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An automated on-line multidimensional HPLC system for protein and peptide mapping with integrated sample preparation

Wagner, K; Miliotis, T; Marko-Varga, György LU ; Bischoff, R and Unger, K K (2002) In Analytical Chemistry 74(4). p.809-820
Abstract
A comprehensive on-line two-dimensional 2D-HPLC system with integrated sample preparation was developed for the analysis of proteins and peptides with a molecular weight below 20 kDa. The system setup provided fast separations and high resolving power and is considered to be a complementary technique to 2D gel electrophoresis in proteomics. The on-line system reproducibly resolved similar to1000 peaks within the total analysis time of 96 min and avoided sample losses by off-line sample handling. The low-molecular-weight target analytes were separated from the matrix using novel silica-based restricted access materials (RAM) with ion exchange functionalities. The size-selective sample fractionation step was followed by anion or cation... (More)
A comprehensive on-line two-dimensional 2D-HPLC system with integrated sample preparation was developed for the analysis of proteins and peptides with a molecular weight below 20 kDa. The system setup provided fast separations and high resolving power and is considered to be a complementary technique to 2D gel electrophoresis in proteomics. The on-line system reproducibly resolved similar to1000 peaks within the total analysis time of 96 min and avoided sample losses by off-line sample handling. The low-molecular-weight target analytes were separated from the matrix using novel silica-based restricted access materials (RAM) with ion exchange functionalities. The size-selective sample fractionation step was followed by anion or cation exchange chromatography as the first dimension. The separation mechanism in the subsequent second dimension employed hydrophobic interactions using short reversed-phase (RP) columns. A new column-switching technique, including four parallel reversed-phase columns, was employed in the second dimension for on-line fractionation and separation. Gradient elution and UV detection of two columns were performed simultaneously while loading the third and regenerating the fourth column. The total integrated workstation was operated in an unattended mode. Selected peaks were collected and analyzed off-line by MALDI-TOF mass spectrometry. The system was applied to protein mapping of biological samples of human hemofiltrate as well as of cell lysates originating from a human fetal fibroblast cell line, demonstrating it to be a viable alternative to 2D gel electrophoresis for mapping peptides and small proteins. (Less)
Please use this url to cite or link to this publication:
author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Analytical Chemistry
volume
74
issue
4
pages
809 - 820
publisher
The American Chemical Society
external identifiers
  • pmid:11866061
  • wos:000173915300011
  • scopus:0037084079
ISSN
1520-6882
DOI
10.1021/ac010627f
language
English
LU publication?
yes
id
3b26d4c6-6681-4f91-be95-213cc694a6ff (old id 343294)
date added to LUP
2007-11-19 14:42:58
date last changed
2017-11-19 03:39:42
@article{3b26d4c6-6681-4f91-be95-213cc694a6ff,
  abstract     = {A comprehensive on-line two-dimensional 2D-HPLC system with integrated sample preparation was developed for the analysis of proteins and peptides with a molecular weight below 20 kDa. The system setup provided fast separations and high resolving power and is considered to be a complementary technique to 2D gel electrophoresis in proteomics. The on-line system reproducibly resolved similar to1000 peaks within the total analysis time of 96 min and avoided sample losses by off-line sample handling. The low-molecular-weight target analytes were separated from the matrix using novel silica-based restricted access materials (RAM) with ion exchange functionalities. The size-selective sample fractionation step was followed by anion or cation exchange chromatography as the first dimension. The separation mechanism in the subsequent second dimension employed hydrophobic interactions using short reversed-phase (RP) columns. A new column-switching technique, including four parallel reversed-phase columns, was employed in the second dimension for on-line fractionation and separation. Gradient elution and UV detection of two columns were performed simultaneously while loading the third and regenerating the fourth column. The total integrated workstation was operated in an unattended mode. Selected peaks were collected and analyzed off-line by MALDI-TOF mass spectrometry. The system was applied to protein mapping of biological samples of human hemofiltrate as well as of cell lysates originating from a human fetal fibroblast cell line, demonstrating it to be a viable alternative to 2D gel electrophoresis for mapping peptides and small proteins.},
  author       = {Wagner, K and Miliotis, T and Marko-Varga, György and Bischoff, R and Unger, K K},
  issn         = {1520-6882},
  language     = {eng},
  number       = {4},
  pages        = {809--820},
  publisher    = {The American Chemical Society},
  series       = {Analytical Chemistry},
  title        = {An automated on-line multidimensional HPLC system for protein and peptide mapping with integrated sample preparation},
  url          = {http://dx.doi.org/10.1021/ac010627f},
  volume       = {74},
  year         = {2002},
}