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Top‐down mass spectrometry of intact membrane protein complexes reveals oligomeric state and sequence information in a single experiment
Author(s) -
Konijnenberg Albert,
Bannwarth Ludovic,
Yilmaz Duygu,
Koçer Armağan,
VenienBryan Catherine,
Sobott Frank
Publication year - 2015
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.2703
Subject(s) - chemistry , fragmentation (computing) , mass spectrometry , dissociation (chemistry) , collision induced dissociation , tandem mass spectrometry , monomer , membrane , ion channel , membrane protein , biophysics , ion , analytical chemistry (journal) , chromatography , biochemistry , organic chemistry , biology , ecology , receptor , polymer
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.