A fully automated 2-D LC-MS method utilizing online continuous pH and RP gradients for global proteome analysis

Authors

  • Hu Zhou,

    1. Key Laboratory of Systems Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Shanghai, P. R. China
    Search for more papers by this author
  • Jie Dai,

    1. Key Laboratory of Systems Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Shanghai, P. R. China
    Search for more papers by this author
  • Quan-Hu Sheng,

    1. Key Laboratory of Systems Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Shanghai, P. R. China
    Search for more papers by this author
  • Rong-Xia Li,

    1. Key Laboratory of Systems Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Shanghai, P. R. China
    Search for more papers by this author
  • Chia-Hui Shieh,

    1. Key Laboratory of Systems Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Shanghai, P. R. China
    Search for more papers by this author
  • Andras Guttman,

    1. Horvath Laboratory of Bioseparation Sciences, University of Innsbruck, Innsbruck, Austria
    Search for more papers by this author
  • Rong Zeng Professor

    Corresponding author
    1. Key Laboratory of Systems Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Shanghai, P. R. China
    • Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yueyang Road, Shanghai 200031, P. R. China Fax: +86-21-54920171
    Search for more papers by this author

Abstract

The conventional 2-D LC-MS/MS setup for global proteome analysis was based on online and offline salt gradients (step and continuous) using strong-cation-exchange chromatography in conjunction with RP chromatography and MS. The use of the online system with step salt elution had the possibility of resulting in peptide overlapping across fractions. The offline mode had the option to operate with continuous salt gradient to decrease peak overlap, but exhibited decreased robustness, lower reproducibility, and sample loss during the process. Due to the extensive washing requirement between the chromatography steps, online continuous gradient was not an option for salt elution. In this report, a fully automated, online, and continuous gradient (pH continuous online gradient, pCOG) 2-D LC-MS/MS system is introduced that provided excellent separation and identification power. The pH gradient-based elution provided more basic peptides than that of salt-based elution. Fraction overlap was significantly minimized by combining pH and continuous gradient elutions. This latter approach also increased sequence coverage and the concomitant confidence level in protein identification. The salt and pH elution-based 2-D LC-MS/MS approaches were compared by analyzing the mouse liver proteome.

Ancillary