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Related Experiment Videos

Differential detergent fractionation for non-electrophoretic eukaryote cell proteomics.

Fiona M McCarthy1, Shane C Burgess, Bart H J van den Berg

  • 1College of Veterinary Medicine, PO Box 6100, Mississippi State University, Mississippi 39762, USA.

Journal of Proteome Research
|April 13, 2005
PubMed
Summary

Differential detergent fractionation (DDF) combined with mass spectrometry enhances proteome coverage, identifying more membrane proteins for functional genome annotation.

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Area of Science:

  • Proteomics
  • Cellular Biology
  • Mass Spectrometry

Background:

  • Differential detergent fractionation (DDF) is a technique for sequential protein extraction from eukaryotic cells.
  • DDF has been utilized to improve proteome coverage in 2D-PAGE.
  • Comprehensive analysis of cellular proteomes, especially membrane proteins, remains a challenge.

Purpose of the Study:

  • To evaluate the efficacy of DDF extraction coupled with 2D-LC ESI MS2 for comprehensive cellular proteomics.
  • To assess the yield of membrane proteins, including those with multiple transmembrane domains, using this combined method.
  • To explore the utility of DDF-derived data for functional genome annotation.

Main Methods:

  • Differential detergent fractionation (DDF) for sequential protein extraction.

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  • Liquid chromatography (LC) coupled with electrospray ionization tandem mass spectrometry (ESI MS2).
  • Integration of DDF with 2D-LC ESI MS2 for proteomic analysis.

    • Main Results:

    • DDF extraction in conjunction with 2D-LC ESI MS2 provides comprehensive cellular proteome coverage.
    • The method significantly increases the proportion of identified membrane proteins, including those with multiple transmembrane domains.
    • DDF-based fractionation yields approximately twice the number of membrane proteins compared to some specific membrane protein isolation methods.

    Conclusions:

    • DDF extraction is a valuable method for enhancing proteome coverage in conjunction with mass spectrometry-based proteomics.
    • This approach effectively enriches for membrane proteins, offering insights into their abundance and characteristics.
    • The physicochemistry and localization data from DDF aid in functional genome annotation, crucial for ongoing genome sequencing projects.