Use of sequential chemical extractions to purify nuclear membrane proteins for proteomics identification.
The nuclear envelope (NE) is a double membrane system that is both a part of the endoplasmic reticulum and part of the nucleus. As its constituent proteins tend to be highly complexed with nuclear and cytoplasmic components, it is notoriously difficult to purify. Two methods can reduce this difficulty for the identification of nuclear membrane proteins: comparison to contaminating membranes and chemical extractions to enrich for certain groups of proteins. The purification of nuclear envelopes and contaminating microsomal membranes is described here along with procedures for chemical extraction using salt and detergent, chaotropes, or alkaline solutions. Each extraction method enriches for different combinations of nuclear envelope proteins. Finally, we describe the analysis of these fractions with MudPIT, a proteomics methodology that avoids gel extraction of bands to facilitate identification of minor proteins and membrane proteins that do not resolve well on gels. Together these three approaches can significantly increase the output of proteomics studies aimed at identifying the protein complement of subcellular membrane systems.
Korfali
Nadia
N
The Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, UK.
Fairley
Elizabeth A L
EA
Swanson
Selene K
SK
Florens
Laurence
L
Schirmer
Eric C
EC
D000468:Alkalies / Q000737:chemistry; D002458:Cell Fractionation / Q000379:methods*; D005591:Chemical Fractionation / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D003902:Detergents / Q000737:chemistry; D006801:Humans; D008214:Lymphocytes / Q000737:chemistry / Q000166:cytology; D008565:Membrane Proteins / Q000302:isolation & purification*; D008861:Microsomes / Q000737:chemistry; D009685:Nuclear Envelope / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D012492:Salts / Q000737:chemistry; D012984:Software; D053719:Tandem Mass Spectrometry
D000468:Alkalies; D003902:Detergents; D008565:Membrane Proteins; D012492:Salts