Glycosylation changes in cancer proteins have been associated with malignant transformation. However, techniques for analyzing site-specific glycosylation changes in target proteins obtained from clinical tissue samples are insufficient. To overcome these problems, we developed a targeted N-glycoproteomic approach consisting of immunoprecipitation, glycopeptide enrichment, LC/MS/MS, and structural assignment using commercially available analytical software followed by manual confirmation. This approach was applied to the comparative site-specific glycosylation analysis of lysosome-associated membrane glycoprotein 1 (LAMP1) between breast cancer (BC) tumors and normal tissues adjacent to tumors. Extensive determination of glycan heterogeneity from four N-glycosylation sites (Asn84/103/249/261) in LAMP1 identified 262 glycoforms, and revealed remarkable diversity in tumor glycan structures. A significant increase in N-glycoforms with multiple fucoses and sialic acids at Asn84/249 and high-mannose-type glycans at Asn103/261 were observed in the tumor. Principal component analysis revealed that tumors of different subtypes have independent distributions. This approach enables site-specific glycopeptide analysis of target glycoprotein in breast cancer tissue and become a powerful tool for characterizing tumors with different pathological features by their glycan profiles.

中文翻译：

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乳腺癌组织中 LAMP1 位点特异性 N-糖基化的比较分析

癌症蛋白的糖基化变化与恶性转化有关。然而，分析从临床组织样本获得的靶蛋白的位点特异性糖基化变化的技术还不够。为了克服这些问题，我们开发了一种靶向 N-糖蛋白质组学方法，包括免疫沉淀、糖肽富集、LC/MS/MS 和使用市售分析软件进行结构分配，然后进行手动确认。该方法应用于乳腺癌 (BC) 肿瘤和肿瘤邻近正常组织之间溶酶体相关膜糖蛋白 1 (LAMP1) 的位点特异性糖基化比较分析。对 LAMP1 中四个 N-糖基化位点 (Asn84/103/249/261) 的聚糖异质性进行广泛测定，鉴定出 262 种糖型，并揭示了肿瘤聚糖结构的显着多样性。在肿瘤中观察到 Asn84/249 处具有多个岩藻糖和唾液酸的 N-糖型以及 Asn103/261 处高甘露糖型聚糖的显着增加。主成分分析显示不同亚型的肿瘤具有独立的分布。这种方法能够对乳腺癌组织中的目标糖蛋白进行位点特异性糖肽分析，并成为通过聚糖谱来表征具有不同病理特征的肿瘤的强大工具。