High Recovery HPLC of Lipid Raft Domains with the Agilent mRP-C18 Column – A New Proteomic Strategy for Enabling Membrane Protein Identifications Application Note

Membrane proteins play a critical role in cellular processes and currently account for ~70% of all known pharmaceutical drug targets. However, despite their biological importance and natural abundance, they remain an under-represented subset of studied proteins within proteomics. Lipid rafts are membrane microdomains that are enriched in cholesterol and glycosphingolipids and it is believed that proteins embedded in the membrane localize either to the clusters of sphigolipids and cholesterol or to the intervening fluid regions of glycerolipids. They are extremely important entities of the plasma membrane and have been implicated in diverse cellular processes, including signal transduction, endocytic events, such as viral entry, and cholesterol trafficking. Yet, while analyses of raft function and composition continues to be of great interest, their study has limitations due to the extremely hydrophobic nature of the raft domains. A robust proteomic workflow was developed for the identification of membrane proteins from human brain lipid rafts using a novel macroporous reversed-phase column High Recovery HPLC of Lipid Raft Domains with the Agilent mRP-C18 Column – A New Proteomic Strategy for Enabling Membrane Protein Identifications Application Note (mRP-C18) and optimized chromatographic conditions. The column conditions and special chromatographic material combine to provide high-protein recoveries, excellent separations and reproducible chromatography. The robust column methodology was used in a multidimensional separation strategy to identify membrane proteins found in lipid rafts. The lipid rafts were fractionated by the mRP-C18 column and further resolved by gel electrophoresis. Unique bands were identified and analyzed by mass spectrometry. Using this strategy we have identified 158 proteins (73 membrane) and 35 integral membrane proteins) from 48 excised gel bands.