High-throughput quantitative analysis of plant N-glycan using a DNA sequencer
High-throughput quantitative analysis of plant N-glycan using a DNA sequencer
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DOI：
10.1016/j.bbrc.2009.01.070
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发表时间：
2009-03-06
影响因子：
3.1
通讯作者：
Oh, Doo-Byoung
中科院分区：
文献类型：
--
作者：
Lee, Kyung Jin;Jung, Jin-Hee;Oh, Doo-Byoung
建立了植物n -聚糖的高通量定量分析方法。所有步骤，包括肽n -糖苷酶（PNGase） A处理，聚糖制备和外糖苷酶消化，都经过优化，采用96孔格式程序和DNA测序仪自动分析，达到高通量应用。通过比较PNGase A和PNGase F处理获得的多糖谱，可以区分具有植物特异性核心α(1,3)-聚焦的辣根过氧化物酶的多糖谱。通过α(1,3)-甘露糖苷酶和β(1,2)-木糖糖苷酶的同时处理，可以很容易地量化α(1,3)-焦点（91%）和α(1,3)-焦点（1.2%）的聚糖峰，并表明α(1,3)-甘露糖苷酶对β(1,2)-木糖进行了分切。该优化方法成功地应用于分析植物表达的重组抗体的n -聚糖，该重组抗体被设计成含有少量含有P(1,2)-木糖的聚糖。这些结果表明，我们基于DNA测序仪的方法为植物特异性n -聚糖分析提供了高通量的定量信息，这是以前基于质谱的聚糖分析所没有实现的。(c) 2009爱思唯尔公司版权所有。
High-throughput quantitative analytical method for plant N-glycan has been developed. All steps, including peptide N-glycosidase (PNGase) A treatment, glycan preparation, and exoglycosidase digestion, were optimized for high-throughput applications using 96-well format procedures and automatic analysis on a DNA sequencer. The glycans of horseradish peroxidase with plant-specific core alpha(1,3)-fucose can be distinguished by the comparison of the glycan profiles obtained via PNGase A and F treatments. The peaks of the glycans with (91%) and without (1.2%) alpha(1,3)-fucose could be readily quantified and shown to harbor bisecting beta(1,2)-xylose via simultaneous treatment with alpha(1,3)-mannosidase and beta(1,2)-xylosidase. This optimized method was successfully applied to analyze N-glycans of plant-expressed recombinant antibody, which was engineered to contain a minor amount of glycan harboring P(1,2)-xylose. These results indicate that our DNA sequencer-based method provides quantitative information for plant-specific N-glycan analysis in a high-throughput manner, which has not previously been achieved by glycan profiling based on mass spectrometry. (c) 2009 Elsevier Inc. All rights reserved.