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Sample extraction techniques for enhanced proteomic analysis of plant tissues

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Abstract

Major improvements in proteomic techniques in recent years have led to an increase in their application in all biological fields, including plant sciences. For all proteomic approaches, protein extraction and sample preparation are of utmost importance for optimal results; however, extraction of proteins from plant tissues represents a great challenge. Plant tissues usually contain relatively low amounts of proteins and high concentrations of proteases and compounds that potentially can limit tissue disintegration and interfere with subsequent protein separation and identification. An effective protein extraction protocol must also be adaptable to the great variation in the sets of secondary metabolites and potentially contaminating compounds that occurs between tissues (e.g., leaves, roots, fruit, seeds and stems) and between species. Here we present two basic protein extraction protocols that have successfully been used with diverse plant tissues, including recalcitrant tissues. The first method is based on phenol extraction coupled with ammonium acetate precipitation, and the second is based on trichloroacetic acid (TCA) precipitation. Both extraction protocols can be completed within 2 d.

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Figure 1: Flowchart of protein extraction protocol.
Figure 2: Two-dimensional gel electrophoresis of protein samples from maize root tips.

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References

  1. Wu, W.W., Wang, G., Baek, S.J. & Shen, R.F. Comparative study of three proteomic quantitative methods, DIGE, cICAT, and iTRAQ, using 2D gel- or LC-MALDI TOF/TOF. J. Proteome Res. 5, 651–658 (2006).

    Article  CAS  Google Scholar 

  2. Rose, J.K., Bashir, S., Giovannoni, J.J., Jahn, M.M. & Saravanan, R.S. Tackling the plant proteome: practical approaches, hurdles and experimental tools. Plant J. 39, 715–733 (2004).

    Article  CAS  Google Scholar 

  3. Saravanan, R.S. & Rose, J.K. A critical evaluation of sample extraction techniques for enhanced proteomic analysis of recalcitrant plant tissues. Proteomics 4, 2522–2532 (2004).

    Article  CAS  Google Scholar 

  4. Rabilloud, T. Solubilization of proteins for electrophoretic analyses. Electrophoresis 17, 813–829 (1996).

    Article  CAS  Google Scholar 

  5. Damerval, C., Devienne, D., Zivy, M. & Thiellement, H. Technical improvements in two-dimensional electrophoresis increase the level of genetic-variation detected in wheat-seedling proteins. Electrophoresis 7, 52–54 (1986).

    Article  CAS  Google Scholar 

  6. Santoni, V.R., Bellini, C. & Caboche, M. Use of two-dimensional protein-pattern analysis for the characterization of Arabidopsis thaliana mutants. Planta 192, 557–566 (1994).

    Article  CAS  Google Scholar 

  7. Carpentier, S.C. et al. Preparation of protein extracts from recalcitrant plant tissues: An evaluation of different methods for two-dimensional gel electrophoresis analysis. Proteomics 5, 2497–2507 (2005).

    Article  CAS  Google Scholar 

  8. Wang, W. et al. Protein extraction for two-dimensional electrophoresis from olive leaf, a plant tissue containing high levels of interfering compounds. Electrophoresis 24, 2369–2375 (2003).

    Article  CAS  Google Scholar 

  9. Hurkman, W.J. & Tanaka, C.K. Solubilization of plant membrane proteins for analysis by two-dimensional gel electrophoresis. Plant Physiol. 81, 802–806 (1986).

    Article  CAS  Google Scholar 

  10. Meyer, Y., Grosset, J., Chartier, Y. & Cleyetmarel, J.C. Preparation by two-dimensional electrophoresis of proteins for antibody-production—antibodies against proteins whose synthesis is reduced by auxin in tobacco mesophyll protoplasts. Electrophoresis 9, 704–712 (1988).

    Article  CAS  Google Scholar 

  11. Schuster, A.M. & Davies, E. Ribonucleic acid and protein metabolism in pea epicotyls: I. The aging process. Plant Physiol. 73, 809–816 (1983).

    Article  CAS  Google Scholar 

  12. Usuda, H. & Shimogawara, K. Phosphate deficiency in maize. VI. Changes in the two-dimensional electrophoretic patterns of soluble proteins from second leaf blades associated with induced senescence. Plant Cell Physiol. 36, 1149–1155 (1995).

    Article  CAS  Google Scholar 

  13. Mijnsbrugge, K.V., Meyermans, H., Van Montagu, M., Bauw, G. & Boerjan, W. Wood formation in poplar: identification, characterization, and seasonal variation of xylem proteins. Planta 210, 589–598 (2000).

    Article  Google Scholar 

  14. Alban, A. et al. A novel experimental design for comparative two-dimensional gel analysis: Two-dimensional difference gel electrophoresis incorporating a pooled internal standard. Proteomics 3, 36–44 (2003).

    Article  CAS  Google Scholar 

  15. Unlu, M., Morgan, M.E. & Minden, J.S. Difference gel electrophoresis: A single gel method for detecting changes in protein extracts. Electrophoresis 18, 2071–2077 (1997).

    Article  CAS  Google Scholar 

  16. Gygi, S.P. et al. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat. Biotech. 17, 994–999 (1999).

    Article  CAS  Google Scholar 

  17. Ross, P.L. et al. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol. Cell. Proteomics 3, 1154–1169 (2004).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

Research in this area was supported by a grant to J.K.C.R. from the National Science Foundation (DBI:0606595). T.I. is supported by a Vaadia-BARD Postdoctoral Fellowship Award (No. FI-375-05) from BARD, The United States–Israel Binational Agricultural Research and Development Fund. C.M.B.D. is supported by a CNPq-Brazil Fellowship.

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Authors and Affiliations

  1. Department of Plant Biology, 228 Plant Sciences Building, Cornell University, Ithaca, 14853, New York, USA

    Tal Isaacson, Cynthia M B Damasceno, Ramu S Saravanan, Yonghua He, Carmen Catalá, Montserrat Saladié & Jocelyn K C Rose

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  1. Tal Isaacson
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  2. Cynthia M B Damasceno
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  3. Ramu S Saravanan
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  4. Yonghua He
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  5. Carmen Catalá
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  6. Montserrat Saladié
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  7. Jocelyn K C Rose
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Correspondence to Jocelyn K C Rose.

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Isaacson, T., Damasceno, C., Saravanan, R. et al. Sample extraction techniques for enhanced proteomic analysis of plant tissues. Nat Protoc 1, 769–774 (2006). https://doi.org/10.1038/nprot.2006.102

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