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Differential ProteoTope Radioactive Quantification of Protein Abundance Ratios

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The Protein Protocols Handbook

Part of the book series: Springer Protocols Handbooks ((SPH))

Abstract

2D-PAGE remains a widely employed proteomics method because it provides the highest separating resolution of intact polypeptide isoforms that are amenable to subsequent characterization (1, 2). ProteoTope is a differential radioactive detection method that we developed based upon temporally separated exposures of iodinated proteins on 2D-PAGE gels (3, 4). We have also developed systems for extremely high resolution 2D-PAGE over 54 cm or longer, which were designed for optimal quantification of radioactively labeled proteins (5, 6). These methods provide synergistic improvement in data quality for obtaining quality results from limited samples, such as typical valuable clinical tissue samples (7–9).

Here we provide a comprehensive technical description of differential 2D-radioactive ProteoTope quantification using highly standardized samples from the ABRF PRG2006 study (10). Our method is a novel application of the widespread Imaging Plate radioactive detection method (11, 12). The twofold study aims for multiple ABRF study participants were to blindly identify the eight unknown standard proteins in the two sample mixtures, and to determine a best estimate of their relative amounts in each sample by multiple measurements of mixtures of the two samples at a ratio of 1:1 (10). 2D-PAGE and Proteo-Tope differential radioactive imaging provided excellent relative quantification data of even subtle differences between the two samples. However our inverse symmetrical replicate experimental design and precise relative quantifications revealed that the quantities of proteins in both samples were apparently altered during sample resolubilization in our laboratory. Thus this case study demonstrates both the excellent quantification of limited protein samples achievable by differential radioactive imaging, and the requirement for stringently optimized sample-specific handling protocols which were not developed for this one-off time-restricted experiment.

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References

  1. Van den, B. G., Arckens, L. (2005) Recent advances in 2D electrophoresis: an array of possibilities. Expert. Rev. Proteomics 2, 243–252.

    Article  Google Scholar 

  2. Zolg, J. W., Langen, H. (2004) How industry is approaching the search for new diagnostic markers and biomarkers. Mol. Cell Proteomics 3, 345–354.

    Article  CAS  PubMed  Google Scholar 

  3. Cahill, M. A., Wozny, W., Schwall, G., Schroer, K., Holzer, K., Poznanovic, S., Hunzinger, C., Vogt, J. A., Stegmann, W., Matthies, H., Schrattenholz, A. (2003) Analysis of relative isotopologue abundances for quantitative profiling of complex protein mixtures labelled with the acrylamide/D3-acrylamide alkylation tag system. Rapid Commun. Mass Spectrom. 17, 1283–1290.

    Article  CAS  PubMed  Google Scholar 

  4. Schrattenholz, A., Wozny, W., Klemm, M., Schroer, K., Stegmann, W., Cahill, M. A. (2005) Differential and quantitative molecular analysis of ischemia complexity reduction by isotopic labeling of proteins using a neural embryonic stem cell model. J. Neurol. Sci. 229–230, 261–267.

    Google Scholar 

  5. Poland, J., Cahill, M. A., Sinha, P. (2003) Isoelectric focusing in long immobilized pH gradient gels to improve protein separation in proteomic analysis. Electrophore-sis 24, 1271–1275.

    Article  CAS  Google Scholar 

  6. Poznanovic, S., Schwall, G., Zengerling, H., Cahill, M. A. (2005) Isoelectric focusing in serial immobilized pH gradient gels to improve protein separation in proteomic analysis. Electrophoresis 26, 3185–3190.

    Article  CAS  PubMed  Google Scholar 

  7. Neubauer, H., Clare, S. E., Kurek, R., Fehm, T., Wallwiener, D., Sotlar, K., Nordheim, A., Wozny, W., Schwall, G. P., Poznanovic, S., Sastri, C., Hunzinger, C., Stegmann, W., Schrattenholz, A., Cahill, M. A. (2006) Breast cancer proteomics by laser capture microdissection, sample pooling, 54-cm IPG IEF, and differential iodine radioiso-tope detection. Electrophoresis 27, 1840–1852.

    Article  CAS  PubMed  Google Scholar 

  8. Poznanovic, S., Wozny, W., Schwall, G. P., Sastri, C., Hunzinger, C., Stegmann, W., Schrattenholz, A., Buchner, A., Gangnus, R., Burgemeister, R., Cahill, M. A. (2005) Differential radioactive proteomic analysis of microdissected renal cell carcinoma tissue by 54 cm isoelectric focusing in serial immobilized pH gradient gels. J. Proteome. Res. 4, 2117–2125.

    Article  CAS  PubMed  Google Scholar 

  9. Wozny, W., Schroer, K., Schwall, G. P., Poznanovic, S., Stegmann, W., Dietz, K., Rogatsch, H., Schaefer, G., Huebl, H., Klocker, H., Schrattenholz, A., Cahill, M. A. (2007) Differential radioactive quantification of protein abundance ratios between benign and malignant prostate tissues: cancer association of annexin A3. Proteomics 7, 313–322.

    Article  CAS  PubMed  Google Scholar 

  10. Turck, C. W., Falick, A. M., Kowalak, J. A., Lane, W. S., Lilley, K. S., Phinney, B. S., Weintraub, S. T., Witkowska, H. E., Yates, N. A. (2007) ABRF-PRG2006 study: Relative protein qQuantitation. Mol. Cell Proteomics 6, 1291–1298.

    Article  CAS  PubMed  Google Scholar 

  11. Amemiya, Y., Miyahara, J. (1988) Imaging plate illuminates many fields. Nature 336, 89–90.

    Article  CAS  PubMed  Google Scholar 

  12. Miyahara, J. (1999) The imaging plate: a new radiation image sensor. Chem. Today (Japan) 223, 29–36.

    Google Scholar 

  13. Gorg, A., Postel, W., Gunther, S. (1988) The current state of two-dimensional elec-trophoresis with immobilized pH gradients. Electrophoresis 9, 531–546.

    Article  CAS  PubMed  Google Scholar 

  14. Gorg, A. (1993) Two-dimensional electrophoresis with immobilized pH gradients: current state. Biochem. Soc. Trans. 21, 130–132.

    CAS  PubMed  Google Scholar 

  15. Gorg, A., Obermaier, C., Boguth, G., Harder, A., Scheibe, B., Wildgruber, R., Weiss, W. (2000) The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 21, 1037–1053.

    Article  CAS  PubMed  Google Scholar 

  16. Gorg, A., Weiss, W., Dunn, M. J. (2004) Current two-dimensional electrophoresis technology for proteomics. Proteomics 4, 3665–3685.

    Article  PubMed  Google Scholar 

  17. Gorg, A., Weiss, W. (1999) Analytical IPG-Dalt. Methods Mol. Biol. 112, 189–195.

    CAS  PubMed  Google Scholar 

  18. Rabilloud, T. (1996) Solubilization of proteins for electrophoretic analyses. Elec-trophoresis 17, 813–829.

    Article  CAS  Google Scholar 

  19. Vuong, G. L., Weiss, S. M., Kammer, W., Priemer, M., Vingron, M., Nordheim, A., Cahill, M. A. (2000) Improved sensitivity proteomics by postharvest alkylation and radioactive labelling of proteins. Electrophoresis 21, 2594–2605.

    Article  CAS  PubMed  Google Scholar 

  20. Bonardi, M. L., Birattari, C., Groppi, F., Gini, L., Mainardi, H. S. C. (2004) Cyclotron production and quality control of “High Specific Activity” radionuclides in “No Carrier Added” form for radioanalytical applications in life sciences. J. Radio-analyt. Nucl. Chem. 259, 415–419.

    Article  CAS  Google Scholar 

  21. Johnston, R. F., Pickett, S. C., Barker, D. L. (1990) Autoradiography using storage phosphor technology. Electrophoresis 11, 355–360.

    Article  CAS  PubMed  Google Scholar 

  22. Monribot-Espagne, C., Boucherie, H. (2002) Differential gel exposure, a new methodology for the two-dimensional comparison of protein samples. Proteomics 2, 229–240.

    Article  CAS  PubMed  Google Scholar 

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Author information

Authors and Affiliations

  1. ProteoSys AG, Mainz, Germany

    Wojciech Wozny, Gerhard P. Schwall, Chaturvedula S. Sastri, Slobodan Poznanović, Werner Stegmann, Christian Hunzinger & Karlfried Groebe

  2. School of Biomedical Sciences, Charles Sturt University, Wagga, NSW, Australia

    Michael A. Cahill

Authors
  1. Wojciech Wozny
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  2. Gerhard P. Schwall
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  3. Chaturvedula S. Sastri
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  4. Slobodan Poznanović
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  5. Werner Stegmann
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  6. Christian Hunzinger
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  7. Karlfried Groebe
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  8. Michael A. Cahill
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Editor information

Editors and Affiliations

  1. School of Life Sciences, University of Hertfordshire, Hatfield, Hertfordshire, UK

    John M. Walker

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© 2009 Springer Science+Business Media, LLC

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Wozny, W. et al. (2009). Differential ProteoTope Radioactive Quantification of Protein Abundance Ratios. In: Walker, J.M. (eds) The Protein Protocols Handbook. Springer Protocols Handbooks. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-198-7_42

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  • DOI: https://doi.org/10.1007/978-1-59745-198-7_42

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-60327-474-6

  • Online ISBN: 978-1-59745-198-7

  • eBook Packages: Springer Protocols

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