A bioanalytical method for the proteome wide display and analysis of protein complexes from whole plant cell lysates.
While protein interaction studies and protein network modeling come to the forefront, the isolation and identification of protein complexes in a cellular context remains a major challenge for plant science. To this end, a nondenaturing extraction procedure was optimized for plant whole cell matrices and the combined use of gel filtration and BN-PAGE for the separation of protein complexes was studied. Hyphenation to denaturing electrophoresis and mass spectrometric analysis allows for the simultaneous identification of multiple (previously unidentified) protein interactions in single samples. The reliability and efficacy of the technique was confirmed (i) by the identification of well-studied plant protein complexes, (ii) by the presence of nonplant interologs for several of the novel complexes (iii) by presenting physical evidence of previously hypothetical plant protein interactions and (iv) by the confirmation of found interactions using co-IP. Furthermore practical issues concerning the use of this 2-D BN/SDS-PAGE display method for the analysis of protein-protein interactions are discussed.
Remmerie
Noor
N
Center for Proteome Analysis and Mass Spectrometry, University of Antwerp, Antwerp, Belgium.
Roef
Luc
L
Van De Slijke
Eveline
E
Van Leene
Jelle
J
Persiau
Geert
G
Eeckhout
Dominique
D
Stals
Hilde
H
Laukens
Kris
K
Lemière
Filip
F
Esmans
Eddy
E
Van Onckelen
Harry
H
Inzé
Dirk
D
De Jaeger
Geert
G
Witters
Erwin
E
D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D047468:Immunoprecipitation; D010940:Plant Proteins / Q000032:analysis*; D020543:Proteome / Q000032:analysis*
D010940:Plant Proteins; D020543:Proteome