Protocol

The Nucleus

Volume 463 of the series Methods in Molecular Biology pp 77-92

High-Yield Isolation and Subcellular Proteomic Characterization of Nuclear and Subnuclear Structures from Trypanosomes

  • Jeffrey A. DeGrasseAffiliated withLaboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University
  • , Brian T. ChaitAffiliated withLaboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University
  • , Mark C. FieldAffiliated withDepartment of Pathology, University of Cambridge
  • , Michael P. RoutAffiliated withLaboratory of Cellular and Structural Biology, The Rockefeller University

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Abstract

The vast evolutionary distance between the Opisthokonta (animals and yeast) and the excavata (a major group of protists, including Giardia and Trypanosoma) presents a significant challenge to in silico functional genomics and ortholog identification. Subcellular proteomic identification of the constituents of highly enriched organelles can alleviate this problem by both providing localization evidence and yielding a manageably sized proteome for detailed in silico functional assignment. We describe a method for the high-yield isolation of nuclei from the kinetoplastid Trypanosoma brucei. We also describe the subsequent purification of subnuclear compartments, including the nuclear envelope and nucleolus. Finally, using several proteomic strategies, we survey the proteome of a subcellular structure or organelle, using the nuclear pore complex as an example.

Keywords

Nuclear isolation Trypanosoma brucei Subcellular fractionation Proteomics Sucrose density gradient centrifugation Nuclear envelope Nucleolus Nuclear pore complex