Enrichment of hydrophobic proteins via Triton X-114 phase partitioning andhydroxyapatite column chromatography for mass spectrometry

Membrane proteins are the starting point of several signal transduction pat hways. Therefore, the separation and identification of these proteins are o f 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 extrem ely hydrophobic proteins and demonstrate the advantages of special presepar ation procedures for the identification of proteins which have very similar M-r and p/. Using the example of the integral membrane protein very low de nsity lipoprotein (VLDL) receptor (NCBI Acc. #1730111) and the soluble heat shock protein (HSP) 90 (NCBI Acc. # 386786) we present the applicability o f a phase-separation system with Triton X-114. Using matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) of th e protein spots after 2-D separation of the hydrophilic and the strongly hy drophobic protein fraction of human endothelial cells (ECV cell line), we w ere able to distinguish both proteins.