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Research paper : Development of basic tools for glycoscience and their application to cancer diagnosis (H. Narimatsu)−208−Synthesiology - English edition Vol.5 No.3 (2012) database (DB). A researcher to identify an unknown glycan structure should analyze the target glycan up to MS2 and refer to the DB. The DB will suggest to the researcher which fragment should be subjected to MS3. The researcher will refer to the DB with the obtained MS3. In most of the cases, the structure would be determined at this point, but sometimes the DB would direct the researcher to MS4.This MSn method for the glycan structure identification system was developed collaboratively by Kameyama, Narimatsu, and other members of AIST, Shimadzu Corporation, and Mitsui Knowledge Industry Co., Ltd., and marketed by Shimadzu Corporation.Elementary technology 7: Antibody-overlay lectin microarrayTo determine the alteration of glycan structures on glycoproteins along with disease development in biological samples, “high throughput, highly sensitive, highly reproducible, and rapid” comparative analytical technology is necessary. The most suitable system is the antibody-overlay lectin microarray developed by Kuno and Hirabayashi et al. of RCMG.[38] The lectin microarray consists of 43 lectins with a variety of specificity solidified on a glass base plate, which can analyze several samples simultaneously by one plate. In the antibody overlay method, the glycoproteins as the objective samples are applied on the lectin micro array without labeling or any other preprocessing, and the reacted glycoproteins binding to the lectins on the plate are detected by the fluorescence labeled antibody recognizing core proteins. By the excitation light radiated from the glass fragment and its total reflection, about 200-m thick of evanescent wave is generated around the glass surface. We designated the system so that only the labeled substances within this layer are signaling. This system is highly sensitive and useful even for detection of only a slight amount of glycans (Fig.5). The preceding glycan analysis methods by liquid chromatography or mass chromatography require a considerable amount of preprocessing and time to release glycans from proteins and to label them. In comparison to these methods, this is a progressive method enabling easy detection. Although the sensitivity relies on the quality of antibodies, the amount of the target glycoprotein samples required for western blotting (a few nanograms) is sufficient for this method. Moreover, as the binding signal of the target glycoprotein is detected specifically by the antibody, the only sample preparation required is simple purification by immunoprecipitation or similar methods. In fact, we were successful in comparative analyses of more than 50 glycoproteins in about a 10-ng level of samples efficiently enriched from the serum or tissue samples and cell culture supernatants by the antibody overlay lectin micro array. This technology is applied for verification of candidate glycoproteins for biomarkers and contributed to establishing the development pipeline of useful glycobiomarkers.[31][33][38][39] The detail of this developmental scheme was published as literature.[40] The lectin array glycan profiling system was developed by Kuno and Hirabayashi et al. of AIST and GP Bioscience, and made publically available by GP Bioscience. Fig. 5 Application example of lectin microarraySelection of useful lectins from enriched glycoproteins in slight pieces of histological samples by comparative analysis Lectin array baseDeveloped by AISTExcitation lightFluorescence detectionEvanescent fieldfluorescence< 200 nm Evanescent-field fluorescence-assisted scanner Co-developed with GP Biosciences Ltd. Fluorescent labeled glycan or glycoproteinCy3/Cy5/TMR/Alexa488ICCD Camera Histological analysis(>1 ㎜2 )Histological specimensCanceroustissueNormaltissueCanceroustissueNormaltissueCanceroustissueNormaltissueLectinNormalized signalScanned dataScan by GlycoStation®

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