title: 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.
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.
authors.affiliation: Institute of Clinical Chemistry, University Medical Center Hamburg-Eppendorf (UKE), Martinistraße 52, D-20246 Hamburg, Germany. trusch@chemie.uni-hamburg.de
authors.forename: Maria
authors.initials: M
authors.lastname: Trusch
authors.forename: Kati
authors.initials: K
authors.lastname: Tillack
authors.forename: Marcel
authors.initials: M
authors.lastname: Kwiatkowski
authors.forename: Andreas
authors.initials: A
authors.lastname: Bertsch
authors.forename: Robert
authors.initials: R
authors.lastname: Ahrends
authors.forename: Oliver
authors.initials: O
authors.lastname: Kohlbacher
authors.forename: Roland
authors.initials: R
authors.lastname: Martin
authors.forename: Mireia
authors.initials: M
authors.lastname: Sospedra
authors.forename: Hartmut
authors.initials: H
authors.lastname: Schlüter
mesh_terms: D002852:Chromatography, Ion Exchange / Q000295:instrumentation / Q000379:methods*; D056148:Chromatography, Reverse-Phase / Q000295:instrumentation / Q000379:methods*; D003600:Cytosol / Q000737:chemistry; D030562:Databases, Protein; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D009504:Neutrophils / Q000737:chemistry; D010446:Peptide Fragments / Q000032:analysis; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods*
chemical_list: D010446:Peptide Fragments; D020543:Proteome