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Difficult Proteins

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Book cover Two-Dimensional Electrophoresis Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 519))

Summary

The degree of protein diversity and dynamic range within organisms means that even the simplest proteome cannot be captured by any single extraction and separation step. New techniques have focused on major protein classes often under-represented in proteome analysis; low abundance, membrane, and alkaline proteins. The last decade has seen considerable technology development in fractionation tools aimed at complexity reduction in many forms. The key outcome of complexity reduction is that each fraction, or sub-proteome, can be studied in more detail, and proteins which would have remained undetected in a total extract are present in sufficient quantities. However, the tools available are fractionations, not amplifications, and like all mining for rare and difficult items, a large amount of starting material is often required. The key shortcomings of many proteome analysis techniques are now well documented. With this knowledge, the best modern proteomics ‘platform’ involves combining multiple protein extractions, gel and chromatographic separations, and multiple MS analysis methods.

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References

  1. Gygi, S.P., Corthals, G.L., Zhang, Y., Rochon, Y.,and Aebersold, R. (2000). Evaluation of two-dimensional electrophoresis-based proteome analysis. Proc. Natl. Acad. Sci. U.S.A. 97, 9390–9395

    Article  PubMed  CAS  Google Scholar 

  2. Washburn, M.P., Wolters, D., and Yates, J.R.(2001) Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat. Biotechnol. 19, 242–247

    Article  PubMed  CAS  Google Scholar 

  3. Patterson, S.D. (2000) Proteomics: the industrialization of protein chemistry. Curr. Opin. Biotechnol. 11, 413–418

    Article  PubMed  CAS  Google Scholar 

  4. 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

    Article  PubMed  CAS  Google Scholar 

  5. Futcher, B., Latter, G.I., Monardo, P., McLaughlin, C.S., and Garrells, J.I. (1999) A sampling of the yeast proteome. Mol. Cell. Biol. 19, 7357–7368

    PubMed  CAS  Google Scholar 

  6. Ghaemmaghami, S., Huh, W.K., Bower, K., Howson, R.W., Belle, A., Dephoure, N. et al. (2003). Global analysis of protein expression in yeast. Nature 425, 737–741

    Article  PubMed  CAS  Google Scholar 

  7. Kurland, C.G. (1991) Codon bias and gene expression. FEBS Lett. 285, 165–169

    Article  Google Scholar 

  8. Herbert, B., and Righetti, P.G. (2000) A turning point in proteome analysis: sample prefractionation via multicompartment electrolysers with isoelectric membranes. Electrophoresis 21, 3639–3648

    Article  PubMed  CAS  Google Scholar 

  9. Fey, S.J., and Larsen, P.M. (2001) 2-D or not 2-D. Two-dimensional gel electrophoresis. Curr. Opin. Chem. Biol. 5, 26–33

    Article  PubMed  CAS  Google Scholar 

  10. Righetti, P.G., Castagna, A., and Herbert, B. (2001) Prefractionation techniques in proteome analysis. Anal. Chem. 73, 320A–326A

    Article  PubMed  CAS  Google Scholar 

  11. Zuo, X., and Speicher, D.W. (2000) A method for global analysis of complex proteomes using sample prefractionation by solution isoelectrofocusing prior to two-dimensional electrophoresis. Anal. Biochem. 284, 266–278

    Article  PubMed  CAS  Google Scholar 

  12. Molloy, M.P., Herbert, B.R., Walsh, B.J., Tyler, M.I., Traini, M., Sanchez, J.C. et al. (1998) Extraction of membrane proteins by differential solubilization for separation using two-dimensional gel electrophoresis. Electrophoresis 19, 837–844

    Article  PubMed  CAS  Google Scholar 

  13. Corthals, G.L., Wasinger, V.C., Hochstrasser, D.F., and Sanchez, J.C. (2000) The dynamic range of protein expression: a challenge for proteomic research. Electrophoresis 21, 1104–1115

    Article  PubMed  CAS  Google Scholar 

  14. Fountoulakis, M., Takács, M.F., Berndt, P., Langen, H., and Takács, B. (1999) Enrichment of low abundance proteins of Escherichia coli by hydroxyapatite chromatography. Electrophoresis 20, 2181–2195

    Article  PubMed  CAS  Google Scholar 

  15. Fountoulakis, M., Takács, M.F., and Takács, B.(1999) Enrichment of low-copy-number gene products by hydrophobic interaction chromatography. J. Chromatogr. A 833, 157–168

    Article  PubMed  CAS  Google Scholar 

  16. Richmond, C.S., Glasner, J.D., Mau, R., Jin, H., and Blattner, F.R. (1999) Genome-wide expression profiling in Escherichia coli K-12. Nucleic Acids Res. 27, 3821–3385

    Article  PubMed  CAS  Google Scholar 

  17. Velculescu, V.E., Zhang, L., Zhou, W., Vogelstein, J., Basrai, M.A., Bassett, D.E. Jr. et al. (1997) Characterization of the yeast transcriptome. Cell, 88, 243–251

    Article  PubMed  CAS  Google Scholar 

  18. Heller, M., Ye, M., Michel, P.E., Morier, P., Stalder, D., Jünger, M.A. et al. (2005) Added value for tandem mass spectrometry shotgun proteomics data validation through isoelectric focusing of peptides. J. Proteome Res. 4, 2273–2282

    Article  PubMed  CAS  Google Scholar 

  19. Hannig, K. (1978) Continuous free-flow electrophoresis as an analytical and preparative method in biology. J Chromatogr. 159, 183–191

    Article  PubMed  CAS  Google Scholar 

  20. Hoffmann, P., Ji, H., Moritz, R.L., Connolly, L.M., Frecklington, D.F., Layton, M.J. et al. (2001) Continuous free-flow electrophoresis separation of cytosolic proteins from the human colon carcinoma cell line LIM 1215: a non two-dimensional gel electrophoresis-based proteome analysis strategy. Proteomics 1, 807–818

    Article  PubMed  CAS  Google Scholar 

  21. Xiao, Z., Conrads, T.P., Lucas, D.A., Janini, G.M., Schaefer, C.F., and Buetow, K.H. (2004) Direct ampholyte-free liquid-phase isoelectric peptide focusing: application to the human serum proteome. Electrophoresis 25, 128–133

    Article  PubMed  CAS  Google Scholar 

  22. Righetti, P.G., Wenisch, E., and Faupel, M. (1989) Preparative protein purification in a multi-compartment electrolyzer with immobiline membranes. J. Chromatogr. 475, 293–309

    Article  CAS  Google Scholar 

  23. Righetti, P.G., Wenisch, E., Jungbauer, A., Katinger, H., and Faupel, M. (1990) Preparative purification of human monoclonal antibody isoforms in a multicompartment electrolyzer with Immobiline membranes. J. Chromatogr. 500, 681–696

    Article  PubMed  CAS  Google Scholar 

  24. Pedersen, S.K., Harry, J.L., Sebastian, L., Baker, J., Traini, M.D., McCarthy, J.T. et al. (2003) Unseen proteome: mining below the tip of the iceberg to find low abundance and membrane proteins. J. Proteome Research 2, 303–312

    Article  CAS  Google Scholar 

  25. Fortis, F., Girot, P., Brieau, O., Boschetti, E., Castagna, A., and Righetti, P.G. Amphoteric buffering chromatographic beads for proteome prefractionation. I: Theoretical model. Proteomics 5, 620-628.

    Google Scholar 

  26. Molloy, M.P., Herbert, B.R., Slade, M.B., Rabilloud, T., Nouwens, A.S., Williams, K.L., and Gooley, A.A. (2000) Proteomic analysis of the Escherichia coli outer membrane. Eur. J. Biochem. 267, 2871–2881

    Article  PubMed  CAS  Google Scholar 

  27. Michel, P.E., Reymond, P., Arnaud, I.L., Josserand, J., Girault, H.H., and Rossier, J.S.(2003) Protein fractionation in a multicompartment device using Off-Gel isoelectric focusing. Electrophoresis 24, 3–11

    Article  PubMed  CAS  Google Scholar 

  28. Hörth, P., Miller, C.A., Preckel, T., and Wenz, C. (2006) Efficient fractionation and improved protein identification by peptide OFFGEL electrophoresis. Mol. Cell. Proteomics 10, 1968–1974

    Google Scholar 

  29. Liu, T., Qian, W.J., Gritsenko, M.A., Xiao, W.,Moldawer, L.L., Kaushal, A. et al. (2006) High dynamic range characterization of the trauma patient plasma proteome. Mol. Cell. Proteomics 10, 1899–1913

    Google Scholar 

  30. Shen, Y., Kim, J., Strittmatter, E.F., Jacobs, J.M., Camp II, D.G., Fang, R. et al. (2005) Characterization of the human blood plasma proteome. Proteomics 5, 4034–4045

    Article  PubMed  CAS  Google Scholar 

  31. Colantonio, D.A., Dunkinson, C., Bovenkamp, D.E., and Van Eyk, J.E. (2005) Effective removal of albumin from serum. Proteomics 5, 3831–3835

    Article  PubMed  CAS  Google Scholar 

  32. Thulasiraman, V., Lin, S., Gheorghiu, L., Lathrop, J., Lomas, L., Hammond, D., and Boschetti, E. (2005) Reduction of the concentration difference of proteins in biological liquids using a library of combinatorial ligands. Electrophoresis 26, 3561–3571

    Article  PubMed  CAS  Google Scholar 

  33. Righetti, P.G., Castagna, A., Antonucci, F., Piubelli, C., Cecconi, D., Campostrini, N. et al. (2005) Proteome analysis in the clinical chemistry laboratory: myth or reality? Clin. Chim. Acta 357, 123–139

    Article  PubMed  CAS  Google Scholar 

  34. Righetti, P.G., Boschetti, E., Lomas, L., and Citterio, A. (2006) Protein Equalizer Technology: the quest for a “democratic proteome”. Proteomics 6, 3980–3992

    Article  PubMed  CAS  Google Scholar 

  35. Anderson, L.N., Polanski, M., Pieper, R., Gatlin, T., Tirumalai, R.S., Conrads, T.P. et al. (2004) The human plasma proteome: a nonredundant list developed by combination of four separate sources. Mol. Cell. Proteomics 3, 311–326

    Article  PubMed  CAS  Google Scholar 

  36. Sennels, L., Salek, M., Lomas, L., Boschetti, E., Righetti, P.G., and Rappsilber, J. (2007) Proteomic analysis of human blood serum using peptide library beads. J Proteome Res. 6, 4055–4062

    Google Scholar 

  37. Santoni, V., Molloy, M., and Rabilloud, T. (2000) Membrane proteins and proteomics: un amour impossible? Electrophoresis 21, 1054–1070

    Article  PubMed  CAS  Google Scholar 

  38. Eymann, C., Dreisbach, A., Albrecht, D., Bernhardt, J., Becher, D., Gentner, S. et al. (2004) A comprehensive proteome map of growing Bacillus subtilis cells. Proteomics 4, 2849–2876

    Article  PubMed  CAS  Google Scholar 

  39. Wolff, S., Otto, A., Albrecht, D., Zeng, J.S., Buttner, K., Gluckmann, M. et al. (2006) Gel-free and gel-based proteomics in Bacillus subtilis: a comparative study. Mol. Cell. Proteomics 7, 1183–1192

    Google Scholar 

  40. Herbert, B., Galvani, M., Hamdan, M., Oliveri, E., McCarthy, J., Pedersen, S. et al. (2001) Reduction and alkylation of proteins in preparation of two-dimensional map analysis: why, when and how? Electrophoresis 22, 2046–2057

    Article  PubMed  CAS  Google Scholar 

  41. Galvani, M., Hamdan, M., Herbert, B., and Righetti, P.G. (2001). Reduction and alkylation of proteins in preparation of two-dimensional map analysis: a kinetic study via MALDI-TOF MS. Electrophoresis 22, 2058–2065

    Article  PubMed  CAS  Google Scholar 

  42. Galvani, M., Rovatti, M., Hamdan, M., Herbert, B., and Righetti, P.G. (2001). Protein alkylation in presence/absence of thiourea in proteome analysis: A MALDI-TOF mass spectrometry investigation. Electrophoresis 22, 2066–2074

    Article  PubMed  CAS  Google Scholar 

  43. Luche, S., Diemer, H., Tastet, C., Chevallet, M., Van Dorsselaer, A., Leize-Wagner, E. et al. (2004). About thiol derivatization and resolution of basic proteins in two-dimensional electrophoresis. Proteomics 4, 551–561

    Article  PubMed  CAS  Google Scholar 

  44. Lander, E.S., Linton, L.M., Birren, B., Nusbaum, C., Zody, M.C., Baldwin, J. et al. (2001). Initial sequencing and analysis of the human genome. Nature 409, 860–921

    Article  PubMed  CAS  Google Scholar 

  45. Venter, J.C., Adams, M.D., Myers, E.W., Li, P.W., Mural, R.J., Sutton, G.G. et al. (2001). The sequence of the human genome. Science 291, 1304–1351

    Article  PubMed  CAS  Google Scholar 

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  1. Department of Medical and Molecular Biosciences, University of Technology, Sydney, 15 Broadway, Ultimo, Sydney, NSW 2007, Australia

    Ben Herbert & Elizabeth Harry

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  1. Ben Herbert
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  2. Elizabeth Harry
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Correspondence to Elizabeth Harry .

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    © 2009 Humana Press, a part of Springer Science+Business Media, LLC

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    Herbert, B., Harry, E. (2009). Difficult Proteins. In: Tyther, R., Sheehan, D. (eds) Two-Dimensional Electrophoresis Protocols. Methods in Molecular Biology, vol 519. Humana Press. https://doi.org/10.1007/978-1-59745-281-6_4

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    • DOI: https://doi.org/10.1007/978-1-59745-281-6_4

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    • Publisher Name: Humana Press

    • Print ISBN: 978-1-58829-937-6

    • Online ISBN: 978-1-59745-281-6

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