title: Towards a membrane proteome in Drosophila: a method for the isolation of plasma membrane.
abstract: BACKGROUND
  The plasma membrane (PM) is a compartment of significant interest because cell surface proteins influence the way in which a cell interacts with its neighbours and its extracellular environment. However, PM is hard to isolate because of its low abundance. Aqueous two-phase affinity purification (2PAP), based on PEG/Dextran two-phase fractionation and lectin affinity for PM-derived microsomes, is an emerging method for the isolation of high purity plasma membranes from several vertebrate sources. In contrast, PM isolation techniques in important invertebrate genetic model systems, such as Drosophila melanogaster, have relied upon enrichment by density gradient centrifugation. To facilitate genetic investigation of activities contributing to the content of the PM sub-proteome, we sought to adapt 2PAP to this invertebrate model to provide a robust PM isolation technique for Drosophila.
  
  
  RESULTS
  We show that 2PAP alone does not completely remove contaminating endoplasmic reticulum and mitochondrial membrane. However, a novel combination of density gradient centrifugation plus 2PAP results in a robust PM preparation. To demonstrate the utility of this technique we isolated PM from fly heads and successfully identified 432 proteins using MudPIT, of which 37% are integral membrane proteins from all compartments. Of the 432 proteins, 22% have been previously assigned to the PM compartment, and a further 34% are currently unassigned to any compartment and represent candidates for assignment to the PM. The remainder have previous assignments to other compartments.
  
  
  CONCLUSIONS
  A combination of density gradient centrifugation and 2PAP results in a robust, high purity PM preparation from Drosophila, something neither technique can achieve on its own. This novel preparation should lay the groundwork for the proteomic investigation of the PM in different genetic backgrounds in Drosophila. Our results also identify two key steps in this procedure: The optimization of membrane partitioning in the PEG/Dextran mixture, and careful choice of the correct lectin for the affinity purification step in light of variations in bulk membrane lipid composition and glycosylation patterns respectively. This points the way for further adaptations into other systems.
authors.affiliation: Department of Biology, The Pennsylvania State University, University Park, PA 16802, USA.
authors.forename: Mansi R
authors.initials: MR
authors.lastname: Khanna
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Stanley
authors.forename: Graham H
authors.initials: GH
authors.lastname: Thomas
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000737:chemistry*; D029721:Drosophila Proteins / Q000032:analysis*; D004331:Drosophila melanogaster / Q000737:chemistry*; D008565:Membrane Proteins / Q000032:analysis*; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods*
chemical_list: D029721:Drosophila Proteins; D008565:Membrane Proteins; D020543:Proteome
references.citation: Plant Cell. 2008 Dec;20(12):3210-26
references.pmid: 19114538
references.citation: Proc Natl Acad Sci U S A. 2007 Jul 3;104(27):11364-9
references.pmid: 17592126
references.citation: J Proteome Res. 2007 Jan;6(1):34-43
references.pmid: 17203946
references.citation: J Cell Sci. 2009 Jul 15;122(Pt 14):2413-23
references.pmid: 19571114
references.citation: Nat Rev Drug Discov. 2006 Dec;5(12):993-6
references.pmid: 17139284
references.citation: Genetics. 2006 Apr;172(4):2025-32
references.pmid: 16636111
references.citation: Proc Natl Acad Sci U S A. 2003 Sep 2;100(18):10219-24
references.pmid: 12923296
references.citation: J Biol Chem. 2001 Jul 27;276(30):28058-67
references.pmid: 11382750
references.citation: Electrophoresis. 2006 Jul;27(13):2747-58
references.pmid: 16739230
references.citation: Genes Brain Behav. 2005 Apr;4(3):147-56
references.pmid: 15810903
references.citation: Cell Biol Int. 2007 Feb;31(2):186-90
references.pmid: 17207647
references.citation: Anal Biochem. 2009 Mar 15;386(2):133-46
references.pmid: 19123999
references.citation: Curr Opin Mol Ther. 2008 Apr;10(2):116-23
references.pmid: 18386223
references.citation: Anal Biochem. 1993 Aug 1;212(2):506-9
references.pmid: 8214593
references.citation: J Biol Chem. 1997 Sep 5;272(36):22583-90
references.pmid: 9278413
references.citation: Mol Cell Proteomics. 2007 Sep;6(9):1638-55
references.pmid: 17533153
references.citation: Nature. 1970 Aug 15;227(5259):680-5
references.pmid: 5432063
references.citation: Proteomics. 2007 Aug;7(16):3010-29
references.pmid: 17654460
references.citation: Proteomics. 2004 Nov;4(11):3536-48
references.pmid: 15378739
references.citation: J Biol Chem. 1998 Jul 31;273(31):19715-21
references.pmid: 9677401
references.citation: Nat Rev Mol Cell Biol. 2008 Feb;9(2):112-24
references.pmid: 18216768
references.citation: Anal Biochem. 1992 Jul;204(1):131-6
references.pmid: 1381154
references.citation: Glycobiology. 2007 Dec;17(12):1388-403
references.pmid: 17893096
references.citation: Curr Opin Cell Biol. 2008 Aug;20(4):378-85
references.pmid: 18606534
references.citation: J Proteome Res. 2008 Sep;7(9):3661-7
references.pmid: 18700793
references.citation: Biochim Biophys Acta. 2001 Apr 30;1531(3):209-21
references.pmid: 11325612
references.citation: Electrophoresis. 2000 Oct;21(16):3427-40
references.pmid: 11079563
references.citation: Biochem J. 1991 Jan 1;273(Pt 1):173-7
references.pmid: 1703408
references.citation: Glycobiology. 2009 May;19(5):527-36
references.pmid: 19218400
references.citation: J Mol Biol. 1982 May 5;157(1):105-32
references.pmid: 7108955
references.citation: Nat Cell Biol. 2005 Jun;7(6):612-8
references.pmid: 15908943
references.citation: Lipids. 1993 Jan;28(1):23-8
references.pmid: 8446007
references.citation: Mol Biol Rep. 1986;11(1):19-24
references.pmid: 3003563
references.citation: Mol Cell Proteomics. 2006 Feb;5(2):390-400
references.pmid: 16249173
references.citation: Proteomics. 2008 Oct;8(19):4066-82
references.pmid: 18763707
references.citation: J Biol Chem. 1999 Apr 23;274(17):12049-54
references.pmid: 10207028
references.citation: Biochem J. 1993 Aug 15;294 ( Pt 1):1-14
references.pmid: 8363559
references.citation: Anal Biochem. 2003 Feb 1;313(1):1-8
references.pmid: 12576051
references.citation: J Cell Sci. 1979 Apr;36:401-12
references.pmid: 457815
references.citation: Proteomics. 2006 Oct;6(20):5409-17
references.pmid: 16972286
references.citation: J Neurochem. 1995 Jul;65(1):434-43
references.pmid: 7540667
references.citation: Anal Biochem. 1989 Aug 1;180(2):205-10
references.pmid: 2817350
references.citation: Mol Cell Proteomics. 2006 Jun;5(6):1082-94
references.pmid: 16543254
references.citation: Nature. 2000 Mar 23;404(6776):394-8
references.pmid: 10746727