Relative Quantitative Analysis of Site-Specific N-Linked Glycosylation in Hyperglycosylated R27T via Mass Spectrometry

Glycosylation is a key determinant of the efficacy, stability, and pharmacological behavior of therapeutic proteins. The presence of two N-glycosylation sites (Asn25 and Asn80) on R27T, an engineered variant of interferon-β1a, increases its structural complexity and analytical demands. In this study, we established an integrated workflow for comprehensive glycan profiling of R27T by refining existing analytical tools. For total glycan analysis, optimized fluorescent labeling was performed using procainamide, which provided enhanced sensitivity and broader glycan coverage than conventional 2-aminobenzamide. To evaluate the reliability of site-specific glycan quantification, liquid chromatography-tandem mass spectrometry-based peptide mapping results were systematically compared with fluorescence-based profiling, a widely accepted quantitative standard. The correlation varied with glycan structure due to differences in ionization efficiency associated with sialylation and antennary complexity. Protease digestion conditions were also optimized, and chymotrypsin was most effective for site-specific glycopeptide recovery. Distinct glycan distributions were observed between the two glycosylation sites, underscoring the functional importance of site-specific profiling. Furthermore, molecular modeling revealed that the additional glycan at Asn25 may enhance structural stability and receptor-binding affinity by reducing aggregation and strengthening interactions with interferon alpha/beta receptor subunit 2. Together, this study presents a refined approach for glycan analysis in multi-site glycoproteins, facilitating accurate assessment of glycosylation-related critical quality attributes in biotherapeutic development.Keywords: Interferon beta; N-linked glycan; Site-specific glycosylation; Relative quantitative analysis; R27T糖基化（Glycosylation）是决定治疗性蛋白疗效、稳定性及药理学行为的关键因素。干扰素-β1a的工程化变体R27T上存在两个N-糖基化位点（N-glycosylation sites）：天冬酰胺25（Asn25）与天冬酰胺80（Asn80），这使其结构复杂度与分析难度均有所提升。本研究通过优化现有分析工具，建立了一套用于R27T全面糖谱分析的整合分析流程。在总糖链分析中，本研究采用普鲁卡因胺（procainamide）进行优化的荧光标记，相较于传统的2-氨基苯甲酰胺（2-aminobenzamide），该方法可获得更高的灵敏度与更广泛的糖链覆盖范围。为评估位点特异性糖链定量的可靠性，本研究将基于液相色谱-串联质谱（liquid chromatography-tandem mass spectrometry）的肽图分析结果，与当前广泛认可的定量金标准——基于荧光的糖谱分析结果进行了系统对比。由于唾液酸化（sialylation）与天线型糖链结构复杂度相关的电离效率差异，两种方法的相关性随糖链结构不同而有所变化。本研究同时优化了蛋白酶酶解条件，结果显示胰凝乳蛋白酶（chymotrypsin）对于位点特异性糖肽的回收效果最佳。两个糖基化位点的糖链分布存在显著差异，这凸显了位点特异性糖谱分析的功能重要性。此外，分子建模结果显示，天冬酰胺25（Asn25）上额外的糖链可通过减少蛋白聚集、增强与干扰素α/β受体亚基2（interferon alpha/beta receptor subunit 2）的相互作用，提升R27T的结构稳定性与受体结合亲和力。综上，本研究建立了一套针对多位点糖蛋白的糖链分析优化方法，可为生物治疗药物开发过程中糖基化相关关键质量属性（critical quality attributes）的精准评估提供助力。 关键词：干扰素β；N-连接糖链；位点特异性糖基化；相对定量分析；R27T