A Systematic Strategy for Proteomic Analysis of Chloroplast Protein Complexes in Wheat

Abstract

A systematic strategy was developed for the proteomic analysis of wheat chloroplast protein complexes. First, comprehensive centrifugation methods were utilized for the exhaustive isolation of thylakoid, envelope, and stromal fractions. Second, 1% n-dodecyl-β-D-maltoside was selected from a series of detergents as the optimal detergent to dissolve protein complexes effectively from membranes. Then, blue native polyacrylamide gel electrophoresis (BN–PAGE) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) were improved to separate and analyze the protein complexes. By this systematic strategy, envelopes, thylakoids, and stromata were enriched effectively from chloroplasts in the same process, and more than 18 complexes were obtained simultaneously by BN–PAGE. Finally, thylakoid protein complexes were further analyzed by BN/SDS–PAGE, and nine complex bands and 40 protein spots were observed on BN–PAGE and SDS–PAGE respectively. Our results indicate that this new strategy can be used efficiently to analyze the proteome of chloroplast protein complexes and can be applied conveniently to the analysis of other subcellular protein complexes.