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Nanodisc-based co-immunoprecipitation for mass spectrometric identification of membrane-interacting proteins.

Authors
  • Borch, Jonas
  • Roepstorff, Peter
  • Møller-Jensen, Jakob
Type
Published Article
Journal
Molecular & Cellular Proteomics
Publisher
American Society for Biochemistry and Molecular Biology
Publication Date
Jul 01, 2011
Volume
10
Issue
7
Identifiers
DOI: 10.1074/mcp.O110.006775
PMID: 21532009
Source
Medline
License
Unknown

Abstract

Proteomic identification of protein interactions with membrane associated molecules in their native membrane environment pose a challenge because of technical problems of membrane handling. We investigate the possibility of employing membrane nanodiscs for harboring the membrane associated molecule to tackle the challenges. Nanodiscs are stable, homogenous pieces of membrane with a discoidal shape. They are stabilized by an encircling amphipatic protein with an engineered epitope tag. In the present study we employ the epitope tag of the nanodiscs for detection and co-immunoprecipitation of interaction partners of the glycolipid ganglioside GM1 harbored by nanodiscs. Highly specific binding activity for nanodisc-GM1 immobilized on sensorchips was observed by surface plasmon resonance in culture media from enterotoxigenic Escherischia coli. To isolate the interaction partner(s) from enterotoxigenic Escherischia coli, GM1-nanodiscs were employed for co-immunoprecipitation. The B subunit of heat labile enterotoxin was identified as a specific interaction partner by mass spectrometry, thus demonstrating that nanodisc technology is useful for highly specific detection and identification of interaction partners to specific lipids embedded in a membrane bilayer.

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