Two-Dimensional Nanoflow Liquid Chromatography-Tandem Mass Spectrometry of Proteins Extracted from Rice Leaves and Roots

  • Linda Breci
  • Paul A. Haynes
Part of the Methods in Molecular Biology book series (MIMB, volume 355)


In this chapter we present a detailed protocol for the large-scale identification of proteins present in rice leaf and root tissue samples using 2D liquid chromatography-tandem mass spectrometry of protein extracts. This is performed using biphasic (strong cation exchange/reversed phase) columns with integral electrospray emitters operating at nanoliter flow rates, a technique known by the acronym Mudpit (for multidimensional protein identification technique). The protocol involves harvesting of leaves and roots from rice plants, preparing protein extracts from the harvested tissues, preparing proteolytic digests of the extracted proteins, making a biphasic capillary column with an integral electrospray emitter, performing two-dimensional chromatographic separation of peptides with data-dependent tandem mass spectrometry, and the use of database searching of the acquired tandem mass spectra to identify peptides and proteins. This protocol is adaptable for use with a wide variety of plant materials and can be used to identify large numbers of proteins present in a specific tissue, organ, organelle, or other subcellular fraction. In addition to the detailed protocol, we also present the results of a representative experiment showing the identification of more than 1000 distinct proteins from rice leaf and root samples in two Mupdit experiments.

Key Words

Nanoflow liquid chromatography-tandem mass spectrometry two-dimensional nanoflow chromatography multidimensional protein identification technique (Mudpit) plant protein extraction rice leaf rice root 


  1. 1.
    Link, A. J., Eng, J., Schieltz, D. M., et al. (1999) Direct analysis of protein complexes using mass spectrometry. Nat. Biotechnol. 17, 676–682.CrossRefPubMedGoogle Scholar
  2. 2.
    Washburn, M. P., Wolters, D., and Yates, J. R. III (2001) Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat. Biotechnol. 19, 242–247.CrossRefPubMedGoogle Scholar
  3. 3.
    Haynes, P. A. and Yates, J. R. III (2000) Proteome profiling-pitfalls and progress. Yeast 17, 81–87.CrossRefPubMedGoogle Scholar
  4. 4.
    Opiteck, G. J. and Jorgenson, J. W. (1997) Two-dimensional SEC/RPLC coupled to mass spectrometry for the analysis of peptides. Anal. Chem. 69, 2283–2291.CrossRefPubMedGoogle Scholar
  5. 5.
    Opiteck, G. J., Lewis, K. C., Jorgenson, J. W., and Anderegg, R. J. (1997) Comprehensive on-line LC/LC/MS of proteins. Anal. Chem. 69, 1518–1524.CrossRefPubMedGoogle Scholar
  6. 6.
    Washburn, M. P., Ulaszek, R., Deciu, C., Schieltz, D. M., and Yates, J. R. III (2002) Analysis of quantitative proteomic data generated via multidimensional protein identification technology. Anal. Chem. 74, 1650–1657.CrossRefPubMedGoogle Scholar
  7. 7.
    Wu, C. C., MacCoss, M. J., Howell, K. E., Matthews, D. E., and Yates, J. R. 3rd (2004) Metabolic labeling of mammalian organisms with stable isotopes for quantitative proteomic analysis. Anal. Chem. 76, 4951–4959.CrossRefPubMedGoogle Scholar
  8. 8.
    Froehlich, J. E., Wilkerson, C. G., Ray, W. K., et al. (2003) Proteomic study of the Arabidopsis thaliana chloroplastic envelope membrane utilizing alternatives to traditional two-dimensional electrophoresis. J. Proteome Res. 2, 413–425.CrossRefPubMedGoogle Scholar
  9. 9.
    Andon, N. L., Hollingworth, S., Koller, A., Greenland, A. J., Yates, J. R. III, and Haynes, P. A. (2002) Proteomic characterization of wheat amyloplasts using identification of proteins by tandem mass spectrometry. Proteomics 2, 1156–1168.CrossRefPubMedGoogle Scholar
  10. 10.
    Peltier, J. B., Ytterberg, A. J., Sun, Q., and van Wijk, K. J. (2004) New functions of the thylakoid membrane proteome of Arabidopsis thaliana revealed by a simple, fast, and versatile fractionation strategy. J. Biol. Chem. 279, 49367–4983.CrossRefPubMedGoogle Scholar
  11. 11.
    Heinemeyer, J., Eubel, H., Wehmhoner, D., Jansch, L., and Braun, H. P. (2004) Proteomic approach to characterize the supramolecular organization of photosystems in higher plants. Phytochemistry 65, 1683–1692.CrossRefPubMedGoogle Scholar
  12. 12.
    Koller, A., Washburn, M. P., Lange, B. M., et al. (2002) Proteomic survey of metabolic pathways in rice. Proc. Natl. Acad. Sci. USA 99, 11969–11974.CrossRefPubMedGoogle Scholar
  13. 13.
    Chen, M., Presting, G., Barbazuk, W. B., et al. (2002) An integrated physical and genetic map of the rice genome. Plant Cell 14, 537–545.CrossRefPubMedGoogle Scholar
  14. 14.
    Yu, J., Hu, S., Wang, J., et al. (2002) A draft sequence of the rice genome (Oryza sativa L. ssp. indica). Science 296, 79–92.CrossRefPubMedGoogle Scholar
  15. 15.
    Goff, S. A., Ricke, D., Lan, T. H., et al. (2002) A draft sequence of the rice genome (Oryza sativa L. ssp. japonica). Science 296, 92–100.CrossRefPubMedGoogle Scholar
  16. 16.
    Initiative, A. G. (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408, 796–815.CrossRefGoogle Scholar
  17. 17.
    Salanoubat, M., Lemcke, K., Rieger, M., et al.. (2000) Sequence and analysis of chromosome 3 of the plant Arabidopsis thaliana. Nature 408, 820–822.CrossRefPubMedGoogle Scholar
  18. 18.
    Tabata, S., Kaneko, T., Nakamura, Y., et al. (2000) Sequence and analysis of chromosome 5 of the plant Arabidopsis thaliana. Nature 408, 823–826.CrossRefPubMedGoogle Scholar
  19. 19.
    Lunde, C. F., Morrow, D. J., Roy, L. M., and Walbot, V. (2003) Progress in maize gene discovery: a project update. Funct. Integr. Genomics 3, 25–32.PubMedGoogle Scholar
  20. 20.
    Damerval, C., de Vienne, D., Zivy, M., and Thiellement, H. (1986) Technical improvements in two-dimensional electrophoresis increase the level of genetic variation detected in wheat-seedling proteins. Electrophoresis 7, 52–54.CrossRefGoogle Scholar
  21. 21.
    Porubleva, L., Vander Velden, K., Kothari, S., Oliver, D. J., and Chitnis, P. R. (2001) The proteome of maize leaves: use of gene sequences and expressed sequence tag data for identification of proteins with peptide mass fingerprints. Electrophoresis 22, 1724–1738.Google Scholar
  22. 22.
    Zuo, X., Echan, L., Hembach, P., et al. (2001) Towards global analysis of mammalian proteomes using sample prefractionation prior to narrow pH range two-dimensional gels and using one-dimensional gels for insoluble and large proteins. Electrophoresis 22, 1603–1615.CrossRefPubMedGoogle Scholar
  23. 23.
    Blonder, J., Hale, M. L., Lucas, D. A., et al. (2004) Proteomic analysis of detergent-resistant membrane rafts. Electrophoresis 25, 1307–1318.CrossRefPubMedGoogle Scholar
  24. 24.
    Guina, T., Wu, M., Miller, S. I., et al. (2003) Proteomic analysis of Pseudomonas aeruginosa grown under magnesium limitation. J. Am. Soc. Mass Spectrom. 14, 742–751.CrossRefPubMedGoogle Scholar
  25. 25.
    Gatlin, C. L., Kleemann, G. R., Hays, L. G., Link, A. J., and Yates, J. R. 3rd (1998) Protein identification at the low femtomole level from silver-stained gels using a new fritless electrospray interface for liquid chromatography-microspray and nanospray mass spectrometry. Anal. Biochem. 263, 93–101.CrossRefPubMedGoogle Scholar
  26. 26.
    Eng, J., McCormack, A. L., and Yates, J. R. III (1994) An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J. Am. Mass Spectrom. 5, 976–989.CrossRefGoogle Scholar
  27. 27.
    Yates, J. R. III, Eng, J. K., McCormack, A. L., and Schieltz, D. (1995) A method to correlate tandem mass spectra of modified peptides to amino acid sequences in the protein database. Anal. Chem. 67, 1426–1436.CrossRefPubMedGoogle Scholar
  28. 28.
    Perkins, D. N., Pappin, D. J., Creasy, D. M., and Cottrell, J. S. (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20, 3551–3567.CrossRefPubMedGoogle Scholar
  29. 29.
    Creasy, D. M. and Cottrell, J. S. (2002) Error tolerant searching of uninterpreted tandem mass spectrometry data. Proteomics 2, 1426–1434.CrossRefPubMedGoogle Scholar
  30. 30.
    Craig, R. and Beavis, R. C. (2003) A method for reducing the time required to match protein sequences with tandem mass spectra. Rapid Commun. Mass Spectrom. 17, 2310–2316.CrossRefPubMedGoogle Scholar
  31. 31.
    Craig, R. and Beavis, R. C. (2004) TANDEM: matching proteins with tandem mass spectra. Bioinformatics 20, 1466–1467.CrossRefPubMedGoogle Scholar
  32. 32.
    Nesvizhskii, A. I., Keller, A., Kolker, E., and Aebersold, R. (2003) A statistical model for identifying proteins by tandem mass spectrometry. Anal. Chem. 75, 4646–4658.CrossRefPubMedGoogle Scholar
  33. 33.
    Sadygov, R. G., Liu, H., and Yates, J. R. (2004) Statistical models for protein validation using tandem mass spectral data and protein amino acid sequence databases. Anal. Chem. 76, 1664–1671.CrossRefPubMedGoogle Scholar
  34. 34.
    Peng, J., Elias, J. E., Thoreen, C. C., Licklider, L. J., and Gygi, S. P. (2003) Evaluation of multidimensional chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for large-scale protein analysis: the yeast proteome. J. Proteome Res. 2, 43–50.CrossRefPubMedGoogle Scholar
  35. 35.
    Elias, J. E., Gibbons, F. D., King, O. D., Roth, F. P., and Gygi, S. P. (2004) Intensity-based protein identification by machine learning from a library of tandem mass spectra. Nat. Biotechnol. 22, 214–219.CrossRefPubMedGoogle Scholar
  36. 36.
    Breci, L., Hattrup, E., Keeler, M., Letarte, J., Johnson, R., and Haynes, P. A. (2005) Comprehensive proteomics in yeast using chromatographic fractionation, gas phase fractionation, protein gel electrophoresis, and isoelectric focusing. Proteomics 5, 2018–2028.CrossRefPubMedGoogle Scholar
  37. 37.
    Hall, N., Karras, M., Raine, J. D., et al. (2005) A comprehensive survey of the Plasmodium life cycle by genomic, transcriptomic, and proteomic analyses. Science 307, 82–86.CrossRefPubMedGoogle Scholar
  38. 38.
    Vitali, B., Wasinger, V., Brigidi, P., and Guilhaus, M. (2005) A proteomic view of Bifidobacterium infantis generated by multi-dimensional chromatography coupled with tandem mass spectrometry. Proteomics 5, 1859–1867.CrossRefPubMedGoogle Scholar
  39. 39.
    Durr, E., Yu, J., Krasinska, K. M., et al. (2004) Direct proteomic mapping of the lung microvascular endothelial cell surface in vivo and in cell culture. Nat. Biotechnol. 22, 985–992.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2007

Authors and Affiliations

  • Linda Breci
    • 1
  • Paul A. Haynes
    • 2
    • 3
  1. 1.Department of ChemistryThe University of ArizonaTucson
  2. 2.Bio5 Institute for Collaborative BioresearchThe University of ArizonaTucson
  3. 3.Department of Biochemistry and Molecular Biophysics1The University of ArizonaTucson

Personalised recommendations