Abstract
Mucins are difficult to handle for their identification and characterization via proteomic applications due to their heavily glycosylated nature (up to 90%), high molecular weight (200 kDa–200 MDa), and size (Rg 10–300 nm). Their core proteins are extremely large and highly substituted with oligosaccharides, which only allow access to a highly restricted portion of their protein. For this reason, conventional 1D or 2D polyacrylamide gel-based proteomic approaches are not effective for identification and characterization of mucin molecules. In this chapter, we present our current protocol employing a modified shotgun proteomic approach to identify these complex glycoproteins.
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References
Hovenberg, H. W., Davies, J. R., and Carlstedt, I. (1996) Different mucins are produced by the surface epithelium and the submucosa in human trachea: identification of MUC5AC as a major mucin from the goblet cells, Biochem J 318 (Pt 1), 319–324.
Buisine, M. P., Desseyn, J. L., Porchet, N., Degand, P., Laine, A., and Aubert, J. P. (1998) Genomic organization of the 3′-region of the human MUC5AC mucin gene: additional evidence for a common ancestral gene for the 11p15.5 mucin gene family, Biochem J 332 (Pt 3), 729–738.
Desseyn, J. L., Buisine, M. P., Porchet, N., Aubert, J. P., and Laine, A. (1998) Genomic organization of the human mucin gene MUC5B. cDNA and genomic sequences upstream of the large central exon, J Biol Chem 273, 30157–30164.
Hattrup, C. L., and Gendler, S. J. (2008) Structure and function of the cell surface (tethered) mucins, Annu Rev Physiol 70, 431–457.
Kesimer, M., Kirkham, S., Pickles, R. J., Henderson, A. G., Alexis, N. E., Demaria, G., Knight, D., Thornton, D. J., and Sheehan, J. K. (2009) Tracheobronchial air-liquid interface cell culture: a model for innate mucosal defense of the upper airways?, Am J Physiol Lung Cell Mol Physiol 296, L92–L100.
Nicholas, B., Skipp, P., Mould, R., Rennard, S., Davies, D. E., O’Connor, C. D., and Djukanovic, R. (2006) Shotgun proteomic analysis of human-induced sputum, Proteomics 6, 4390–4401.
Utleg, A. G., Yi, E. C., Xie, T., Shannon, P., White, J. T., Goodlett, D. R., Hood, L., and Lin, B. (2003) Proteomic analysis of human prostasomes, Prostate 56, 150–161.
Wu, C. C., and MacCoss, M. J. (2002) Shotgun proteomics: tools for the analysis of complex biological systems, Curr Opin Mol Ther 4, 242–250.
Lu, B., Xu, T., Park, S. K., McClatchy, D. B., Liao, L., and Yates, J. R., 3 rd. (2009) Shotgun protein identification and quantification by mass spectrometry in neuroproteomics, Methods Mol Biol 566, 229–259.
Link, A. J. (2002) Multidimensional peptide separations in proteomics, Trends Biotechnol 20, S8–13.
Washburn, M. P., Ulaszek, R. R., and Yates, J. R., 3 rd. (2003) Reproducibility of quantitative proteomic analyses of complex biological mixtures by multidimensional protein identification technology, Anal Chem 75, 5054–5061.
Vissers, J. P., Blackburn, R. K., and Moseley, M. A. (2002) A novel interface for variable flow nanoscale LC/MS/MS for improved proteome coverage, J Am Soc Mass Spectrom 13, 760–771.
Thornton, D. J., Carlstedt, I., Howard, M., Devine, P. L., Price, M. R., and Sheehan, J. K. (1996) Respiratory mucins: identification of core proteins and glycoforms, Biochem J 316 (Pt 3), 967–975.
Lohrig, K., and Wolters, D. (2009) Multidimensional protein identification technology, Methods Mol Biol 564, 143–153.
Frohlich, T., and Arnold, G. J. (2009) A newcomer’s guide to nano-liquid-chromatography of peptides, Methods Mol Biol 564, 123–141.
Acknowledgment
This work was supported by National Heart, Lung, and Blood Institute/National Institutes of Health (NHLBI/NIH) grants HL103940 (MK) and HL084934 (JKS).
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Kesimer, M., Sheehan, J.K. (2012). Mass Spectrometric Analysis of Mucin Core Proteins. In: McGuckin, M., Thornton, D. (eds) Mucins. Methods in Molecular Biology, vol 842. Humana Press. https://doi.org/10.1007/978-1-61779-513-8_4
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DOI: https://doi.org/10.1007/978-1-61779-513-8_4
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