Global Identification And Differential Distribution Analysis Of Glycans In Subcellular Fractions Of Bladder Cells

The bladder cancer is the most common malignancy tumor which is closely associated with alterations of glycan expression. The extraction of subcellular protein fractions is advantageous to the low abundance protein extraction and reduces the complexity of the whole proteomics. The research of subcellular glycomics is beneficial to extract more kinds of sugar chain and lays a foundation for the study of glycoprotein functional diversity.In the experiment, We used differential centrifugation techniques to isolate four subcellular protein fractions from homogenate of metastatic bladder YTS1 cells, low grade non-muscle invasive bladder cancer KK47cells and normal bladder epithelia HCV29 cells. An integrated strategy combining lectin microarray and mass spectrometry (MS) analysis was then applied to evaluate protein glycosylation of the four fractions.1. Four subcellular proteins in the YTS1, KK47 and HCV29 is extracted by the improved differential centrifugation and validated by SDS-PAGE, silver stain, western-blot.2. Difference of the N-linked glycosylation content of four kinds of subcellular protein fractions is compared. Lectin microarray analysis revealed significant differences among the four fractions in terms of glycan binding to the lectins for LCA, AAL, MPL, WGA, PWM in YTS1, STL, VVA, LCA, LTL, EEL, WGA in KK47 and ConA, GNA, VVA, ACA in HCV29. The striking differences were observed for LCA-binding structures in Mito vs. Nuc, AAL-binding structures in Mito vs. Cyto, and MPL-binding structures in Nuc vs. Cyto in YTS1. The striking differences were observed for STL-binding structures in Mito vs. Nuc, AAL-binding structures in Mic vs. Cyto in KK47. The striking differences were observed for GNA-binding structures in Mic vs. Nuc in HCV29.3. The glycotype distribution in four subcellular fractions is compared. Among a total of 40, 32 and 15 N-glycan in four fractions of three cells are detected by MS analysis. High-mannose and fucosylated glycoprotein structures were predominant in three cells.10 N-glycan in YTS1,5 N-glycan in KK47 and 7 N-glycan in HCV29 were present in all four fractions and 10 N-glycan in YTS1,16 N-glycan in KK47, and 3 N-glycan in HCV29 were present in only one fraction. Glycan in the latter category are considered potential markers for the corresponding organelles.4. The filter auxiliary chemical method to extract the N-linked glycosylation is established and optimized in the level of standard samples of protein, serum and cell.It is the first time to establish glycomics research method in the level of subcellular fractions which provides the help for explain the diversity of glycoprotein function. The integrated strategy allow detailed research of subcellular glycan with high resolution and sensitivity, and will be useful for elucidation of the function of glycan and corresponding glycosylated proteins in distinct organelles.