Abstract
In this study we present a new technology to detect stable oligomeric protein complexes in membranes. The technology is based on the ability of small membrane-active alcohols to dissociate the highly stable homotetrameric potassium channel KcsA. It is shown via a proteomics approach, using diagonal electrophoresis and nano-flow liquid chromatography coupled to tandem mass spectrometry, that a large number of both integral and peripheral Escherichia coli inner membrane proteins are part of stable oligomeric complexes that can be dissociated by small alcohols. This study gives insight into the composition and stability of these complexes.
Highlights
In this study we present a new technology to detect stable oligomeric protein complexes in membranes
It is shown via a proteomics approach, using diagonal electrophoresis and nano-flow liquid chromatography coupled to tandem mass spectrometry, that a large number of both integral and peripheral Escherichia coli inner membrane proteins are part of stable oligomeric complexes that can be dissociated by small alcohols
The search for protein interaction partners has received much attention recently and extensive interaction networks have been established for water-soluble proteins [4, 5], little attention has been focused so far on the search for interaction partners of membrane proteins
Summary
It is shown via a proteomics approach, using diagonal electrophoresis and nano-flow liquid chromatography coupled to tandem mass spectrometry, that a large number of both integral and peripheral Escherichia coli inner membrane proteins are part of stable oligomeric complexes that can be dissociated by small alcohols. Off-diagonal spots are digested by trypsin and identified by nano-flow liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) Using this method we observed many off-diagonal spots that contained membrane proteins, either integral with one or more transmembrane segments or peripheral with a membrane-interactive domain. The method is complementary to traditional two-dimensional electrophoresis methods of blue-native PAGE in combination with SDS-PAGE [12], which allows detection of protein complexes that are not stable in SDS
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