N-连接聚糖释放效率：次氯酸钠（NaOCl）与肽-N-糖苷酶F（PNGase F）释放方案的定量比较
-linked Glycan Release Efficiency: A Quantitative Comparison between NaOCl and PNGase F Release Protocols.
作者信息
Fischler David A, Orlando Ron
机构信息
Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30605, USA.
出版信息
J Biomol Tech. 2019 Dec;30(4):58-63. doi: 10.7171/jbt.19-3004-001.
Abstract
There are several methods, both chemical and enzymatic, to release -linked glycans for structural characterization. One of the most common enzymatic release methods is the use of peptide:-glycosidase F (PNGase F). A less expensive and quicker alternative has been reported for the release of -linked glycans chemically using sodium hypochlorite (NaOCl), which hydrolyzes the peptide-glycan bond, yielding the intact glycan with a free reducing terminus. Here, we quantitatively analyzed the efficiency of the NaOCl release protocol compared with the PNGaseF release protocol for small-scale analysis (300 µg) using liquid chromatography-single reaction monitoring-mass spectrometry. We determined that the relative glycan composition of released -linked glycans from the NaOCl protocol is similar to a typical PNGase F protocol, but the absolute recovery of -linked glycans is significantly lower with the chemical procedure.
摘要
有几种化学和酶促方法可用于释放N-连接聚糖以进行结构表征。最常见的酶促释放方法之一是使用肽：N-糖苷酶F（PNGase F）。据报道，有一种成本较低且速度更快的替代方法，即使用次氯酸钠（NaOCl）化学释放N-连接聚糖，它能水解肽聚糖键，产生具有游离还原末端的完整聚糖。在此，我们使用液相色谱 - 单反应监测 - 质谱法对次氯酸钠释放方案与PNGaseF释放方案在小规模分析（300μg）中的效率进行了定量分析。我们确定，次氯酸钠方案释放的N-连接聚糖的相对聚糖组成与典型的PNGase F方案相似，但化学方法中N-连接聚糖的绝对回收率显著较低。
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