Glycoproteins, proteins that are co- and posttranslationally modified by sugars (glycans), have significant roles in pathophysiology of many different diseases. One of the main steps in sample preparation for free N-glycan analysis is deglycosylation or glycan removal. The aim of this study was to compare different peptide N-glycosidase F (PNGase F) enzymes (Rapid PNGase F and two recombinant versions) for deglycosylation of total human plasma glycoproteins and different amounts of human immunoglobulin G (IgG). Deglycosylation with different PNGase F enzymes resulted in different IgG and plasma N-glycosylation hydrophilic interaction liquid chromatography ultra-performance liquid chromatography profiles. Additionally, one recombinant version of PNGase F is more efficient in deglycosylation of complex N-glycans compared with Rapid PNGase F and recombinant version of PNGase F from a different manufacturer. In terms of chromatographic peak intensities and coefficient of variation %Area values, all tested versions of PNGase F enzymes were very reproducible and on the similar level when used in optimal conditions. However, care should be taken in terms of which enzyme is used with which protocol, particularly when scaling up.

中文翻译：

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在免疫球蛋白 G 和总血浆 N-聚糖分析中评估不同的 PNGase F 酶。

糖蛋白是由糖（聚糖）共同和翻译后修饰的蛋白质，在许多不同疾病的病理生理学中具有重要作用。游离 N-聚糖分析样品制备的主要步骤之一是去糖基化或聚糖去除。本研究的目的是比较不同的肽 N-糖苷酶 F (PNGase F) 酶（快速 PNGase F 和两种重组版本）对总人血浆糖蛋白和不同量的人免疫球蛋白 G (IgG) 的去糖基化。用不同的 PNGase F 酶去糖基化导致不同的 IgG 和血浆 N-糖基化亲水相互作用液相色谱超高效液相色谱图谱。此外，与来自不同制造商的 Rapid PNGase F 和重组版本的 PNGase F 相比，一种重组版本的 PNGase F 在复杂 N-聚糖的去糖基化方面更有效。在色谱峰强度和变异系数 % 面积值方面，所有测试版本的 PNGase F 酶在最佳条件下使用时都具有非常高的重现性和相似的水平。然而，应注意哪种酶与哪种协议一起使用，特别是在放大时。