Two-dimensional Blue Native/sodium dodecyl sulfate gel electrophoresis for analysis of multimeric proteins in platelets.
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.
Claeys
Dirk
D
Department of Pharmacology and Toxicology, University Lausanne, Rue du Bugnon 27, CH-1005 Lausanne, Switzerland. dirk.claeys@unil.ch
Geering
Käthi
K
Meyer
Beat J
BJ
D000818:Animals; D001792:Blood Platelets / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006801:Humans; D008565:Membrane Proteins / Q000097:blood* / Q000302:isolation & purification; D008861:Microsomes / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism; D020836:Protein Structure, Quaternary; D020543:Proteome / Q000302:isolation & purification*; D014981:Xenopus
D008565:Membrane Proteins; D020543:Proteome