SpringerProtocols: Abstract: 13. Purification of Basolateral Integral Membrane Proteins by Cationic Colloidal Silica-Based Apical Membrane Subtraction

13. Purification of Basolateral Integral Membrane Proteins by Cationic Colloidal Silica-Based Apical Membrane Subtraction

By: Robert J.A. Goode³, Richard J. Simpson⁴

Abstract

Full Text

Download PDF (543K)

Epithelial cell polarity mediates many essential biological functions and perturbation of the apical/basolateral divide is a hallmark of epithelial to mesenchymal transition in carcinoma. Therefore, correct targeting of proteins to the apical and basolateral surfaces is essential to proper epithelial cell function. However, proteomic characterisation of apical/basolateral sorting has been largely ignored, due to ineffectual separation techniques and contamination of plasma-membrane preparations with housekeeping proteins. Here we describe a method that strips the apical membrane from the adherent cells and releases the intracellular contents, thereby leaving the basolateral membrane available for stringent washes and collection. Analysis of the basolateral membrane of an adherent colon adenocarcinoma cell line resulted in 66% of identified proteins being integral membrane proteins, which possessed either a transmembrane domain or lipid modification, including 35 CD antigens. Based on the abundance of peptides from basolateral marker proteins, this method efficiently captures basolateral integral membrane proteins, with minimal contamination from other membranes and basic proteins.

Affiliation(s): (3) Joint Proteomics Laboratory, Ludwig Institute for Cancer Research & The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia
(4) Joint ProteomicS Laboratory, Ludwig Institute for Cancer Research & The Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Parkville, VIC, Australia

Book Title: Membrane Proteomics: Methods and Protocols

Series: Methods in Molecular Biology | Volume: 528 | Pub. Date: Jul-01-2009 | Page Range: 177-187 | DOI: 10.1007/978-1-60327-310-7_13

Subject: Cell Biology

Key Words: Membrane - cationic colloidal silica - basolateral - integral membrane proteins - plasma membrane - epithelial cell polarity

References

Download References

1.	Rabilloud, T. (2003). Membrane proteins ride shotgun. Nat. Biotechnology 21, 508–10.

2.	Yates, J. R., 3rd, Gilchrist, A., Howell, K. E., and Bergeron, J. J. (2005). Proteomics of organelles and large cellular structures. Nat Rev Mol Cell Biol 6, 702–14.

3.	Goode, R. J. A., and Simpson, R. J. (2005) Handling membrane proteins in “Encyclopedia of genetics, genomics, proteomics, and bioinformatics” (Dunn, M., Jorde, L. B., Little, D., Subramanian, S. Eds.), John Wiley & Sons Ltd., Hoboken, N.J.

4.	Shin, B. K., Wang, H., Yim, A. M., Le Naour, F., Brichory, F., Jang, J. H., Zhao, R., Puravs, E., Tra, J., Michael, C. W., Misek, D. E., and Hanash, S. M. (2003). Global profiling of the cell surface proteome of cancer cells uncovers an abundance of proteins with chaperone function. J Biol Chem 278, 7607–16.

5.	Simpson, R. J., Connolly, L. M., Eddes, J. S., Pereira, J. J., Moritz, R. L., and Reid, G. E. (2000). Proteomic analysis of the human colon carcinoma cell line (LIM 1215): development of a membrane protein database. Electrophoresis 21, 1707–32.

6.	Adam, P. J., Boyd, R., Tyson, K. L., Fletcher, G. C., Stamps, A., Hudson, L., Poyser, H. R., Redpath, N., Griffiths, M., Steers, G., Harris, A. L., Patel, S., Berry, J., Loader, J. A., Townsend, R. R., Daviet, L., Legrain, P., Parekh, R., and Terrett, J. A. (2003). Comprehensive proteomic analysis of breast cancer cell membranes reveals unique proteins with potential roles in clinical cancer. J Biol Chem 278, 6482–89.

7.	Jang, J. H., and Hanash, S. (2003). Profiling of the cell surface proteome. Proteomics 3, 1947–54.

8.	Chen, W. N., Yu, L. R., Strittmatter, E. F., Thrall, B. D., Camp, D. G., and Smith, R. D. (2003). Detection of in situ labeled cell surface proteins by mass spectrometry: application to the membrane subproteome of human mammary epithelial cells. Proteomics 3, 1647–51.

9.	Le Bivic, A., Sambuy, Y., Mostov, K., and Rodriguez-Boulan, E. (1990). Vectorial targeting of an endogenous apical membrane sialoglycoprotein and uvomorulin in MDCK cells. J Cell Biol 110, 1533–9.

10.	Peirce, M. J., Wait, R., Begum, S., Saklatvala, J., and Cope, A. P. (2004). Expression profiling of lymphocyte plasma membrane proteins. Mol Cell Proteomics 3, 56–65.

11.	Chaney, L. K., and Jacobson, B. S. (1983). Coating cells with colloidal silica for high yield isolation of plasma membrane sheets and identification of transmembrane proteins. J Biol Chem 258, 10062–72.

12.	Jacobson, B. S., Schnitzer, J. E., McCaffery, M., and Palade, G. E. (1992). Isolation and partial characterization of the luminal plasmalemma of microvascular endothelium from rat lungs. Eur J Cell Biol 58, 296–306.

13.	Jacobson, B. S., Stolz, D. B., and Schnitzer, J. E. (1996). Identification of endothelial cell-surface proteins as targets for diagnosis and treatment of disease. Nat Med 2, 482–84.

14.	Oh, P., Li, Y., Yu, J., Durr, E., Krasinska, K. M., Carver, L. A., Testa, J. E., and Schnitzer, J. E. (2004). Subtractive proteomic mapping of the endothelial surface in lung and solid tumours for tissue-specific therapy. Nature 429, 629–35.

15.	Schnitzer, J. E., McIntosh, D. P., Dvorak, A. M., Liu, J., and Oh, P. (1995). Separation of caveolae from associated microdomains of GPI-anchored proteins. Science 269, 1435–39.

16.	Schnitzer, J. E., Oh, P., Jacobson, B. S., and Dvorak, A. M. (1995). Caveolae from luminal plasmalemma of rat lung endothelium: microdomains enriched in caveolin, Ca(2+)-ATPase, and inositol trisphosphate receptor. Proc Nat. Acad. Sci. USA 92, 1759–63.

17.	Spector, D. L., Goldman, R. D., and Leinwand, L. A. (1998) Cells: A Laboratory Manual 1, 35.1–35.14.

18.	Rahbar, A. M., and Fenselau, C. (2004). Integration of Jacobson’s pellicle method into proteomic strategies for plasma membrane proteins. J Proteome Res 3, 1267–77.

19.	Fujiki, Y., Hubbard, A. L., Fowler, S., and Lazarow, P. B. (1982). Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum. J Cell Biol 93, 97–102.

20.	Wu, C. H., Apweiler, R., Bairoch, A., Natale, D. A., Barker, W. C., Boeckmann, B., Ferro, S., Gasteiger, E., Huang, H., Lopez, R., Magrane, M., Martin, M. J., Mazumder, R., O’Donovan, C., Redaschi, N., and Suzek, B. (2006). The Universal Protein Resource (UniProt): an expanding universe of protein information. Nucleic Acids Res 34, D187–91.

21.	Krogh, A., Larsson, B., von Heijne, G., and Sonnhammer, E. L. (2001). Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 305, 567–80.

22.	Bosman, F. T. (1993). Integrins: cell adhesives and modulators of cell function. Histochem J 25, 469–77.

23.	Amerongen, H. M., Mack, J. A., Wilson, J. M., and Neutra, M. R. (1989). Membrane domains of intestinal epithelial cells: distribution of Na+,K+-ATPase and the membrane skeleton in adult rat intestine during fetal development and after epithelial isolation. J Cell Biol 109, 2129–38.

24.	Green, K. J., and Gaudry, C. A. (2000). Are desmosomes more than tethers for intermediate filaments? Nat Rev Mol Cell Biol 1, 208–16.

25.	Martosella, J., Zolotarjova, N., Liu, H., Moyer, S. C., Perkins, P. D., and Boyes, B. E. (2006). High recovery HPLC separation of lipid rafts for membrane proteome analysis. J Proteome Res 5, 1301–12.

Comments (Loading...)

Post comment/View all comments