Analysis of N-glycans from recombinant immunoglobulin G by on-line reversed-phase high-performance liquid chromatography/mass spectrometry
Analysis of N-glycans from recombinant immunoglobulin G by on-line reversed-phase high-performance liquid chromatography/mass spectrometry
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DOI：
10.1016/j.ab.2007.08.012
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发表时间：
2007-11-15
影响因子：
2.9
通讯作者：
Flynn, Gregory C.
中科院分区：
文献类型：
--
作者：
Chen, Xiaoyu;Flynn, Gregory C.
在线反相（RP）高效液相色谱/质谱（MS）方法已被开发用于分析和表征重组免疫球蛋白G抗体的N-聚糖。在该方法中，释放的N-聚糖在其还原端用2-氨基苯甲酰胺（2AB）衍生化，并在RP柱上通过在线荧光和MS检测进行分离。该方法实现了所有主要聚糖的良好分离度，并将聚糖类型（高甘露糖、杂合和复合）分离到色谱图的不同区域，从而仅根据荧光信号准确定量N-聚糖。此外，所使用的移动的相允许高质量的在线MS检测。2AB标记的N-聚糖在电喷雾中表现出良好的电离效率，并主要产生双电荷[M+2 H]（2+）离子。质量和结构信息可以很容易地从在线MS和串联MS数据中获得。可以检测和鉴定低至70 fmol的聚糖种类。(c)2007爱思唯尔公司All rights reserved.
An on-line reversed-phase (RP) high-performance liquid chromatography/mass spectrometry (MS) method has been developed for profiling and characterizing N-glycans from recombinant immunoglobutin G antibodies. In this method, released N-glycans are derivatized at their reducing end with 2-aminobenzamide (2AB) and separated on a RP column with on-line fluorescence and MS detection. The method achieves good resolution of all major glycans and segregates glycan types (high-mannose, hybrid, and complex) to different regions of the chromatogram, thus allowing accurate quantification of N-glycans from the fluorescent signal alone. Moreover, the mobile phase used allows high quality on-line MS detection. The 2AB-labeled N-glycans demonstrate good ionization efficiency in electrospray and generate primarily doubly charged [M+2H](2+) ions. The mass and structural information can be readily obtained from the on-line MS and tandem MS data. As little as 70 fmol glycan species can be detected and identified. (c) 2007 Elsevier Inc. All rights reserved.