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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 orcid ; 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
; ; ; ; ; ; ; and
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
2016-04-01 16:12:24
date last changed
2022-01-28 18:06:41
@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}},
  keywords     = {{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}},
  doi          = {{10.1016/j.chroma.2003.12.016}},
  volume       = {{1029}},
  year         = {{2004}},
}