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Displacement chromatography as first separating step in online two-dimensional liquid chromatography coupled to mass spectrometry analysis of a complex protein sample--the proteome of neutrophils.
- Source :
-
Journal of chromatography. A [J Chromatogr A] 2012 Apr 06; Vol. 1232, pp. 288-94. Date of Electronic Publication: 2012 Feb 18. - Publication Year :
- 2012
-
Abstract
- Displacement chromatography provides some advantages over elution chromatography such as the opportunity to enrich trace amounts of molecules and to elute molecules in highest concentrations achievable with liquid chromatography. In a previous study we demonstrated that displacement chromatography is a well-suited alternative to gradient elution in an offline two-dimensional (2D-)LC-MS approach for the analysis of proteomes. In this study we present a method for applying displacement chromatography in an online 2D-LC-MS system including a cation exchange (CEX) column and a reversed phase column. We circumvented the problem of determining the sample capacity of the CEX column by repeated injection (pulses) of sample aliquots monitored by an LC-MS analysis of each flow-through fraction of the CEX column. Elution of tryptic peptides from the CEX column was achieved by repeated injection (pulses) of the displacer spermine. Pulsed displacer injections offer the advantage through physical separation of preventing post-column mixing of already separated compounds. As a proof of principle we analyzed the cytosolic proteome of human neutrophils.<br /> (Copyright © 2012 Elsevier B.V. All rights reserved.)
- Subjects :
- Chromatography, Ion Exchange instrumentation
Chromatography, Reverse-Phase instrumentation
Cytosol chemistry
Databases, Protein
Humans
Mass Spectrometry methods
Neutrophils chemistry
Peptide Fragments analysis
Chromatography, Ion Exchange methods
Chromatography, Reverse-Phase methods
Proteome analysis
Proteomics methods
Subjects
Details
- Language :
- English
- ISSN :
- 1873-3778
- Volume :
- 1232
- Database :
- MEDLINE
- Journal :
- Journal of chromatography. A
- Publication Type :
- Academic Journal
- Accession number :
- 22391494
- Full Text :
- https://doi.org/10.1016/j.chroma.2012.02.029