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‐β‐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.