Tandem application of cationic colloidal silica and Triton X-114 for plasma membrane protein isolation and purification: towards developing an MDCK protein database

Proteomics. 2011 Apr;11(7):1238-53. doi: 10.1002/pmic.201000591. Epub 2011 Feb 17.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Blotting, Western
  • Cations / metabolism
  • Cell Adhesion Molecules / genetics*
  • Cell Adhesion Molecules / isolation & purification*
  • Cell Adhesion Molecules / metabolism
  • Cell Fractionation
  • Cell Membrane / chemistry
  • Cell Membrane / genetics*
  • Cell Membrane / metabolism
  • Cells, Cultured
  • Colloids / metabolism
  • Databases, Protein
  • Dogs
  • Female
  • Hydrophobic and Hydrophilic Interactions
  • Ion Transport / genetics
  • Kidney / cytology
  • Mass Spectrometry
  • Membrane Proteins / genetics*
  • Membrane Proteins / isolation & purification*
  • Membrane Proteins / metabolism
  • Octoxynol
  • Polyethylene Glycols / metabolism
  • Protein Array Analysis / methods
  • Protein Structure, Tertiary
  • Proteome / chemistry
  • Proteome / genetics
  • Proteome / metabolism
  • Proteomics / methods*
  • Silicon Dioxide / metabolism


  • Cations
  • Cell Adhesion Molecules
  • Colloids
  • Membrane Proteins
  • Proteome
  • Polyethylene Glycols
  • Silicon Dioxide
  • Octoxynol
  • Nonidet P-40