NIBRT Dublin-Oxford Glycobiology Laboratory, NIBRT - The National Institute for Bioprocessing Research and Training, UCD Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland.
J Proteome Res. 2011 Aug 5;10(8):3820-9. doi: 10.1021/pr200371s. Epub 2011 Jul 8.
N-glycans attached to the C(H)2 domains of the Fc or the antigen binding regions of IgG play an important role in stabilizing and modulating antibody activity. Exhaustive elucidation of 32 IgG N-glycans using a combination of weak anion exchange enrichment and exoglycosidase array digestion with subsequent profiling exceeded 48 h. Pursuing increased throughput and associated structural annotation confidence, we compared the 1.7 μm hydrophilic interaction phase for UPLC with CE-LIF for the rapid and comprehensive characterization of N-glycans released from healthy human serum polyclonal IgG. Combination of the data individually generated using each technique demonstrated that complete structural annotation was possible within a total analysis time of 20 min due to the advantageous orthogonality of the separation mechanisms. The parallel use of both analytical techniques provides a powerful platform for rapid and comprehensive analysis of IgG N-glycosylation present on therapeutic antibodies or on antibodies of biomedical or pathological significance.
IgG 的 Fc 或抗原结合区域的 C(H)2 结构域上附着的 N-糖链在稳定和调节抗体活性方面发挥着重要作用。使用弱阴离子交换富集和外切糖苷酶阵列消化的组合，以及随后的分析，详尽阐明 32 个 IgG N-糖链的结构需要超过 48 小时。为了提高通量和相关结构注释的置信度，我们比较了 UPLC 的 1.7μm 亲水相互作用相和 CE-LIF 用于快速和全面表征从健康人血清多克隆 IgG 释放的 N-糖链。两种技术分别产生的数据的组合表明，由于分离机制的有利正交性，在总共 20 分钟的分析时间内可以实现完整的结构注释。两种分析技术的并行使用为快速全面分析治疗性抗体或具有生物医学或病理学意义的抗体上存在的 IgG 糖基化提供了强大的平台。
