title: Tandem application of cationic colloidal silica and Triton X-114 for plasma membrane protein isolation and purification: towards developing an MDCK protein database.
abstract: Plasma membrane (PM) proteins are attractive therapeutic targets because of their accessibility to drugs. Although genes encoding PM proteins represent 20-30% of eukaryotic genomes, a detailed characterisation of their encoded proteins is underrepresented, due, to their low copy number and the inherent difficulties in their isolation and purification as a consequence of their high hydrophobicity. We describe here a strategy that combines two orthogonal methods to isolate and purify PM proteins from Madin Darby canine kidney (MDCK) cells. In this two-step method, we first used cationic colloidal silica (CCS) to isolate adherent (Ad) and non-adherent (nAd) PM fractions, and then subjected each fraction to Triton X-114 (TX-114) phase partitioning to further enrich for hydrophobic proteins. While CCS alone identified 255/757 (34%) membrane proteins, CCS/TX-114 in combination yielded 453/745 (61%). Strikingly, of those proteins unique to CCS/TX-114, 277/393 (70%) had membrane annotation. Further characterisation of the CCS/TX-114 data set using Uniprot and transmembrane hidden Markov model revealed that 306/745 (41%) contained one or more transmembrane domains (TMDs), including proteins with 25 and 17 TMDs. Of the remaining proteins in the data set, 69/439 (16%) are known to contain lipid modifications. Of all membrane proteins identified, 93 had PM origin, including proteins that mediate cell adhesion, modulate transmembrane ion transport, and cell-cell communication. These studies reveal that the application of CCS to first isolate Ad and nAd PM fractions, followed by their detergent-phase TX-114 partitioning, to be a powerful method to isolate low-abundance PM proteins, and a useful adjunct for in-depth cell surface proteome analyses.
authors.affiliation: Ludwig Institute for Cancer Research, Parkville, Victoria, Australia.
authors.forename: Rommel A
authors.initials: RA
authors.lastname: Mathias
authors.forename: Yuan-Shou
authors.initials: YS
authors.lastname: Chen
authors.forename: Robert J A
authors.initials: RJ
authors.lastname: Goode
authors.forename: Eugene A
authors.initials: EA
authors.lastname: Kapp
authors.forename: Suresh
authors.initials: S
authors.lastname: Mathivanan
authors.forename: Robert L
authors.initials: RL
authors.lastname: Moritz
authors.forename: Hong-Jian
authors.initials: HJ
authors.lastname: Zhu
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Simpson
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002412:Cations / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D002458:Cell Fractionation; D002462:Cell Membrane / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002478:Cells, Cultured; D003102:Colloids / Q000378:metabolism; D030562:Databases, Protein; D004285:Dogs; D005260:Female; D057927:Hydrophobic and Hydrophilic Interactions; D017136:Ion Transport / Q000235:genetics; D007668:Kidney / Q000166:cytology; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000378:metabolism; D040081:Protein Array Analysis / Q000379:methods; D017434:Protein Structure, Tertiary; D020543:Proteome / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D012822:Silicon Dioxide / Q000378:metabolism
chemical_list: D002412:Cations; D015815:Cell Adhesion Molecules; D003102:Colloids; D008565:Membrane Proteins; D020543:Proteome; D011092:Polyethylene Glycols; D012822:Silicon Dioxide; D017830:Octoxynol; C010615:Nonidet P-40