Summary
All techniques needed for proteomic analyses of plant plasma membranes are described in detail, from isolation of plasma membranes to protein identification by mass spectrometry (MS). Plasma membranes are isolated by aqueous two-phase partitioning yielding vesicles with a cytoplasmic side-in orientation and a purity of about 95%. These vesicles are turned inside-out by treatment with Brij 58, which removes soluble contaminating proteins enclosed in the vesicles as well as loosely attached proteins. The final plasma membrane preparation thus retains all integral proteins and many peripheral proteins. Proteins are separated by one-dimensional sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE), and protein bands are excised and digested with trypsin. Peptides in tryptic digests are separated by nanoflow liquid chromatography and either fed directly into an ESI-MS or spotted onto matrix-assisted laser desorption ionization (MALDI) plates for analysis with MALDI-MS. Finally, data processing and database searching are used for protein identification to define a plasma membrane proteome.
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References
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Acknowledgments
We thank Gerhard Saalbach for his help and comments on the ESI-MS section. Work in the authors’ laboratories was supported by grants from the Swedish Foundation for Strategic Research (C.L.), the Swedish Research Council (C.L.), the Knut and Alice Wallenberg Foundation (C.L.), and Formas (P.K.).
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© 2008 Humana Press, a part of Springer Science+Business Media, LLC
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Alexandersson, E., Gustavsson, N., Bernfur, K., Karlsson, A., Kjellbom, P., Larsson, C. (2008). Purification and Proteomic Analysis of Plant Plasma Membranes. In: Pflieger, D., Rossier, J. (eds) Organelle Proteomics. Methods in Molecular Biology™, vol 432. Humana Press. https://doi.org/10.1007/978-1-59745-028-7_11
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DOI: https://doi.org/10.1007/978-1-59745-028-7_11
Publisher Name: Humana Press
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