title: An efficient method for the extraction of chloroplast proteins compatible for 2-DE and MS analysis.
abstract: Comparative proteomic analysis of chloroplast by 2-DE has received significant attention in recent years. However, the complication of membrane systems in chloroplast made it challenging to elucidate entire chloroplast proteome by 2-DE. Here, we developed an efficient method for extracting chloroplast proteins, and produced excellent 2-DE profiles from both Arabidopsis thaliana and Salicornia europaea. Comparison of this method with another two protocols for the extraction of A. thaliana chloroplast proteins showed that our method obtained higher protein yields and produced more protein spots on both pH 3-10 and 4-7 2-DE gels. Moreover, this method recovered more proteins in the basic and high M(r) regions, thereby offering the best extraction of chloroplast proteins. Identification of 15 specific chloroplast-targeted proteins on our gels by MALDI-TOF MS revealed that this method was compatible with MS, and recovered more chloroplast membrane proteins than the commonly used methods. This protocol is expected to have a wide application in future chloroplast proteomic analysis.
authors.affiliation: Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing, P. R. China.
authors.forename: Pengxiang
authors.initials: P
authors.lastname: Fan
authors.affiliation: Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing, P. R. China.
authors.forename: Xuchu
authors.initials: X
authors.lastname: Wang
authors.affiliation: Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing, P. R. China.
authors.forename: Tingyun
authors.initials: T
authors.lastname: Kuang
authors.affiliation: Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing, P. R. China.
authors.forename: Yinxin
authors.initials: Y
authors.lastname: Li
mesh_terms: D017360:Arabidopsis / Q000737:chemistry; D019663:Chenopodiaceae / Q000737:chemistry; D002736:Chloroplasts / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry / Q000379:methods*; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods
chemical_list: D010940:Plant Proteins
references.citation: Jorrin, J. V., Maldonado, A. M., Castillejo, M. A., Proteomics 2007, 7, 2947-2962.
references.citation: Warnock, D. E., Fahy, E., Taylor, S. W., Mass Spectrom. Rev. 2004, 23, 259-280.
references.citation: Taylor, S. W., Fahy, E., Ghosh, S. S., Trends Biotechnol. 2003, 21, 82-88.
references.citation: Baginsky, S., Gruissem, W., J. Exp. Bot. 2004, 55, 1213-1220.
references.citation: Schroder, W. P., Kieselbach, T., Photosynth. Res. 2003, 78, 181-193.
references.citation: van Wijk, K. J., Plant Physiol. Biochem. 2004, 42, 963-977.
references.citation: Ferro, M., Salvi, D., Riviere-Rolland, H., Vermat, T., Seigneurin-Berny, D., Grunwald, D., Garin, J. et al., Proc. Natl. Acad. Sci. USA 2002, 99, 11487-11492.
references.citation: Seigneurin-Berny, D., Rolland, N., Garin, J., Joyard, J., Plant J. 1999, 19, 217-228.
references.citation: Ferro, M., Salvi, D., Brugiere, S., Miras, S., Kowalski, S., Louwagie, M., Garin, J. et al., Mol. Cell. Proteomics 2003, 2, 325-345.
references.citation: Froehlich, J. E., Wilkerson, C. G., Ray, W. K., McAndrew, R. S., Osteryoung, K. W., Gage, D. A., Phinney, B. S., J. Proteome Res. 2003, 2, 413-425.
references.citation: Balmer, Y., Koller, A., del Val, G., Manieri, W., Schurmann, P., Buchanan, B. B., Proc. Natl. Acad. Sci. USA 2003, 100, 370-375.
references.citation: Motohashi, K., Kondoh, A., Stumpp, M. T., Hisabori, T., Proc. Natl. Acad. Sci. USA 2001, 98, 11224-11229.
references.citation: Yamaguchi, K., Subramanian, A. R., J. Biol. Chem. 2000, 275, 28466-28482.
references.citation: Peltier, J. B., Cai, Y., Sun, Q., Zabrouskov, V., Giacomelli, L., Rudella, A., Ytterberg, A. J. et al., Mol. Cell. Proteomics 2006, 5, 114-133.
references.citation: Peltier, J. B., Emanuelsson, O., Kalume, D. E., Ytterberg, J., Friso, G., Rudella, A., Liberles, D. A. et al., Plant Cell 2002, 14, 211-236.
references.citation: Friso, G., Giacomelli, L., Ytterberg, A. J., Peltier, J. B., Rudella, A., Sun, Q., van Wijk, K. J., Plant Cell 2004, 16, 478-499.
references.citation: Hippler, M., Klein, J., Fink, A., Allinger, T., Hoerth, P., Plant J. 2001, 28, 595-606.
references.citation: Schubert, M., Petersson, U. A., Haas, B. J., Funk, C., Schroder, W. P., Kieselbach, T., J. Biol. Chem. 2002, 277, 8354-8365.
references.citation: Kieselbach, T., Bystedt, M., Hynds, P., Robinson, C., Schroder, W. P., FEBS Lett. 2000, 480, 271-276.
references.citation: Kieselbach, T., Schroder, W. P., Photosynth. Res. 2003, 78, 249-264.
references.citation: Goulas, E., Schubert, M., Kieselbach, T., Kleczkowski, L. A., Gardestrom, P., Schroder, W., Hurry, V., Plant J. 2006, 47, 720-734.
references.citation: Valcu, C. M., Schlink, K., Proteomics 2006, 6, 4166-4175.
references.citation: Vincent, D., Wheatley, M. D., Cramer, G. R., Electrophoresis 2006, 27, 1853-1865.
references.citation: Yao, Y., Yang, Y. W., Liu, J. Y., Electrophoresis 2006, 27, 4559-4569.
references.citation: Wang, X. C., Li, X. F., Deng, X., Han, H. P., Shi, W. L., Li, Y. X., Electrophoresis 2007, 28, 3976-3987.
references.citation: Gomez-Vidal, S., Tena, M., Lopez-Llorca, L. V., Salinas, J., Electrophoresis 2008, 29, 448-456.
references.citation: Pirovani, C. P., Carvalho, H. A. S., Machado, R. C. R., Gomes, D. S., Alvim, F. C., Pomella, A. W. V., Gramacho, K. P. et al., Electrophoresis 2008, 29, 2391-2401.
references.citation: Wilson, K. A., McManus, M. T., Gordon, M. E., Jordan, T. W., Proteomics 2002, 2, 1114-1122.
references.citation: Kubis, S., Baldwin, A., Patel, R., Razzaq, A., Dupree, P., Lilley, K., Kurth, J. et al., Plant Cell 2003, 15, 1859-1871.
references.citation: Lonosky, P. M., Zhang, X. S., Honavar, V. G., Dobbs, D. L., Fu, A., Rodermel, S. R., Plant Physiol. 2004, 134, 560-574.
references.citation: Phee, B. K., Cho, J. H., Park, S., Jung, J. H., Lee, Y. H., Jeon, J. S., Bhoo, S. H. et al., Proteomics 2004, 4, 3560-3568.
references.citation: Kleffmann, T., Russenberger, D., von Zychlinski, A., Christopher, W., Sjolander, K., Gruissem, W., Baginsky, S., Curr. Biol. 2004, 14, 354-362.
references.citation: Bhushan, D., Pandey, A., Chattopadhyay, A., Choudhary, M. K., Chakraborty, S., Datta, A., Chakraborty, N., J. Proteome Res. 2006, 5, 1711-1720.
references.citation: Bradford, M. M., Anal. Biochem. 1976, 72, 248-254.
references.citation: Wang, X. C., Li, X. F., Li, Y. X., Biotechnol. Lett. 2007, 29, 1599-1603.
references.citation: Emanuelsson, O., Nielsen, H., Brunak, S., von Heijne, G., J. Mol. Biol. 2000, 300, 1005-1016.
references.citation: Krogh, A., Larsson, B., von Heijne, G., Sonnhammer, E. L. L., J. Mol. Biol. 2001, 305, 567-580.
references.citation: Kyte, J., Doolittle, R. F., J. Mol. Biol. 1982, 157, 105-132.
references.citation: Sun, Q., Zybailov, B., Majeran, W., Friso, G., Olinares, P. D. B., van Wijk, K. J., Nucleic Acids Res. 2009, 37, D969-D974.
references.citation: Marmagne, A., Rouet, M. A., Ferro, M., Rolland, N., Alcon, C., Joyard, J., Garin, J. et al., Mol. Cell. Proteomics 2004, 3, 675-691.
references.citation: Saravanan, R. S., Rose, J. K. C., Proteomics 2004, 4, 2522-2532.
references.citation: Gorg, A., Weiss, W., Dunn, M. J., Proteomics 2004, 4, 3665-3685.
references.citation: Carpentier, S. C., Witters, E., Laukens, K., Deckers, P., Swennen, R., Panis, B., Proteomics 2005, 5, 2497-2507.
references.citation: Nandakumar, M. P., Shen, J., Raman, B., Marten, M. R., J. Proteome Res. 2003, 2, 89-93.
references.citation: Espagne, C., Martinez, A., Valot, B., Meinnel, T., Giglione, C., Proteomics 2007, 7, 3788-3799.
references.citation: Zybailov, B., Rutschow, H., Friso, G., Rudella, A., Emanuelsson, O., Sun, Q., van Wijk, K. J., PLoS ONE 2008, 3, e1994.