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Laser‐induced liquid bead ion desorption‐MS of protein complexes from blue‐native gels, a sensitive top‐down proteomic approach
Author(s) -
Sokolova Lucie,
Wittig Ilka,
Barth HansDieter,
Schägger Hermann,
Brutschy Bernhard,
Brandt Ulrich
Publication year - 2010
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200900756
Subject(s) - multiprotein complex , chromatography , chemistry , elution , centrifugation , differential centrifugation , mass spectrometry , protein subunit , proteome , electrophoresis , proteomics , resolution (logic) , desorption , analytical chemistry (journal) , biochemistry , organic chemistry , adsorption , artificial intelligence , computer science , gene
Abstract We have developed an experimental approach that combines two powerful methods for proteomic analysis of large membrane protein complexes: blue native electrophoresis (BNE or BN‐PAGE) and laser‐induced liquid bead ion desorption (LILBID) MS. Protein complexes were separated by BNE and eluted from the gel. The masses of the constituents of the multiprotein complexes were obtained by LILBID MS, a detergent‐tolerant method that is especially suitable for the characterisation of membrane proteins. High sensitivity and small sample volumes required for LILBID MS resulted in low demands on sample quantity. Eluate from a single band allowed assessing the mass of an entire multiprotein complex and its subunits. The method was validated with mitochondrial NADH:ubiquinone reductase from Yarrowia lipolytica . For this complex of 947 kDa, typically 30 μg or 32 pmol were sufficient to obtain spectra from which the subunit composition could be analysed. The resolution of this electrophoretic small‐scale approach to the purification of native complexes was improved markedly by further separation on a second dimension of BNE. Starting from a subcellular fraction obtained by differential centrifugation, this allowed the purification and analysis of the constituents of a large multiprotein complex in a single LILBID spectrum.