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Protein pre-fractionation in detergent-polymer aqueous two-phase systems for facilitated proteomic studies of membrane proteins

Everberg, Henrik LU ; Sivars, Ulf; Emanuelsson, Cecilia LU ; Persson, Cecilia; Englund, A K; Haneskog, L; Lipniunas, P; Jornten-Karlsson, M and Tjerneld, Folke LU (2004) In Journal of Chromatography A 1029(1-2). p.113-124
Abstract
Pre-fractionation of a complex mixture of proteins increases the resolution in analytical separations of proteins from cells, tissues or organisms. Here we demonstrate a novel method for pre-fractionation of membrane proteins by a detergent-based aqueous two-phase system. Membrane proteins are strongly under-represented in proteomic studies based on two-dimensional electrophoresis (2-DE). As a model system, we have isolated mitochondria from the yeast Saccharomyces cerevisiae. Mitochondrial proteins were fractionated in an aqueous two-phase system consisting of the polymer poly(ethylene glycol) and either of two commonly used non-ionic detergents, Triton X-114 or dodecyl maltoside (DDM). Soluble proteins partitioned mainly to the polymer... (More)
Pre-fractionation of a complex mixture of proteins increases the resolution in analytical separations of proteins from cells, tissues or organisms. Here we demonstrate a novel method for pre-fractionation of membrane proteins by a detergent-based aqueous two-phase system. Membrane proteins are strongly under-represented in proteomic studies based on two-dimensional electrophoresis (2-DE). As a model system, we have isolated mitochondria from the yeast Saccharomyces cerevisiae. Mitochondrial proteins were fractionated in an aqueous two-phase system consisting of the polymer poly(ethylene glycol) and either of two commonly used non-ionic detergents, Triton X-114 or dodecyl maltoside (DDM). Soluble proteins partitioned mainly to the polymer phase while membrane proteins were enriched in the detergent phase, as identified from one-dimensional electrophoresis (I-DE) and/or 2-DE followed by mass spectrometric analysis. Pre-fractionation was further enhanced by addition of an anionic detergent, sodium dodecyl sulfate, or a chaotropic salt, NaClO4, and by raising the pH in the system. The two-phase system pre-fractionation was furthermore combined with an alternative two-dimensional high-resolution separation method, namely ion-exchange chromatography and 1-DE. By this approach a larger number of membrane proteins could be identified compared to separation with conventional 2-DE. Thus, pre-fractionation of complex protein mixtures using the aqueous two-phase systems developed here will help to disclose larger proportions of membrane proteins in different proteomes. (C) 2004 Elsevier B.V. All rights reserved. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
surfactants, aqueous two-phase systems, proteomics, proteins
in
Journal of Chromatography A
volume
1029
issue
1-2
pages
113 - 124
publisher
Elsevier
external identifiers
  • wos:000189076000014
  • pmid:15032356
  • scopus:1042298936
ISSN
0021-9673
DOI
10.1016/j.chroma.2003.12.016
language
English
LU publication?
yes
id
8ca25539-dae9-4430-aefa-277b332891d1 (old id 120881)
date added to LUP
2007-07-05 08:40:11
date last changed
2017-01-01 06:58:49
@article{8ca25539-dae9-4430-aefa-277b332891d1,
  abstract     = {Pre-fractionation of a complex mixture of proteins increases the resolution in analytical separations of proteins from cells, tissues or organisms. Here we demonstrate a novel method for pre-fractionation of membrane proteins by a detergent-based aqueous two-phase system. Membrane proteins are strongly under-represented in proteomic studies based on two-dimensional electrophoresis (2-DE). As a model system, we have isolated mitochondria from the yeast Saccharomyces cerevisiae. Mitochondrial proteins were fractionated in an aqueous two-phase system consisting of the polymer poly(ethylene glycol) and either of two commonly used non-ionic detergents, Triton X-114 or dodecyl maltoside (DDM). Soluble proteins partitioned mainly to the polymer phase while membrane proteins were enriched in the detergent phase, as identified from one-dimensional electrophoresis (I-DE) and/or 2-DE followed by mass spectrometric analysis. Pre-fractionation was further enhanced by addition of an anionic detergent, sodium dodecyl sulfate, or a chaotropic salt, NaClO4, and by raising the pH in the system. The two-phase system pre-fractionation was furthermore combined with an alternative two-dimensional high-resolution separation method, namely ion-exchange chromatography and 1-DE. By this approach a larger number of membrane proteins could be identified compared to separation with conventional 2-DE. Thus, pre-fractionation of complex protein mixtures using the aqueous two-phase systems developed here will help to disclose larger proportions of membrane proteins in different proteomes. (C) 2004 Elsevier B.V. All rights reserved.},
  author       = {Everberg, Henrik and Sivars, Ulf and Emanuelsson, Cecilia and Persson, Cecilia and Englund, A K and Haneskog, L and Lipniunas, P and Jornten-Karlsson, M and Tjerneld, Folke},
  issn         = {0021-9673},
  keyword      = {surfactants,aqueous two-phase systems,proteomics,proteins},
  language     = {eng},
  number       = {1-2},
  pages        = {113--124},
  publisher    = {Elsevier},
  series       = {Journal of Chromatography A},
  title        = {Protein pre-fractionation in detergent-polymer aqueous two-phase systems for facilitated proteomic studies of membrane proteins},
  url          = {http://dx.doi.org/10.1016/j.chroma.2003.12.016},
  volume       = {1029},
  year         = {2004},
}