title: Improved recovery and identification of membrane proteins from rat hepatic cells using a centrifugal proteomic reactor.
abstract: Despite their importance in many biological processes, membrane proteins are underrepresented in proteomic analysis because of their poor solubility (hydrophobicity) and often low abundance. We describe a novel approach for the identification of plasma membrane proteins and intracellular microsomal proteins that combines membrane fractionation, a centrifugal proteomic reactor for streamlined protein extraction, protein digestion and fractionation by centrifugation, and high performance liquid chromatography-electrospray ionization-tandem MS. The performance of this approach was illustrated for the study of the proteome of ER and Golgi microsomal membranes in rat hepatic cells. The centrifugal proteomic reactor identified 945 plasma membrane proteins and 955 microsomal membrane proteins, of which 63 and 47% were predicted as bona fide membrane proteins, respectively. Among these proteins, >800 proteins were undetectable by the conventional in-gel digestion approach. The majority of the membrane proteins only identified by the centrifugal proteomic reactor were proteins with ≥ 2 transmembrane segments or proteins with high molecular mass (e.g. >150 kDa) and hydrophobicity. The improved proteomic reactor allowed the detection of a group of endocytic and/or signaling receptor proteins on the plasma membrane, as well as apolipoproteins and glycerolipid synthesis enzymes that play a role in the assembly and secretion of apolipoprotein B100-containing very low density lipoproteins. Thus, the centrifugal proteomic reactor offers a new analytical tool for structure and function studies of membrane proteins involved in lipid and lipoprotein metabolism.
authors.affiliation: Ottawa Institute of Systems Biology, Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, Canada.
authors.forename: Hu
authors.initials: H
authors.lastname: Zhou
authors.forename: Fangjun
authors.initials: F
authors.lastname: Wang
authors.forename: Yuwei
authors.initials: Y
authors.lastname: Wang
authors.forename: Zhibin
authors.initials: Z
authors.lastname: Ning
authors.forename: Weimin
authors.initials: W
authors.lastname: Hou
authors.forename: Theodore G
authors.initials: TG
authors.lastname: Wright
authors.forename: Meenakshi
authors.initials: M
authors.lastname: Sundaram
authors.forename: Shumei
authors.initials: S
authors.lastname: Zhong
authors.forename: Zemin
authors.initials: Z
authors.lastname: Yao
authors.forename: Daniel
authors.initials: D
authors.lastname: Figeys
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002498:Centrifugation / Q000379:methods; D005591:Chemical Fractionation; D002853:Chromatography, Liquid; D022781:Hepatocytes / Q000737:chemistry* / Q000378:metabolism; D050356:Lipid Metabolism; D008074:Lipoproteins / Q000302:isolation & purification / Q000378:metabolism; D008565:Membrane Proteins / Q000032:analysis* / Q000302:isolation & purification; D008861:Microsomes / Q000737:chemistry / Q000378:metabolism; D059748:Proteolysis; D020543:Proteome / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D051381:Rats; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D008074:Lipoproteins; D008565:Membrane Proteins; D020543:Proteome