High Recovery HPLC Separation of Lipid Rafts for Membrane Proteome Analysis

Proteomic analysis of complex samples can be facilitated by protein fractionation prior to enzymatic or chemical fragmentation combined with MS-based identification of peptides. Although aqueous soluble protein fractionation by liquid chromatography is relatively straightforward, membrane protein separations have a variety of technical challenges. Reversed-phase high performance liquid chromatography (RP−HPLC) separations of membrane proteins often exhibit poor recovery and bandwidths, and generally require extensive pretreatment to remove lipids and other membrane components. Human brain tissue lipid raft protein preparations have been used as a model system to develop RP−HPLC conditions that are effective for protein fractionation, and are compatible with downstream proteomic analytical workflows. By the use of an appropriate RP column material and operational conditions, human brain membrane raft proteins were successfully resolved by RP−HPLC and some of the protein components, including specific integral membrane proteins, identified by downstream SDS-PAGE combined with in-gel digestion, or in-solution digestion and LC−MS/MS analysis of tryptic fragments. Using the described method, total protein recovery was high, and the repeatability of the separation maintained after repeated injections of membrane raft preparations. Keywords: proteomics • pre-fractionation • reversed-phase chromatography • integral membrane proteins • lipid rafts • mass spectroscopy • gel electrophoresis • HPLC