The analysis of N-linked glycans using liquid chromatography and mass spectrometry (LC-MS) presents significant challenges, particularly owing to their hydrophilic nature. To address these difficulties, a variety of derivatization methods have been developed to facilitate improved ionization and detection sensitivity. One such method, the Individuality Normalization when Labeling with Isotopic Glycan Hydrazide Tags (INLIGHT)™ strategy for labeling glycans, has previously been utilized in the analysis of N- and O-linked glycans in biological samples. To assess the maximum sensitivity and separability of the INLIGHT™ preparation and analysis pipeline, several critical steps were investigated. First, recombinant and nonrecombinant sources of PNGase F were compared to assess variations in the released glycans. Second, modifications in the INLIGHT™ derivatization step were evaluated including temperature optimization, solvent composition changes, reaction condition length and tag concentration. Optimization of the modified method resulted in 20–100 times greater peak areas for the detected N-linked glycans in fetuin and horseradish peroxidase compared with the standard method. Furthermore, the identification of low-abundance glycans, such as (Fuc)₁(Gal)₂(GlcNAc)₄(Man)₃(NeuAc)₁ and (Gal)₃(GlcNAc)₅(Man)₃(NeuAc)₃, was possible. Finally, the optimal LC setup for the INLIGHT™ derivatized N-linked glycan analyses was found to be a C18 reverse-phase (RP) column with mobile phases typical of RPLC.

使用液相色谱和质谱 (LC-MS)分析N连接聚糖面临着巨大的挑战，特别是由于其亲水性。为了解决这些困难，已经开发了多种衍生方法以促进提高电离和检测灵敏度。其中一种方法是用同位素聚糖酰肼标签进行标记时的个体标准化 (INLIGHT)™ 策略，用于标记聚糖，此前已用于分析生物样品中的N连接和O连接聚糖。为了评估 INLIGHT™ 制备和分析流程的最大灵敏度和可分离性，研究了几个关键步骤。首先，比较 PNGase F 的重组和非重组来源，以评估释放的聚糖的变化。其次，评估了 INLIGHT™ 衍生化步骤的修改，包括温度优化、溶剂成分变化、反应条件长度和标签浓度。与标准方法相比，改进方法的优化导致胎球蛋白和辣根过氧化物酶中检测到的N连接聚糖的峰面积增加了 20-100 倍。此外，低丰度聚糖的鉴定，例如 (Fuc) ₁ (Gal) ₂ (GlcNAc) ₄ (Man) ₃ (NeuAc) ₁和 (Gal) ₃ (GlcNAc) ₅ (Man) ₃ (NeuAc) ₃，是可能的。最后，我们发现用于 INLIGHT™ 衍生化N连接聚糖分析的最佳 LC 设置是采用 RPLC 典型流动相的 C18 反相 (RP) 色谱柱。