Abstract
Improved understanding of dental enamel development will benefit not only dentistry but also biomedicine more generally. Rat and mouse models of enamel development are relatively well characterized and experimentally powerful. However, the diminutive size of murine teeth makes them difficult to study using standard proteomic approaches. Here we describe gel-based proteomic methods that enable parallel quantification, identification, and functional characterization of proteins from developing rat and mouse teeth. These refined methods are also likely to be applicable to other scarce samples.
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References
Hubbard, M. J. (1996) Abundant calcium homeostasis machinery in rat dental enamel cells. Up-regulation of calcium store proteins during enamel mineralization implicates the endoplasmic reticulum in calcium transcytosis. Eur. J. Biochem. 239, 611–623.
Hubbard, M. J. (2000) Calcium transport across the dental enamel epithelium. Crit. Rev. Oral Biol. Med. 11, 437–466.
Franklin, I. K., Winz, R. A., and Hubbard, M. J. (2001) Endoplasmic reticulum Ca2+-ATPase pump is up-regulated in calcium-transporting dental enamel cells: a non-housekeeping role for SERCA2b. Biochem. J. 358, 217–224.
Turnbull, C. I., Looi, K., Mangum, J. E., Meyer, M., Sayer, R. J., and Hubbard, M. J. (2004) Calbindin independence of calcium transport in developing teeth contradicts the calcium ferry dogma. J. Biol. Chem. 279, 55850–55854.
Hubbard, M. J., and McHugh, N. J. (1995) Calbindin28kDa and calbindin30kDa (calretinin) are substantially localised in the particulate fraction of rat brain. FEBS Lett. 374, 333–337.
Hubbard, M. J. (1995) Calbindin28kDa and calmodulin are hyperabundant in rat dental enamel cells. Identification of the protein phosphatase calcineurin as a principal calmodulin target and of a secretion-related role for calbindin28kDa. Eur. J. Biochem. 230, 68–79.
Hubbard, M. J., and McHugh, N. J. (1996) Mitochondrial ATP synthase F1-beta-subunit is a calcium-binding protein. FEBS Lett. 391, 323–329.
Hubbard, M. J. (1998) Enamel cell biology. Towards a comprehensive biochemical understanding. Connect. Tissue Res. 38, 17–32.
Hubbard, M. J. (1998) Proteomic analysis of enamel cells from developing rat teeth: big returns from a small tissue. Electrophoresis 19, 1891–1900.
Sayer, R. J., Turnbull, C. I., and Hubbard, M. J. (2000) Calbindin28kDa is specifically associated with extranuclear constituents of the dense particulate fraction. Cell Tissue Res. 302, 171–180.
Demmer, J., Zhou, C., and Hubbard, M. J. (1997) Molecular cloning of ERp29, a novel and widely expressed resident of the endoplasmic reticulum. FEBS Lett. 402, 145–150.
Hubbard, M. J., and McHugh, N. J. (2000) Human ERp29: isolation, primary structural characterisation and two-dimensional gel mapping. Electrophoresis 21, 3785–3796.
Hubbard, M. J., McHugh, N. J., and Carne, D. L. (2000) Isolation of ERp29, a novel endoplasmic reticulum protein, from rat enamel cells: Evidence for a unique role in secretory-protein synthesis. Eur. J. Biochem. 267, 1945–1957.
Hubbard, M. J., Mangum, J. E., and McHugh, N. J. (2004) Purification and biochemical characterisation of native ERp29 from rat liver. Biochem. J. 383, 589–598.
Hermann, V. M., Cutfield, J. F., and Hubbard, M. J. (2005) Biophysical characterization of ERp29. Evidence for a key structural role of cysteine 125. J. Biol. Chem. 280, 13529–13537.
Hubbard, M. J., Faught, M. J., Carlisle, B. H., and Stockwell, P. A. (2001) ToothPrint, a proteomic database for dental tissues. Proteomics 1, 132–135.
Hubbard, M. J., and Kon, J. C. (2002) Proteomic analysis of dental tissues. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 771, 211–220.
Mangum, J. E., Farlie, P. G., and Hubbard, M. J. (2005) Proteomic profiling of facial development in chick embryos. Proteomics 5, 2542–2550.
Mangum, J. E., Veith, P. D., Reynolds, E. C., and Hubbard, M. J. (2006) Towards second-generation proteome analysis of murine enamel-forming cells. Eur. J. Oral Sci. 114(Suppl. 1), 259–265.
Kardos, T. B., and Hubbard, M. J. (1981) Rapid dissection of rodent molar-tooth germs. Lab. Anim. 15, 371–373.
Lanne, B., and Panfilov, O. (2005) Protein staining influences the quality of mass spectra obtained by peptide mass fingerprinting after separation on 2-d gels. A comparison of staining with coomassie brilliant blue and sypro ruby. J. Proteome Res. 4, 175–179.
Shnyder, S. D., Mangum, J. E., and Hubbard, M. J. (2008) Triplex profiling of functionally distinct chaperones (ERp29/PDI/BiP) reveals marked heterogeneity of the endoplasmic reticulum proteome in cancer. J. Proteome Res. 7, 3364–3372.
Hubbard, M. J., and Klee, C. B. (1987) Calmodulin binding by calcineurin. Ligand-induced renaturation of protein immobilized on nitrocellulose. J. Biol. Chem. 262, 15062–15070.
James, G. T. (1978) Inactivation of the protease inhibitor phenylmethylsulfonyl fluoride in buffers. Anal. Biochem. 86, 574–579.
McCarthy, J., Hopwood, F., Oxley, D., Laver, M., Castagna, A., Righetti, P. G., Williams, K., and Herbert, B. (2003) Carbamylation of proteins in 2-D electrophoresis – myth or reality? J. Proteome Res. 2, 239–242.
Biedermann, K., Jepsen, P. K., Riise, E., and Svendsen, I. (1989) Purification and characterization of a Serratia marcescens nuclease produced by Escherichia coli. Carlsberg Res. Commun. 54, 17–27.
Hochstrasser, D. F., Harrington, M. G., Hochstrasser, A. C., Miller, M. J., and Merril, C. R. (1988) Methods for increasing the resolution of two-dimensional protein electrophoresis. Anal. Biochem. 173, 424–435.
Acknowledgments
We thank Nicola McHugh for skillfully assisting with development of the 2DGE procedures described here. This work was supported by the Melbourne Research Unit for Facial Disorders, the National Health and Medical Research Council of Australia, and the Health Research Council of New Zealand.
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Mangum, J.E., Kon, J.C., Hubbard, M.J. (2010). Proteomic Analysis of Dental Tissue Microsamples. In: Seymour, G., Cullinan, M., Heng, N. (eds) Oral Biology. Methods in Molecular Biology, vol 666. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-820-1_19
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DOI: https://doi.org/10.1007/978-1-60761-820-1_19
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