Diagnosis of bladder cancer, one of the most common types of human cancer, at an early (nonmuscle-invasive) stage is the best way to reduce the mortality rate. Tumor malignancy in general is closely associated with alterations of glycan expression. Glycosylation status, particularly global glycomes, in bladder cancer has not been well studied. We integrated lectin microarray and mass spectrometry (MS) methods to quantitatively analyze and compare glycan expression in four bladder cancer cell lines (KK47, YTS1, J82, T24) and one normal bladder mucosa cell line (HCV29). Glycopattern alterations were analyzed using lectin microarray analysis and confirmed by lectin staining and lectin blotting. Associations of glycopatterns with diverging stages were evaluated by lectin histochemistry on tissue microarrays. N-Glycans were derivatized by amidation of sialylated glycans with acetohydrazide and reductive amination with the stable isotope tags [12C6]- and [13C6]-aniline, and were quantitatively analyzed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). N-Glycan biosynthesis-associated proteins were quantitatively analyzed by a stable isotope labeling by amino acids in cell culture (SILAC) proteomics method, which revealed significant differences in expression of 13 glycosyltransferases and 4 glycosidases. Our findings indicate that sialyl Lewis X (sLex), terminal GalNAc and Gal, and high mannose-type N-glycans were more highly expressed in bladder cancer cells and tissues than in normal cells. Bladder cancer cells showed high expression of core-fucosylated N-glycans but low expression of terminally fucosylated N-glycans. Each of these glycome changes may be directly related to bladder cancer progression.Keywords: bladder cancer; lectin microarray analysis; mass spectrometry; N-glycans; quantitative glycomics; SILAC method;

•A lectin-based strategy with depth-of-coverage and sensitivity was established.•The strategy facilitates identification of intact N-glycans in different samples.•The strategy can be extended to detailed analysis of O-glycome or glycoproteome.Glycomics provides an increasingly useful research tool as the genomes and proteomes of more and more animal species are elucidated. In view of the general complexity and heterogeneity of glycans, improved depth-of-coverage and sensitivity are required for glycosylation analysis. In this study, we established the lectin-based isolation/enrichment strategy for total glycomic information. Specific lectins are added onto the filter to capture corresponding glycans prior to release of N-glycans by peptide N-glycosidase F (PNGase F). Non-bound glycans and bound glycans are released and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF–MS), respectively. Application of the strategy to chicken ovalbumin, normal mouse mammary epithelial cells (NMuMG), and human serum resulted in detection of 5, 6, and 11 additional N-glycan structures, respectively. The strategy facilitates identification of intact N-glycans in biological samples, and can be extended to detailed analysis of O-glycome or glycoproteome.

Epithelial-to-mesenchymal transition (EMT) is an essential biological process that occurs in embryonic development, metastatic diseases, and cancer progression. Altered expression of glycans is known to be associated with cancer progression. No studies to date have presented global analysis of the precise variation of N-glycans in EMT. We describe here the profile of N-glycans and glycogene expression in the EMT process induced by transforming growth factor-β1 (TGFβ1) in a normal mouse mammary gland epithelial (NMuMG) cell model. An integrated strategy with a combination of mass spectrometry, glycogene microarray analysis, and lectin microarray analysis was applied, and results were confirmed by lectin histochemistry and quantitative real-time PCR. In TGFβ-induced EMT, levels of high-mannose-type N-glycans were enhanced, antennary N-glycans, and fucosylation were suppressed, and bisecting GlcNAc N-glycans were greatly suppressed. The expression of seven N-glycan-related genes was significantly changed. The products of glycogenes ALG9, MGAT3, and MGAT4B appeared to contribute to the observed alteration of N-glycans. The findings indicate that dysregulation of N-glycan synthesis plays a role in the EMT process. Systematic glycomic analysis based on the combination of techniques described here is expected to facilitate the discovery of the aberrant N-glycosylation in tumor progression and provide essential information in systems glycobiology.