title: Proteomic characterization of integral membrane proteins using thermostatted liquid chromatography coupled with tandem mass spectrometry.
abstract: Due to the hydrophobicity and localization of integral membrane proteins, they are difficult to study using conventional biochemical methods that are compatible with proteomic analyses. This chapter describes the coupling of multiple crucial steps that lead to the optimized shotgun proteomic analysis of integral membrane proteins while maintaining empirical topology information. Namely, a membrane shaving method is utilized to separate protease accessible peptides from membrane embedded peptides and elevated temperatures during chromatographic separation is utilized to augment the recovery of hydrophobic peptides for in-line analysis using tandem mass spectrometry. This combination of steps facilitates increased identification of membrane proteins while also maintaining information regarding protein topology.
authors.affiliation: Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
authors.forename: Sarah M
authors.initials: SM
authors.lastname: Moore
authors.forename: Christine C
authors.initials: CC
authors.lastname: Wu
mesh_terms: D002462:Cell Membrane / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods*; D002918:Chymotrypsin / Q000378:metabolism; D019286:Endopeptidase K / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D008565:Membrane Proteins; D010455:Peptides; D002918:Chymotrypsin; D019286:Endopeptidase K