Electrophoretically driven SDS removal and protein fractionation in the shotgun analysis of membrane proteomes

Authors

  • Yi Liu,

    1. Key Laboratory of Protein Chemistry and Developmental Biology of National Education Committee, College of Life Sciences, Hunan Normal University, Changsha, P. R. China
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    • These authors contributed equally to this work.

  • Yong Lin,

    1. Key Laboratory of Protein Chemistry and Developmental Biology of National Education Committee, College of Life Sciences, Hunan Normal University, Changsha, P. R. China
    2. National Research Center of Engineering & Technology for Utilization of Botanical Functional Ingredients, College of Horticulture and Landscape, Hunan Agricultural University, Changsha, P. R. China
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    • These authors contributed equally to this work.

  • Yizhong Yan,

    1. Key Laboratory of Protein Chemistry and Developmental Biology of National Education Committee, College of Life Sciences, Hunan Normal University, Changsha, P. R. China
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  • Jianjun Li,

    1. Key Laboratory of Protein Chemistry and Developmental Biology of National Education Committee, College of Life Sciences, Hunan Normal University, Changsha, P. R. China
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  • Quanze He,

    1. Key Laboratory of Protein Chemistry and Developmental Biology of National Education Committee, College of Life Sciences, Hunan Normal University, Changsha, P. R. China
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  • Ping Chen,

    1. Key Laboratory of Protein Chemistry and Developmental Biology of National Education Committee, College of Life Sciences, Hunan Normal University, Changsha, P. R. China
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  • Xianchun Wang,

    Corresponding author
    1. Key Laboratory of Protein Chemistry and Developmental Biology of National Education Committee, College of Life Sciences, Hunan Normal University, Changsha, P. R. China
    • College of Life Sciences, Hunan Normal University, Changsha City, Hunan 410081, P. R. China Fax: +86-731-8886-1304
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  • Songping Liang

    1. Key Laboratory of Protein Chemistry and Developmental Biology of National Education Committee, College of Life Sciences, Hunan Normal University, Changsha, P. R. China
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Abstract

SDS is mostly used to enhance the solubilization and extraction of membrane proteins due to its strong detergency and low cost. Nevertheless, SDS interferes with the subsequent procedures and needs to be removed from the samples. In this work, a special gradient gel electrophoresis (GGE) system was developed to remove SDS from the SDS-solubilized protein samples. As a proof-of-principle experiment, the GGE system was designed to be composed of an agarose loading layer, six polyacrylamide fractionation layers with different concentrations and a high-concentration polyacrylamide sealing layer. The advantages of the GGE system are that it not only can electrophoretically remove SDS efficiently so that the protein loss resulted from the repeated gel washing after electrophoresis was avoided, but also can reduce the complexity of the sample, prevent the precipitation of proteins after loading and avoid the loss of proteins with low molecular weight during the electrophoresis. Using GGE system, about 85% of SDS in the sample and gel was electrophoretically removed and the proteins were fractionated. Compared with the two representative gel-based sample cleanup methods reported in literature, GGE-based strategy significantly improved the identification efficiency of proteins in terms of the number and coverage of the identified proteins.

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