Two-dimensional Blue Native/sodium dodecyl sulfate gel electrophoresis for analysis of multimeric proteins in platelets

Détails

ID Serval
serval:BIB_C465A00E3512
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Titre
Two-dimensional Blue Native/sodium dodecyl sulfate gel electrophoresis for analysis of multimeric proteins in platelets
Périodique
Electrophoresis
Auteur(s)
Claeys  D., Geering  K., Meyer  B. J.
ISSN
0173-0835 (Print)
Statut éditorial
Publié
Date de publication
03/2005
Volume
26
Numéro
6
Pages
1189-99
Notes
Journal Article
Research Support, Non-U.S. Gov't --- Old month value: Mar
Résumé
Two-dimensional (2-D) Blue Native/SDS gel electrophoresis combines a first-dimensional separation of monomeric and multimeric proteins in their native state with a second denaturing dimension. These high-resolution 2-D gels aim at identifying multiprotein complexes with respect to their subunit composition. We applied this method for the first time to analyze two human platelet subproteomes: the cytosolic and the microsomal membrane protein fraction. Solubilization of platelet membrane proteins was achieved with the nondenaturing detergent n-dodecyl-beta-D-maltoside. To validate native solubilization conditions, we demonstrated the correct assembly of the Na,K-ATPase, a functional multimeric transmembrane protein, when expressed in Xenopus oocytes. We identified 63 platelet proteins after in-gel tryptic digestion of 58 selected protein spots and liquid chromatography-coupled tandem mass spectrometry. Nine proteins were detected for the first time in platelets by a proteomic approach. We also show that this technology efficiently resolves several known membrane and cytosolic multiprotein complexes. Blue Native/SDS gel electrophoresis is thus a valuable procedure to analyze specific platelet subproteomes, like the membrane(-bound) protein fraction, by mass spectrometry and immunoblotting and could be relevant for the study of protein-protein interactions generated following platelet activation.
Mots-clé
Animals Blood Platelets/*chemistry Electrophoresis, Gel, Two-Dimensional/*methods Electrophoresis, Polyacrylamide Gel/*methods Humans Membrane Proteins/*blood/isolation & purification Microsomes/metabolism Oocytes/metabolism Protein Structure, Quaternary Proteome/*isolation & purification Xenopus
Pubmed
Web of science
Création de la notice
24/01/2008 13:28
Dernière modification de la notice
03/03/2018 21:13
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