Analysis of <i>O</i>‑Glycans as 9‑Fluorenylmethyl Derivatives and Its Application to the Studies on Glycan Array

A method is proposed for the analysis of <i>O</i>-glycans as 9-fluorenylmethyl (Fmoc) derivatives. After releasing the <i>O</i>-glycans from the protein backbone in the presence of ammonia-based media, the glycosylamines thus formed are conveniently labeled with Fmoc-Cl and analyzed by HPLC and MALDI-TOF MS after easy purification. Fmoc labeled <i>O</i>-glycans showed 3.5 times higher sensitivities than those labeled with 2-aminobenzoic acid in fluorescent detection. Various types of <i>O</i>-glycans having sialic acids, fucose, and/or sulfate residues were successfully labeled with Fmoc and analyzed by HPLC and MALDI-TOF MS. The method was applied to the comprehensive analysis of <i>O</i>-glycans expressed on MKN45 cells (human gastric adenocarcinoma). In addition, Fmoc-derivatized <i>O</i>-glycans were easily converted to free hemiacetal or glycosylamine-form glycans that are available for fabrication of glycan array and neoglycoproteins. To demonstrate the availability of our methods, we fabricate the glycan array with Fmoc labeled glycans derived from mucin samples and cancer cells. The model studies using the glycan array showed clear interactions between immobilized glycans and some lectins

Common Glycoproteins Expressing Polylactosamine-Type Glycans on Matched Patient Primary and Metastatic Melanoma Cells Show Different Glycan Profiles

Recently, we reported comparative analysis of glycoproteins which express cancer-specific <i>N</i>-glycans on various cancer cells and identified 24 glycoproteins having polylactosamine (polyLacNAc)-type <i>N</i>-glycans that are abundantly present in malignant cells [Mitsui et al., J. Pharm. Biomed. Anal. 2012, 70, 718−726]. In the present study, we applied the technique to comparative studies on common glycoproteins present in the matched patient primary and metastatic melanoma cell lines. Metastatic melanoma cells (WM266-4) contained a large amount of polyLacNAc-type <i>N</i>-glycans in comparison with primary melanoma cells (WM115). To identify the glycoproteins expressing these <i>N</i>-glycans, glycopeptides having polyLacNAc-type <i>N</i>-glycans were captured by a Datura stramonium agglutinin (DSA)-immobilized agarose column. The captured glycopeptides were analyzed by LC/MS after removing <i>N</i>-glycans, and some glycoproteins such as basigin, lysosome-associated membrane protein-1 (LAMP-1), and chondroitin sulfate proteoglycan 4 (CSPG4) were identified in both WM115 and WM266-4 cells. The expression level of polyLacNAc of CSPG4 in WM266-4 cells was significantly higher than that in WM115 cells. In addition, sulfation patterns of chondroitin sulfate (CS) chains in CSPG4 showed dramatic changes between these cell lines. These data show that characteristic glycans attached to common proteins observed in different stages of cancer cells will be useful markers for determining degree of malignancies of tumor cells

Analysis of Nonhuman <i>N</i>-Glycans as the Minor Constituents in Recombinant Monoclonal Antibody Pharmaceuticals

Minor N-linked glycans containing <i>N</i>-glycolylneuraminic acid residues and/or α-Gal epitopes (i.e., galactose-α1,3-galactose residues) have been reported to be present in recombinant monoclonal antibody (mAb) therapeutics. These contaminations are due to their production processes using nonhuman mammalian cell lines in culture media containing animal-derived materials. In case of the treatment of tumors, we inevitably use such mAbs by careful risk–benefit considerations to prolong patients’ lives. However, expanding their clinical applications such as for rheumatism, asthma, and analgesia demands more careful evaluation of the product characteristics. The present work for detailed evaluations of <i>N</i>-glycans demonstrates the methods using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) and a combination of high-performance liquid chromatography and electrospray ionization time-of-flight mass spectrometry. The CE-LIF method provides excellent separation of both major and minor <i>N</i>-glycans from six commercial mAb pharmaceuticals within 30 min and clearly indicates that a possible trigger of immunogenicity in humans due to the presence of nonhuman <i>N</i>-glycans is present. We strongly believe that the proposed method will be a powerful tool for the analysis of <i>N</i>-glycans of recombinant mAb products in various development stages, such as clone selection, process control, and routine release testing to ensure safety and efficacy of the products

Common Glycoproteins Expressing Polylactosamine-Type Glycans on Matched Patient Primary and Metastatic Melanoma Cells Show Different Glycan Profiles

Recently, we reported comparative analysis of glycoproteins which express cancer-specific <i>N</i>-glycans on various cancer cells and identified 24 glycoproteins having polylactosamine (polyLacNAc)-type <i>N</i>-glycans that are abundantly present in malignant cells [Mitsui et al., J. Pharm. Biomed. Anal. 2012, 70, 718−726]. In the present study, we applied the technique to comparative studies on common glycoproteins present in the matched patient primary and metastatic melanoma cell lines. Metastatic melanoma cells (WM266-4) contained a large amount of polyLacNAc-type <i>N</i>-glycans in comparison with primary melanoma cells (WM115). To identify the glycoproteins expressing these <i>N</i>-glycans, glycopeptides having polyLacNAc-type <i>N</i>-glycans were captured by a Datura stramonium agglutinin (DSA)-immobilized agarose column. The captured glycopeptides were analyzed by LC/MS after removing <i>N</i>-glycans, and some glycoproteins such as basigin, lysosome-associated membrane protein-1 (LAMP-1), and chondroitin sulfate proteoglycan 4 (CSPG4) were identified in both WM115 and WM266-4 cells. The expression level of polyLacNAc of CSPG4 in WM266-4 cells was significantly higher than that in WM115 cells. In addition, sulfation patterns of chondroitin sulfate (CS) chains in CSPG4 showed dramatic changes between these cell lines. These data show that characteristic glycans attached to common proteins observed in different stages of cancer cells will be useful markers for determining degree of malignancies of tumor cells

Common Glycoproteins Expressing Polylactosamine-Type Glycans on Matched Patient Primary and Metastatic Melanoma Cells Show Different Glycan Profiles

Recently, we reported comparative analysis of glycoproteins which express cancer-specific <i>N</i>-glycans on various cancer cells and identified 24 glycoproteins having polylactosamine (polyLacNAc)-type <i>N</i>-glycans that are abundantly present in malignant cells [Mitsui et al., J. Pharm. Biomed. Anal. 2012, 70, 718−726]. In the present study, we applied the technique to comparative studies on common glycoproteins present in the matched patient primary and metastatic melanoma cell lines. Metastatic melanoma cells (WM266-4) contained a large amount of polyLacNAc-type <i>N</i>-glycans in comparison with primary melanoma cells (WM115). To identify the glycoproteins expressing these <i>N</i>-glycans, glycopeptides having polyLacNAc-type <i>N</i>-glycans were captured by a Datura stramonium agglutinin (DSA)-immobilized agarose column. The captured glycopeptides were analyzed by LC/MS after removing <i>N</i>-glycans, and some glycoproteins such as basigin, lysosome-associated membrane protein-1 (LAMP-1), and chondroitin sulfate proteoglycan 4 (CSPG4) were identified in both WM115 and WM266-4 cells. The expression level of polyLacNAc of CSPG4 in WM266-4 cells was significantly higher than that in WM115 cells. In addition, sulfation patterns of chondroitin sulfate (CS) chains in CSPG4 showed dramatic changes between these cell lines. These data show that characteristic glycans attached to common proteins observed in different stages of cancer cells will be useful markers for determining degree of malignancies of tumor cells