title: High-Efficiency Isolation of Nuclear Envelope Protein Complexes from Trypanosomes.
abstract: Functional understanding of the nuclear envelope requires the identification of its component proteins and their interactions. Trypanosomes cause human and livestock diseases worldwide but are so divergent from animals and fungi that in silico searches for homologs of proteins are frequently of low value. Here we describe a strategy for the straightforward identification of nuclear envelope proteins from trypanosomes that classifies proteins and their interaction networks in the nuclear pore complex. Milling frozen whole cells into a powder and rapid screening of buffer conditions for optimization of complex isolation is described. The method is inexpensive and potentially applicable to many organisms, providing fast access to functional information.
authors.affiliation: Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, 1230 York Avenue, New York, NY, USA.
authors.forename: Samson O
authors.initials: SO
authors.lastname: Obado
authors.affiliation: Division of Biological Chemistry and Drug Discovery, University of Dundee, Dundee, DD1 5EH, UK. m.c.field@dundee.ac.uk.
authors.forename: Mark C
authors.initials: MC
authors.lastname: Field
authors.affiliation: Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, 1230 York Avenue, New York, NY, USA.
authors.forename: Brian T
authors.initials: BT
authors.lastname: Chait
authors.affiliation: Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, 1230 York Avenue, New York, NY, 10065, USA. rout@mail.rockefeller.edu.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Rout
mesh_terms: D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D008565:Membrane Proteins / Q000302:isolation & purification*; D046912:Multiprotein Complexes / Q000302:isolation & purification*; D009685:Nuclear Envelope / Q000378:metabolism*; D015800:Protozoan Proteins / Q000302:isolation & purification*; D014345:Trypanosoma / Q000378:metabolism*
chemical_list: D008565:Membrane Proteins; D046912:Multiprotein Complexes; D015800:Protozoan Proteins
keywords: Affinity isolation; Cryomilling; Molecular evolution; Nuclear envelope; Nuclear pore complex; Proteomics; Trypanosoma