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Restricted access chromatographic sample preparation of low mass proteins expressed in human fibroblast cells for proteomics analysis

Welinder, Charlotte LU ; Lindberg, C and Marko-Varga, György LU (2001) In Journal of Chromatography A 909(2). p.279-288
Abstract
Two-dimensional electrophoresis and modern image analysis systems have made it possible to study protein expression and regulation of proteins in biological systems. Proteins in the molecular mass region of 20-120 kDa are well investigated and described. However, proteins with masses below 20 kDa are the least investigated as they are rarely seen on 2D-PAGE due to fast migrations in the electric field and lack of staining efficiency. This paper describes a technique that enriches proteins in the lower mass region using solid-phase extraction. The purification step is carried out using C18 functionalised "restricted access" affinity chromatography whereby simultaneous trace enrichment and sample clean up is achieved. In this study... (More)
Two-dimensional electrophoresis and modern image analysis systems have made it possible to study protein expression and regulation of proteins in biological systems. Proteins in the molecular mass region of 20-120 kDa are well investigated and described. However, proteins with masses below 20 kDa are the least investigated as they are rarely seen on 2D-PAGE due to fast migrations in the electric field and lack of staining efficiency. This paper describes a technique that enriches proteins in the lower mass region using solid-phase extraction. The purification step is carried out using C18 functionalised "restricted access" affinity chromatography whereby simultaneous trace enrichment and sample clean up is achieved. In this study expression patterns of TGF-beta stimulated and non-stimulated fibroblasts were compared after the solid-phase fractionation procedure. An increased expression pattern was obtained whereby 400 protein spots could be detected by image analysis in the <20-kDa region. Out of these, specific regulations of 14 spots were found by quantitative image analysis and spots of interest were identified with MALDI TOF-MS. The regulated and identified proteins were triosephosphate isomerase, cofilin and heat shock 27-kDa protein. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Journal of Chromatography A
volume
909
issue
2
pages
279 - 288
publisher
Elsevier
external identifiers
  • pmid:11269527
  • scopus:0035895793
ISSN
0021-9673
DOI
10.1016/S0021-9673(00)01103-1
language
English
LU publication?
yes
id
4a727745-84df-4759-b802-6a27886a8564 (old id 1121968)
date added to LUP
2008-06-25 13:49:45
date last changed
2018-05-29 10:30:51
@article{4a727745-84df-4759-b802-6a27886a8564,
  abstract     = {Two-dimensional electrophoresis and modern image analysis systems have made it possible to study protein expression and regulation of proteins in biological systems. Proteins in the molecular mass region of 20-120 kDa are well investigated and described. However, proteins with masses below 20 kDa are the least investigated as they are rarely seen on 2D-PAGE due to fast migrations in the electric field and lack of staining efficiency. This paper describes a technique that enriches proteins in the lower mass region using solid-phase extraction. The purification step is carried out using C18 functionalised "restricted access" affinity chromatography whereby simultaneous trace enrichment and sample clean up is achieved. In this study expression patterns of TGF-beta stimulated and non-stimulated fibroblasts were compared after the solid-phase fractionation procedure. An increased expression pattern was obtained whereby 400 protein spots could be detected by image analysis in the &lt;20-kDa region. Out of these, specific regulations of 14 spots were found by quantitative image analysis and spots of interest were identified with MALDI TOF-MS. The regulated and identified proteins were triosephosphate isomerase, cofilin and heat shock 27-kDa protein.},
  author       = {Welinder, Charlotte and Lindberg, C and Marko-Varga, György},
  issn         = {0021-9673},
  language     = {eng},
  number       = {2},
  pages        = {279--288},
  publisher    = {Elsevier},
  series       = {Journal of Chromatography A},
  title        = {Restricted access chromatographic sample preparation of low mass proteins expressed in human fibroblast cells for proteomics analysis},
  url          = {http://dx.doi.org/10.1016/S0021-9673(00)01103-1},
  volume       = {909},
  year         = {2001},
}