Large-scale glycomics for discovering cancer-associated N-glycans by integrating glycoblotting and mass spectrometry.

It has known that the glycosylation plays an important role in the biological states, such as development, aging, and diseases. Although genomic and proteomic approaches have been intensively studied for diagnosis and disease treatment, glycomics have been laggard compared to them due to the hardness of the purification procedure from crude biological materials. Recently, we have developed "glycoblotting" method, a high-throughput and quantitative technique for comprehensive glycomics, which enables to enrich and quantify glycans from crude biological materials, such as serum, tissue biopsy, and cell lysate [Niikura, K., Kamitani, R., Kurogochi, M., Uematsu, R., Shinohara, Y., Nakagawa, H., Deguchi, K., Monde, K., Kondo, H., and Nishimuram S.-I. (2005). Versatile glycoblotting nanoparticles for high-throughput protein glycomics. Chem. Eur. J. 11, 3825-3834; Nishimuara, S.-I., Niikura, K., Kurogochi, M., Matsushita, T., Fumoto, M., Hinou, H., Kamitani, R., Nakagawa, H., Deguchi, K., Miura, N., Monde, K., and Kondo, H. (2005). High-throughput protein glycomics: Combined use of chemoselective glycoblotting and MALDI-TOF/TOF mass spectrometry. Angew. Chem. Int. Ed.44, 91-96]. The automated machine for glycoblotting, "SweetBlot," fixed to use optimized protocol allows us to obtain quantitative profile of 40-50 kinds of major glycoforms from 5mul of human serum within 11h. Based on the method, we have detected potential differences of N-glycome between sera from hepatocellular carcinoma (HCC) and healthy donor [Miura, Y., Hato, M., Shinohara, Y., Kuramoto, H., Furukawa, J.-i, Kurogochi, M., Shimaoka, H., Tada, M., Nakanishi, K., Ozaki, M., Todo, S., and Nishimura, S.-I. (2008). BlotGlycoABC(TM), an integrated glycoblotting technique for rapid and large scale clinical glycomics. Mol. Cell. Proteomics7, 370-377]. The method also permitted cellular quantitative N-glycomics to monitor the process of dynamic cellular differentiation of mouse embryonic stem cells into neural cells [Amano, M., Yamaguchi, M., Takegawa, Y., Yamashita, T., Terashima, M., Furukawa, J.-i., Miura, Y., Shinohara, Y., Iwasaki, N., Minami, A., and Nishimura, S.-I. (2010). Threshold in stage-specific embryonic glycotypes uncovered by a full portrait of dynamic N-glycan expression during cell differentiation. Mol. Cell. Proteomics9, 523-537]. In this chapter, we will discuss glycoblotting method including the potentials not only for exploration of glycan-related cancer biomarker but also for detection of cellular differentiation.