基于FFPE组织切片的膀胱癌N-连接糖链原位酶解及分析

目的 研究膀胱癌FFPE组织切片的N-连接糖链，发现膀胱癌FFPE肿瘤组织的异常N-连接糖链修饰情况。方法 发展基于FFPE组织切片原位提取N-连接糖链的实验流程。通过PNGase F酶切FFPE组织解释放N-连接糖链。对N-连接糖链自由端进行全甲基化修饰。通过MALDI-TOF/TOF-MS检测N-连接糖链的相对含量。进行数据库匹配，确定N-连接糖链的可能糖型。ROC分析用于预测显著差异N-连接糖链作为预测膀胱癌生物标志物的准确度。结果 MALDI-TOF/TOF-MS检测泛甲基化修饰N-连接糖链的数据显示，在16例膀胱癌患者的肿瘤和癌旁组织的3次重复实验中，肿瘤组织中蛋白质高甘露糖型N2H6、N2H7、N2H8、N2H9和复杂型N5H6F1糖链修饰水平显著上升，同时高甘露糖型N2H5、杂合型N3H5以及复杂型N3H4、N4H4、N5H6F1S2糖链修饰水平显著下降。ROC分析显示，双天线型N-连接糖链N3H4（AUC=0.90）和N4H4（AUC=0.91）在单独或者共同区分膀胱癌患者肿瘤组织和癌旁组织中都具有很好的可靠性，可能成为膀胱癌的潜在生物标志物。结论 膀胱癌FFPE肿瘤组织中存在蛋白质异常N-糖基化修饰，N-连接糖链N3H4和N4H4或可成为膀胱癌的潜在生物标志物。

In Situ Enzymatic Hydrolysis and Analysis of Bladder Cancer N-glycans Based on FFPE Tissue Sections

Objective To investigated the N-linked glycans in bladder cancer FFPE tissue sections, and explored abnormal N-linked glycans modification in bladder cancer FFPE tumor tissues sections.Methods An experimental procedure for in situ extraction of N-linked glycans based on FFPE tissue sections was developed. FFPE tissue sections were digested by PNGase F and liberated N-linked glycans. Permethylation was performed to modify the free end of the N-linked glycans. The relative intensity of N-linked glycans was detected by MALDI-TOF/TOF-MS. Database matching was performed to determine possible glycoforms of N-linked glycans. The accuracy of significantly different N-linked glycans as a predictive bladder cancer biomarker were predicted by ROC analysis.Results The data of MALDI-TOF/TOF-MS detection of premethylation-modified N-linked glycans showed that, in tumor and peritumoral FFPE tissue sections of 16 patients with bladder cancer, the relative intensity of N-linked glycans N2H6, N2H7, N2H8, N2H9 (high mannose) and N5H6F1 (complex) were increased significantly in the tumor tissues of bladder cancer. At the same time, N2H5 (high mannose), N3H5(hybrid) and type N3H4, N4H4, N5H6F1S2 (complex) N-linked glycans were significantly decreased. ROC analysis showed that the biantennary N-linked glycan N3H4 (AUC=0.90) and N4H4 (AUC= 0.91) were reliable in distinguishing tumor and peritumoral tissue of bladder cancer patients separately or combined together, and these N-linked glycans may become a potential biomarker for bladder cancer.Conclusion Abnormal N-glycosylation of proteins in bladder cancer FFPE tumor tissue, N-linked glycans N3H4 and N4H4 may be potential biomarkers of bladder cancer.