Application of displacement chromatography for the analysis of a lipid raft proteome.
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.
Trusch
Maria
M
Department of Clinical Chemistry, University Medical Center Hamburg-Eppendorf, Campus Forschung, Martinistr. 52, D-20246 Hamburg, Germany. m.trusch@uke.de
Böhlick
Alexandra
A
Hildebrand
Diana
D
Lichtner
Björn
B
Bertsch
Andreas
A
Kohlbacher
Oliver
O
Bachmann
Sebastian
S
Schlüter
Hartmut
H
D000595:Amino Acid Sequence; D000818:Animals; D002411:Cation Exchange Resins; D002853:Chromatography, Liquid / Q000379:methods*; D015151:Immunoblotting; D007668:Kidney / Q000378:metabolism; D008297:Male; D013058:Mass Spectrometry; D021962:Membrane Microdomains / Q000737:chemistry*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification; D020543:Proteome / Q000032:analysis* / Q000737:chemistry; D051381:Rats; D017208:Rats, Wistar
D002411:Cation Exchange Resins; D010455:Peptides; D020543:Proteome