Application of displacement chromatography for the analysis of a lipid raft proteome
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Application of displacement chromatography for the analysis of a lipid raft proteome. / Trusch, Maria; Böhlick, Alexandra; Hildebrand, Diana; Lichtner, Björn; Bertsch, Andreas; Kohlbacher, Oliver; Bachmann, Sebastian; Schlüter, Hartmut.
in: J CHROMATOGR B, Jahrgang 878, Nr. 3-4, 01.02.2010, S. 309-14.Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung
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T1 - Application of displacement chromatography for the analysis of a lipid raft proteome
AU - Trusch, Maria
AU - Böhlick, Alexandra
AU - Hildebrand, Diana
AU - Lichtner, Björn
AU - Bertsch, Andreas
AU - Kohlbacher, Oliver
AU - Bachmann, Sebastian
AU - Schlüter, Hartmut
N1 - 2009 Elsevier B.V. All rights reserved.
PY - 2010/2/1
Y1 - 2010/2/1
N2 - Defining membrane proteomes is fundamental to understand the role of membrane proteins in biological processes and to find new targets for drug development. Usually multidimensional chromatography using step or gradient elution is applied for the separation of tryptic peptides of membrane proteins prior to their mass spectrometric analysis. Displacement chromatography (DC) offers several advantages that are helpful for proteome analysis. However, DC has so far been applied for proteomic investigations only in few cases. In this study we therefore applied DC in a multidimensional LC-MS approach for the separation and identification of membrane proteins located in cholesterol-enriched membrane microdomains (lipid rafts) obtained from rat kidney by density gradient centrifugation. The tryptic peptides were separated on a cation-exchange column in the displacement mode with spermine used as displacer. Fractions obtained from DC were analyzed using an HPLC-chip system coupled to an electrospray-ionization ion-trap mass spectrometer. This procedure yielded more than 400 highly significant peptide spectrum matches and led to the identification of more than 140 reliable protein hits within an established rat kidney lipid raft proteome. The majority of identified proteins were membrane proteins. In sum, our results demonstrate that DC is a suitable alternative to gradient elution separations for the identification of proteins via a multidimensional LC-MS approach.
AB - Defining membrane proteomes is fundamental to understand the role of membrane proteins in biological processes and to find new targets for drug development. Usually multidimensional chromatography using step or gradient elution is applied for the separation of tryptic peptides of membrane proteins prior to their mass spectrometric analysis. Displacement chromatography (DC) offers several advantages that are helpful for proteome analysis. However, DC has so far been applied for proteomic investigations only in few cases. In this study we therefore applied DC in a multidimensional LC-MS approach for the separation and identification of membrane proteins located in cholesterol-enriched membrane microdomains (lipid rafts) obtained from rat kidney by density gradient centrifugation. The tryptic peptides were separated on a cation-exchange column in the displacement mode with spermine used as displacer. Fractions obtained from DC were analyzed using an HPLC-chip system coupled to an electrospray-ionization ion-trap mass spectrometer. This procedure yielded more than 400 highly significant peptide spectrum matches and led to the identification of more than 140 reliable protein hits within an established rat kidney lipid raft proteome. The majority of identified proteins were membrane proteins. In sum, our results demonstrate that DC is a suitable alternative to gradient elution separations for the identification of proteins via a multidimensional LC-MS approach.
KW - Amino Acid Sequence
KW - Animals
KW - Cation Exchange Resins
KW - Chromatography, Liquid
KW - Immunoblotting
KW - Kidney
KW - Male
KW - Mass Spectrometry
KW - Membrane Microdomains
KW - Molecular Sequence Data
KW - Peptides
KW - Proteome
KW - Rats
KW - Rats, Wistar
U2 - 10.1016/j.jchromb.2009.11.035
DO - 10.1016/j.jchromb.2009.11.035
M3 - SCORING: Journal article
C2 - 20015709
VL - 878
SP - 309
EP - 314
JO - J CHROMATOGR B
JF - J CHROMATOGR B
SN - 1570-0232
IS - 3-4
ER -