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Quantitative Analysis of Liver Golgi Proteome in the Cell Cycle

  • Xuequn Chen
  • Philip C. Andrews
  • Yanzhuang WangEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 909)

Abstract

During mitosis, the Golgi membranes in mammalian cells undergo a continuous disassembly process and generate mitotic fragments that are distributed into the daughter cells and reassembled into new Golgi after mitosis. This disassembly and reassembly process is critical for Golgi biogenesis during cell division, but the underlying molecular mechanism is poorly understood. In this study, we have recapitulated this process using an in vitro assay and analyzed the proteins that are associated with interphase and mitotic Golgi membranes using quantitative proteomics that combines the isobaric tags for relative and absolute quantification approach with OFFGEL isoelectric focusing separation and LC-MALDI-MS/MS. A total of 1,193 Golgi-associated proteins were identified and quantified. These included broad functional categories: Golgi structural proteins, Golgi resident enzymes, SNAREs, Rab GTPases, and secretory and cytoskeletal proteins. More importantly, the combination of the quantitative proteomic approach with Western blot analysis allowed us to unveil 86 proteins with significant changes in abundance under the mitotic condition compared to the interphase condition. Altogether, this systematic quantitative proteomic study revealed candidate proteins of the molecular machinery that controls the Golgi disassembly and reassembly processes in the cell cycle.

Key words

Liver Golgi Cell cycle Cell-free assay Quantitative proteomics iTRAQ LC-MALDI-MS/MS 

Notes

Acknowledgements

We gratefully acknowledge Drs. F. Gorelick, K. Gull, T. Kreis, M. Lowe, K. Moremen, A. Price, A. Satoh, J. Seemann, D. Sheff, D. Shields, and G. Warren for generously providing antibodies. We thank J. Williams and D. Tang for suggestions and reagents. We thank Sarah Volk for her assistance in OFFGEL electrophoresis. This work was supported by National Institute of Health grant P41 RR018627 to P. Andrews, and was partially supported by the National Institutes of Health (GM087364) and the American Cancer Society (RGS-09-278-01-CSM) to Y.W.

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Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  • Xuequn Chen
    • 1
  • Philip C. Andrews
    • 2
  • Yanzhuang Wang
    • 3
    Email author
  1. 1.Department of PhysiologyWayne State UniversityDetroitUSA
  2. 2.Department of Biological ChemistryThe University of MichiganAnn ArborUSA
  3. 3.Department of Molecular, Cellular and Developmental BiologyThe University of MichiganAnn ArborUSA

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