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Proteomic Analysis of the Stacked Golgi Complex

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Organelle Proteomics

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 432))

Summary

Although a great deal is known about the structure and function of most mammalian organelles, comprehensive proteomes are necessary to provide a molecular framework to integrate this information. The Golgi complex is the central organelle of the secretory pathway and functions to posttranslationally modify newly synthesized proteins and lipids and to sort them to their sites of function. The methods described in this chapter facilitate the isolation of an enriched stacked Golgi fraction (GF) from rat liver (1,2) and the shotgun proteomic analysis of the fraction using multidimensional protein identification technology (MudPIT) (3,4).

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References

  1. Taylor, R. S., Jones, S. M., Dahl, R. H., Nordeen, M. H., Howell, K. E. (1997) Characterization of the Golgi complex cleared of proteins in transit and examination of calcium uptake activities. Mol. Biol. Cell 8, 1911–1931.

    CAS  PubMed  Google Scholar 

  2. Leelavathi, D. E., Estes, L. W., Feingold, D. S., Lombardi, B. (1970) Isolation of a Golgi-rich fraction from rat liver. Biochim. Biophys. Acta 211, 124–138.

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  4. Wolters, D. A., Washburn, M. P., Yates, J. R. III. (2001) An automated multidimensional protein identification technology for shotgun proteomics. Anal. Chem. 73, 5683–5690.

    Article  CAS  PubMed  Google Scholar 

  5. Marsh, B. J., Mastronarde, D. N., McIntosh, J. R., Howell, K. E. (2001) Structural evidence for multiple transport mechanisms through the Golgi in the pancreatic beta-cell line, HIT-T15. Biochem. Soc. Trans. 29, 461–467.

    Article  CAS  PubMed  Google Scholar 

  6. Ladinsky, M. S., Mastronarde, D. N., McIntosh, J. R., Howell, K. E., Staehelin, L. A. (1999) Golgi structure in three dimensions: functional insights from the normal rat kidney cell.J. Cell Biol. 144, 1135–1149.

    Article  CAS  PubMed  Google Scholar 

  7. Taylor, R. S., Fialka, I., Jones, S. M., Huber, L. A., Howell, K. E. (1997) Two-dimensional mapping of the endogenous proteins of the rat hepatocyte Golgi complex cleared of proteins in transit.Electrophoresis 18, 2601–2612.

    Article  CAS  PubMed  Google Scholar 

  8. Wu, C. C., MacCoss, M. J., Mardones, G., Finnigan, C., Mgelsvang, S., Yates, J. R. III, et al. (2004) Organellar proteomics reveals Golgi arginine dimethylation. Mol. Biol. Cell 15, 2907–2919.

    Article  CAS  PubMed  Google Scholar 

  9. Ehrenreich, J. H., Bergeron, J. J. M., Siekevitz, P., Palade, G. E. (1973) Golgi fractions prepared from rat liver homogenates. I. Isolation procedure and morphological characterization. J. Cell Biol. 59, 45–72.

    Article  CAS  PubMed  Google Scholar 

  10. Howell, K. E., Palade, G. E. (1982) Hepatic Golgi fractions resolved into membrane and content subfractions. J. Cell Biol. 92, 822–832.

    Article  CAS  PubMed  Google Scholar 

  11. Wu, C. C., MacCoss, M. J., Howell, K. E., and Yates, J. R. III (2003) A method for the comprehensive proteomic analysis of membrane proteins. Nat. Biotechnol. 21, 532–538.

    Article  CAS  PubMed  Google Scholar 

  12. Blackler, A. R., Klammer, A. A., MacCoss, M. J., Wu, C. C. (2006) Quantitative comparison of proteomic data quality between a 2D and 3D quadrupole ion trap. Anal. Chem. 78, 1337–1344.

    Article  CAS  PubMed  Google Scholar 

  13. Eng, J. K., McCormack, A. L., Yates III, J. R. (1994) An approach to correlate tandem mass spectra of modified peptides to amino acid sequences in a protein database. J. Am. Soc. Mass Spectrom. 5, 976–989.

    Article  CAS  Google Scholar 

  14. MacCoss, M. J., Wu, C. C., Yates, J. R. III (2002) Probability-based validation of protein identifications using a modified SEQUEST algorithm. Anal. Chem. 74, 5593–5599.

    Article  CAS  PubMed  Google Scholar 

  15. Tabb, D. L., McDonald, W. H., Yates, J. R. III (2002) DTASelect and Contrast: tools for assembling and comparing protein identifications from shotgun proteomics. J. Proteome Res. 1, 21–26.

    Article  CAS  PubMed  Google Scholar 

  16. Speers, A. E., Blackler, A. R., Wu, C. C. (2007). Elevated temperature facilitates shotgun proteomic analysis of membrane proteins. Anal. Chem. 79(12), 4613–4620.

    Article  CAS  PubMed  Google Scholar 

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© 2008 Humana Press, a part of Springer Science+Business Media, LLC

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Lowenthal, M.S., Howell, K.E., Wu, C.C. (2008). Proteomic Analysis of the Stacked Golgi Complex. In: Pflieger, D., Rossier, J. (eds) Organelle Proteomics. Methods in Molecular Biology™, vol 432. Humana Press. https://doi.org/10.1007/978-1-59745-028-7_3

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  • DOI: https://doi.org/10.1007/978-1-59745-028-7_3

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-779-2

  • Online ISBN: 978-1-59745-028-7

  • eBook Packages: Springer Protocols

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