An automated on-line multidimensional HPLC system for protein and peptide mapping with integrated sample preparation
(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:
https://lup.lub.lu.se/record/343294
- author
- Wagner, K ; Miliotis, T ; Marko-Varga, György LU ; Bischoff, R and Unger, K K
- organization
- publishing date
- 2002
- type
- Contribution to journal
- publication status
- published
- subject
- in
- Analytical Chemistry
- volume
- 74
- issue
- 4
- pages
- 809 - 820
- publisher
- The American Chemical Society (ACS)
- external identifiers
-
- pmid:11866061
- wos:000173915300011
- scopus:0037084079
- ISSN
- 1520-6882
- DOI
- 10.1021/ac010627f
- language
- English
- LU publication?
- yes
- additional info
- The information about affiliations in this record was updated in December 2015. The record was previously connected to the following departments: Analytical Chemistry (S/LTH) (011001004)
- id
- 3b26d4c6-6681-4f91-be95-213cc694a6ff (old id 343294)
- date added to LUP
- 2016-04-01 12:34:45
- date last changed
- 2022-03-06 01:27:36
@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 (ACS)}},
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}},
doi = {{10.1021/ac010627f}},
volume = {{74}},
year = {{2002}},
}