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Proteomics of rat liver Golgi complex: minor proteins are identified through sequential fractionation

Academic Article
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Overview

authors

  • Taylor, R. S.
  • Wu, Chunlei
  • Hays, L. G.
  • Eng, J. K.
  • Yates III, John
  • Howell, K. E.

publication date

  • October 2000

journal

  • Electrophoresis  Journal

abstract

  • The discovery of novel proteins resident to the Golgi complex will fuel our future studies of Golgi structure/function and provide justification for proteomic analysis of this organelle. Our approach to Golgi proteomics was to first isolate and characterize the intact organelle free of proteins in transit by use of tissue pretreated with cycloheximide. Then the stacked Golgi fraction was fractionated into biochemically defined subfractions: Triton X-114 insoluble, aqueous, and detergent phases. The aqueous and detergent phases were further fractionated by anion-exchange column chromatography. In addition, radiolabeled cytosol was incubated with stacked Golgi fractions containing proteins in transit, and the proteins bound to the Golgi stacks in an energy-dependent manner were characterized. All fractions were analyzed by two-dimensional (2-D) gel electrophoresis and identification numbers were given to 588 unique 2-D spots. Tandem mass spectrometry was used to analyze 93 of the most abundant 2-D spots taken from preparative Triton X-114 insoluble, aqueous and detergent phase 2-D gels. Fifty-one known and 22 unknown proteins were identified. This study represents the first installment in the mammalian Golgi proteome database. Our data suggest that cell fractionation followed by biochemical dissection of specific classes of molecules provides a significant advantage for the identification of low abundance proteins in organelles.

subject areas

  • Animals
  • Chromatography, High Pressure Liquid
  • Electrophoresis, Gel, Two-Dimensional
  • Golgi Apparatus
  • Liver
  • Mass Spectrometry
  • Polyethylene Glycols
  • Proteins
  • Proteome
  • Rats
  • Silver Staining
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Research

keywords

  • Golgi complex
  • Triton X-114
  • cycloheximide
  • proteomics
  • two-dimensional gel electrophoresis
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Identity

International Standard Serial Number (ISSN)

  • 0173-0835

Digital Object Identifier (DOI)

  • 10.1002/1522-2683(20001001)21:16<3441::aid-elps3441>3.0.co;2-g

PubMed ID

  • 11079564
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Additional Document Info

start page

  • 3441

end page

  • 3459

volume

  • 21

issue

  • 16

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