwerp, Antwerp, Belgium |
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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. |
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Keywords: | mass spectrometry ion channels top-down sequencing integral membrane proteins collision-induced dissociation |
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