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

• Published in: Proteomics (Weinheim) 2011-04, Vol.11 (7), p.1238-1253
• Main Authors: Mathias, Rommel A., Chen, Yuan-Shou, Goode, Robert J. A., Kapp, Eugene A., Mathivanan, Suresh, Moritz, Robert L., Zhu, Hong-Jian, Simpson, Richard J.
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Blotting, Western; Cationic colloidal silica; Cations - metabolism; Cell adhesion; Cell adhesion & migration; Cell Adhesion Molecules - genetics; Cell Adhesion Molecules - isolation & purification; Cell Adhesion Molecules - metabolism; Cell biology; Cell Fractionation; Cell Membrane - chemistry; Cell Membrane - genetics; Cell Membrane - metabolism; Cell surface; Cells, Cultured; Colloids - metabolism; copy number; Data processing; Databases, Protein; Dogs; Drug development; Drugs; Female; Fundamental and applied biological sciences. Psychology; Genomes; hidden Markov models; Hydrophobic and Hydrophilic Interactions; Hydrophobicity; Intercellular signalling; Ion Transport - genetics; Kidney - cytology; Lipids; Mass Spectrometry; MDCK; Membrane proteins; Membrane Proteins - genetics; Membrane Proteins - isolation & purification; Membrane Proteins - metabolism; Miscellaneous; Plasma; Plasma membrane; Plasma membranes; Polyethylene Glycols - metabolism; Protein Array Analysis - methods; protein purification; Protein Structure, Tertiary; Proteins; Proteome - chemistry; Proteome - genetics; Proteome - metabolism; proteomics; Proteomics - methods; Silica; Silicon Dioxide - metabolism; Transmembrane domains; Triton X-114
• ISSN: 1615-9853; 1615-9861; 1615-9861
• DOI: 10.1002/pmic.201000591

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.