Alpha-1,6 fucosylation of N-glycans (core fucosylation, CF) represents a unique form of N-glycans and is widely involved in disease progression. In order to accurately identify CF glycoproteins, several approaches have been developed based on sequential cleavage with different glycosidases to truncate the N-glycans. Since multi-step sample treatments may introduce quantitation bias and affect the practicality of these approaches in large-scale applications. Here, we systematically evaluated the performance of the single-step treatment of intact glycopeptides by endoglycosidase F3 for CF glycoproteome. The single-step truncation (SST) strategy demonstrated higher quantitative stability and higher efficiency compared with previous approaches. The strategy was further practiced on both cell lines and serum samples. The dysregulation of CF glycopeptides between preoperative and postoperative serum from patients with pancreatic ductal adenocarcinoma was revealed, and the CF modifications of BCHE_N369, CDH5_N112 and SERPIND1_N49 were found to be potential prognostic markers. This study thus provides an efficient solution for large-scale quantitative analysis of the CF glycoproteome.

N-聚糖的α-1,6岩藻糖基化（核心岩藻糖基化，CF）代表了N-聚糖的独特形式，广泛参与疾病进展。为了准确鉴定CF糖蛋白，已经开发了几种基于用不同糖苷酶连续切割来截短N-聚糖的方法。由于多步骤样品处理可能会引入定量偏差并影响这些方法在大规模应用中的实用性。在这里，我们系统地评估了 CF 糖蛋白组中糖苷内切酶 F3 单步处理完整糖肽的性能。与以前的方法相比，单步截断（SST）策略表现出更高的定量稳定性和更高的效率。该策略在细胞系和血清样本上得到了进一步实践。揭示了胰腺导管腺癌患者术前和术后血清中 CF 糖肽的失调，并且发现 BCHE_N369、CDH5_N112 和 SERPIND1_N49 的 CF 修饰是潜在的预后标志物。因此，本研究为CF糖蛋白质组的大规模定量分析提供了有效的解决方案。