Abstract
Chlamydomonas is a model organism to study photosynthesis. Thylakoid membranes comprise several proteins belonging to photosystems I and II. In this chapter, we show the accurate proteomic measurements in thylakoid membranes. The chlorophyll-containing membrane protein complexes were precipitated using chloroform/methanol solution. These complexes were separated using two-dimensional gel electrophoresis, and the resolved spots were exercised from the gel matrix and digested with trypsin. These peptide fragments were separated by MALDI-TOF, and the isotopic masses were blasted to a MASCOT server to obtain the protein sequence. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF). The method discussed here would be a useful method for the separation and identification of thylakoid membrane proteins.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Rochaix, J. D. (2002) Chlamydomonas, a model system for studying the assembly and dynamics of photosynthetic complexes. FEBS Lett. 529, 34–38.
Elrad, E. D. and Grossman, A. R. (2004) A genome’s-eye view of the light-harvesting polypeptides of Chlamydomonas reinhardtii. Curr. Genet. 45, 61–75.
Froehlich, J. E., Wilkerson, C. G., Ray, W. K., McAndrew, R. S., Osteryoung, K. W., Gage, D. A., and Phinney, B. S. (2003) Proteomic study of the Arabidopsis thaliana chloroplastic envelope membrane utilizing alternatives to traditional two-dimensional electrophoresis. J. Proteome Res. 2, 413–425.
Julian, P. W. (2003) Thylakoid membrane proteomics. Photosynth. Res. 78, 265–277.
Rolland, N., Ferro, M., Seigneurin-Berny, D., Garin, J., Douce. R., and Joyard, J. (2003) Proteomics of chloroplast envelope membranes. Photosynth. Res. 78, 205–230.
Ephritikhine, G., Ferro, M., and Rolland, N. (2004) Plant membrane proteomics. Plant Physiol. Biochem. 42, 943–962.
Jiang, L., He,L., and Fountoulakis, M. (2004) Comparison of protein precipitation methods for sample preparation prior to proteomic analysis. J. Chromatogr. A 1023, 317–320.
VanWijk, K. J. (2004) Plastid proteomics. Plant Physiol.Biochem. 42, 963–977.
Chen, X., Zhang, W., Xie, Y., Lu, W., and Zhang, R. (2007) Comparative proteomics of thylakoid membrane from a chlorophyll b-less rice mutant and its wild type. Plant Sci. 173, 397–407.
Kashino, Y., Harayama, T., Pakrasi, H., and Satoh, K. (2007) Preparation of membrane proteins for analysis by two-dimensional gel electrophoresis. J. Chromatogr. B 849, 282–292.
Mitra, S. K., Gantt, J. A., Ruby, J. F., Clouse, S. D., and Goshe, M. B. (2007) Membrane proteomic analysis of Arabidopsis thaliana using alternative solubilization techniques. J. Proteome Res. 6, 1933–1950.
Ramesh, V. M. and Webber A. N. (2004) Rapid isolation of photosystem I chlorophyll-binding protein from Chlamydomonas reinhardtii. Meth. Mol. Biol. 274, 19–28.
Arnon, D. I. (1949) Copper enzymes in isolated chloroplasts. polyphenoloxidase in Beta vulgaris. Plant Physiol. 24, 1–15.
Wessel, D. and Fluegge, U. I. (1984) A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal. Biochem. 138, 141–143.
Hippler, M., Klein, J., Fink, A., Allinger, T., and Hoerth, P., (2001). Towards functional proteomics of membrane protein complexes: analysis of thylakoid membranes from Chlamydomonas reinhardtii. Plant J. 28(5), 595–606.
Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Anal. Biochem. 72, 248–254.
Chevallet, M., Luche, S., and Rabillound, T. (2006). Silver staining of proteins in polyacrylamide gels. Nat. protoc. 1, 1852–1857.
Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. (1996) Mass spectrometric sequencing of proteins from silver stained polyacrylamide gels. Anal. Chem. 68, 850–858.
Subramanyam, R., Jolley, C., Brune, D.C., Fromme, P. and Webber, A.N., (2006) Characterization of a novel photosystem I-LHCI supercomplex isolated from Chlamydomonas reinhardtii under anaerobic (State II) conditions. FEBS Lett. 580, 233–238.
Acknowledgments
RS is gratefully acknowledged for the financial support from DST NO.SR/SO/BB-34/2006, DST-FAST track NO SR/FT/L-89/2006 and DB-RGYI, Government of India. VY and SN thank CSIR and UGC India for a fellowship in the form of JRF and SRF. We thank Mark Hunter, Department of Chemistry and Biochemistry, Arizona State University, USA, for critical reading of this chapter. We greatly acknowledge the School of Life Sciences, UOH-CREBB, for providing the facility of MALDI-TOF mass spectrometry at the University of Hyderabad.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Yadavalli, V., Nellaepalli, S., Subramanyam, R. (2011). Proteomic Analysis of Thylakoid Membranes. In: Carpentier, R. (eds) Photosynthesis Research Protocols. Methods in Molecular Biology, vol 684. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-925-3_14
Download citation
DOI: https://doi.org/10.1007/978-1-60761-925-3_14
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60761-924-6
Online ISBN: 978-1-60761-925-3
eBook Packages: Springer Protocols