Enrichment of hydrophobic proteins via Triton X-114 phase partitioning and hydroxyapatite column chromatography for mass spectrometry.
Membrane proteins are the starting point of several signal transduction pathways. Therefore, the separation and identification of these proteins are of great interest in proteome analysis. However, the specific properties of membrane proteins seriously impede their analysis. We present an effective and highly reproducible method for the two-dimensional separation of extremely hydrophobic proteins and demonstrate the advantages of special preseparation procedures for the identification of proteins which have very similar Mr and p/. Using the example of the integral membrane protein very low density lipoprotein (VLDL) receptor (NCBI Acc. # 1730111) and the soluble heat shock protein (HSP) 90 (NCBI Acc. # 386786) we present the applicability of a phase-separation system with Triton X-114. Using matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) of the protein spots after 2-D separation of the hydrophilic and the strongly hydrophobic protein fraction of human endothelial cells (ECV cell line), we were able to distinguish both proteins.
Wissing
J
J
Technical University of Braunschweig, Department of Biochemistry, Germany. jow@gbf.de
Heim
S
S
Flohé
L
L
Bilitewski
U
U
Frank
R
R
D002460:Cell Line; D005591:Chemical Fractionation / Q000379:methods*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002853:Chromatography, Liquid; D004396:Coloring Agents; D003902:Detergents; D017886:Durapatite; D004730:Endothelium, Vascular / Q000737:chemistry; D006801:Humans; D007525:Isoelectric Focusing; D017830:Octoxynol; D011092:Polyethylene Glycols; D011506:Proteins / Q000032:analysis / Q000302:isolation & purification*; D012394:Rosaniline Dyes; D012995:Solubility; D013048:Specimen Handling; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013194:Staining and Labeling; D013696:Temperature
D004396:Coloring Agents; D003902:Detergents; D011506:Proteins; D012394:Rosaniline Dyes; D011092:Polyethylene Glycols; C048139:Coomassie blue; D017830:Octoxynol; C010615:Nonidet P-40; D017886:Durapatite