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Rapid Release of N-Linked Glycans from Glycoproteins by Pressure-Cycling Technology

机译：通过压力循环技术从糖蛋白中快速释放N-连接的聚糖

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摘要

The standard, well-established sample preparation protocol to release N-linked glycans from glycoproteins for downstream analysis requires relatively long deglycosylation times (from several hours to overnight) and relatively high endoglycosidase concentration (from 1:250 to 1:500 enzyme:substrate molar ratio). In this paper, we significantly improve this standard protocol by the use of pressure-cycling technology (PCT) to increase the speed and decrease the relative amount of PNGase F during the release of N-linked glycans from denatured glycoproteins. With the application of pressure cycling from atmospheric to as high as 30 kpsi, >95percent release of the asparagine-linked glycans from bovine ribonuclease B, human transferrin, and polyclonal human immunoglobulin was rapidly achieved in a few minutes using as low as 1:2500 enzyme: substrate molar ratio. The deglycosylation rate was first examined by SDS--PAGE at the protein level. The released glycans were then quantitated by capillary electrophoresis with laser induced fluorescence detection (CE-LIF). This new sample preparation protocol readily supports largescale glycan analysis of biopharmaceuticals with rapid deglycosylation times.
机译：从糖蛋白释放N-连接的聚糖以进行下游分析的标准，完善的样品制备方案需要相对较长的去糖基化时间（从数小时到过夜）和相对较高的糖苷内切酶浓度（从1：250至1：500酶：底物摩尔比）。在本文中，我们通过使用压力循环技术（PCT）显着改进了该标准协议，以提高从变性糖蛋白释放N-连接聚糖的速度和降低PNGase F的相对量。通过从大气压到高达30 kpsi的压力循环，从牛核糖核酸酶B，人转铁蛋白和多克隆人免疫球蛋白中以95％的比例释放了天冬酰胺连接的聚糖，释放率低至1：2500酶：底物摩尔比。首先通过SDS-PAGE在蛋白质水平上检查去糖基化率。然后通过毛细管电泳和激光诱导荧光检测（CE-LIF）对释放的聚糖进行定量。这种新的样品前处理方案可轻松支持以快速的去糖基化时间对生物药物进行大规模的聚糖分析。

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