Comparison of displacement versus gradient mode for separation of a complex protein mixture by anion-exchange chromatography.
Liquid chromatography is often the method of choice for the analysis of proteins in their native state. Nevertheless compared to two-dimensional electrophoresis, the resolution of common chromatographic techniques is low. Liquid chromatography in the displacement mode has previously been shown to offer higher resolution and to elute proteins in the high concentrations. In this study we compared to what extend displacement mode was a suitable alternative to gradient mode for the separation of a complex protein mixture using anion-exchange displacement chromatography and if it is therefore helpful for proteomic investigations. Hence we analyzed the qualitative protein composition of each fraction by tryptic digestion of the proteins, analysis of the tryptic peptides by liquid chromatography coupled to mass spectrometry followed by data base analysis and by measuring the elution profiles of 22 selected proteins with selected reaction monitoring mass spectrometry. In the fractions of displacement mode a significantly higher number of identified proteins (51 versus 16) was yielded in comparison to gradient mode. The resolution of displacement chromatography was slightly lower than of gradient chromatography for many but not for all proteins. The selectivities of displacement mode and gradient mode are very different. In conclusion displacement chromatography is a well suited alternative for top-down proteomic approaches which start with separating intact proteins first prior to mass spectrometric analysis of intact or digested proteins. The significant orthogonality of both modes may be used in the future for combining them in multidimensional fractionation procedures.
University Medical Center Hamburg-Eppendorf, Department of Clinical Chemistry, Mass Spectrometric Proteomics, Campus Forschung, N27 Room 00.08, Martinistr. 52, 20246 Hamburg, Germany.
Robert
R
Ahrends
Björn
B
Lichtner
Friedrich
F
Buck
Diana
D
Hildebrand
Marta
M
Kotasinska
Oliver
O
Kohlbacher
Marcel
M
Kwiatkowski
Moritz
M
Wagner
Maria
M
Trusch
Hartmut
H
Schlüter
D000838:Anions / Q000737:chemistry; D001798:Blood Proteins / Q000737:chemistry / Q000302:isolation & purification*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004553:Electric Conductivity; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet
D000838:Anions; D001798:Blood Proteins; C032309:Cohn fraction IV