Molecular and Cellular Biochemistry

, Volume 298, Issue 1–2, pp 83–92 | Cite as

Extracellular proteome of human hepatoma cell, HepG2 analyzed using two-dimensional liquid chromatography coupled with tandem mass spectrometry

  • Ryo Yamashita
  • Yuko Fujiwara
  • Kohei Ikari
  • Keiko Hamada
  • Asuka Otomo
  • Kazuki Yasuda
  • Mitsuhiko Noda
  • Yasushi KaburagiEmail author
Original paper


Secreted proteins, which may be involved in the regulation of various biological processes, are the potential targets for diagnosis and treatment of diverse diseases. In this study, to identify the human hepatoma HepG2 cells-derived secreted proteins more extensively, we applied the protein sample preparations using the combinations of denaturation methods and molecular-mass cutoff via ultrafiltration to the two-dimensional liquid chromatography coupled with tandem mass spectrometry (2D LC–MS/MS) analysis. We were able to identify a total of 86 proteins containing widely known secreted proteins of HepG2 such as alpha-fetoprotein, of which 73 proteins including 27 signal peptide-containing proteins have never been reported to be secreted from HepG2 cells in other proteomic studies. Among the identified signal peptide-containing proteins, ten proteins such as growth differentiation factor 15, osteopontin and stanniocalcin 2 were discovered as new secreted proteins of HepG2 cells. These observations suggest that the combinations of different sample preparation methods and 2D LC–MS/MS analysis are useful for identifying a wider range of low-abundance proteins and that the secreted proteins from HepG2 identified in this study may be useful as liver-specific biomarkers for diagnosis and treatment.


HepG2 Liquid chromatography Mass spectrometry Proteome Secreted protein 


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This work was supported by a grant for diabetes research (MF-4) from the Organization for Pharmaceutical Safety and Research (to Y. K.), a research grant from the Ministry of Health, Labor, and Welfare of Japan (to Y. K.), and a grant for medical research from Takeda Pharmaceutical Co., Ltd. (to Y. K.). We would like to thank Dr. Takehiko Sasazuki (International Medical Center of Japan) and Dr. Takashi Kadowaki (University of Tokyo) for their helpful suggestions and support.


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Copyright information

© Springer Science+Business Media, LLC 2006

Authors and Affiliations

  • Ryo Yamashita
    • 1
  • Yuko Fujiwara
    • 1
  • Kohei Ikari
    • 1
  • Keiko Hamada
    • 1
  • Asuka Otomo
    • 1
  • Kazuki Yasuda
    • 1
  • Mitsuhiko Noda
    • 1
  • Yasushi Kaburagi
    • 1
    Email author
  1. 1.Department of Metabolic Disorder, Research InstituteInternational Medical Center of JapanTokyoJapan

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