Covance Laboratories Ltd, Harrogate, UK.
Methods Mol Biol. 2021;2271:189-203. doi: 10.1007/978-1-0716-1241-5_14.
Released N-glycan analysis using the fluorescent label 2-AB (2-aminobenzamide) has been the "gold standard" method for released glycan analysis for several years. The more recent RapiFluor-MS™ labeling technique, however, offers enhanced mass spectrometric detection of released N-glycans, improving the sensitivity and detection limits of the method. The optimized multidimensional detection offers increased confidence in glycan identification which can be further supported by an exoglycosidase digestion array (optional). Here we describe the PNGase F release of N-glycans from a typical IgG1 monoclonal antibody (mAb) with subsequent labeling with RapiFluor-MS™ for detection by HILIC-FLR-MS. The method output quantifies the relative proportion of each glycan species including core afucosylation, sialylation, and high-mannose content, and has a limit of detection (LOD) of 0.01% relative abundance.
使用荧光标记 2-AB（2-氨基苯甲酰胺）进行释放的 N-聚糖分析已经是几年来释放糖分析的“金标准”方法。然而，最近的 RapiFluor-MS™标记技术提高了释放的 N-聚糖的质谱检测灵敏度和检测限。优化的多维检测提高了糖识别的可信度，通过外切糖苷酶消化阵列（可选）可以进一步支持糖识别。在这里，我们描述了从典型的 IgG1 单克隆抗体 (mAb) 中用 PNGase F 释放 N-聚糖，然后用 RapiFluor-MS™进行标记，用于 HILIC-FLR-MS 检测。该方法的输出定量了每种聚糖的相对比例，包括核心岩藻糖基化、唾液酸化和高甘露糖含量，检测限 (LOD) 为相对丰度的 0.01%。
