Meng Qingshi, Rao Liqun, Xiang Xiaocong, Zhou Chunxi, Zhang Xinyi, Pan Yinghong
College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha, China.
Biosci Biotechnol Biochem. 2011;75(11):2194-9. doi: 10.1271/bbb.110479. Epub 2011 Nov 7.
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.
开发了一种用于小麦叶绿体蛋白质复合物蛋白质组学分析的系统策略。首先，采用综合离心方法彻底分离类囊体、包膜和基质部分。其次，从一系列去污剂中选择1%的正十二烷基-β-D-麦芽糖苷作为最佳去污剂，以有效地从膜中溶解蛋白质复合物。然后，改进了蓝色天然聚丙烯酰胺凝胶电泳（BN-PAGE）和十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE），以分离和分析蛋白质复合物。通过这种系统策略，在同一过程中从叶绿体中有效地富集了包膜、类囊体和基质，并且通过BN-PAGE同时获得了18种以上的复合物。最后，通过BN/SDS-PAGE对类囊体蛋白质复合物进行进一步分析，在BN-PAGE和SDS-PAGE上分别观察到9条复合带和40个蛋白质斑点。我们的结果表明，这种新策略可有效地用于分析叶绿体蛋白质复合物的蛋白质组，并可方便地应用于其他亚细胞蛋白质复合物的分析。
