MPI for Dynamics of Complex Technical Systems, Sandtorstraße 1, 39106 Magdeburg, Germany.
glyXera GmbH, Brenneckestraße 20, 39120 Magdeburg, Germany.
Molecules. 2023 Feb 15;28(4):1843. doi: 10.3390/molecules28041843.
Glycosylation, especially -glycosylation, is one of the most common protein modifications, with immense importance at the molecular, cellular, and organismal level. Thus, accurate and reliable -glycan analysis is essential in many areas of pharmaceutical and food industry, medicine, and science. However, due to the complexity of the cellular glycosylation process, in-depth glycoanalysis is still a highly challenging endeavor. Contamination of samples with oligosaccharide impurities (OSIs), typically linear glucose homo-oligomers, can cause further complications. Due to their physicochemical similarity to -glycans, OSIs produce potentially overlapping signals, which can remain unnoticed. If recognized, suspected OSI signals are usually excluded in data evaluation. However, in both cases, interpretation of results can be impaired. Alternatively, sample preparation can be repeated to include an OSI removal step from samples. However, this significantly increases sample amount, time, and effort necessary. To overcome these issues, we investigated the option to enzymatically degrade and thereby remove interfering OSIs as a final sample preparation step. Therefore, we screened ten commercially available enzymes concerning their potential to efficiently degrade maltodextrins and dextrans as most frequently found OSIs. Of these enzymes, only dextranase from and glucoamylase P from enabled a degradation of OSIs within only 30 min that is free of side reactions with -glycans. Finally, we applied the straightforward enzymatic degradation of OSIs to -glycan samples derived from different standard glycoproteins and various stem cell lysates.
糖基化，特别是 - 糖基化，是最常见的蛋白质修饰之一，在分子、细胞和机体水平上都具有重要意义。因此，在制药和食品工业、医学和科学的许多领域，准确可靠的 - 聚糖分析是必不可少的。然而，由于细胞糖基化过程的复杂性，深入的糖基分析仍然是一项极具挑战性的工作。样品中寡糖杂质（OSI）的污染，通常是线性葡萄糖同聚物，会导致进一步的并发症。由于它们与 - 聚糖在物理化学性质上相似，OSI 会产生潜在的重叠信号，这些信号可能会被忽略。如果被识别到，可疑的 OSI 信号通常会在数据评估中被排除。然而，在这两种情况下，结果的解释都可能受到影响。或者，可以重复样品制备过程，包括从样品中去除 OSI 的步骤。然而，这会显著增加所需的样品量、时间和工作量。为了解决这些问题，我们研究了在最终样品制备步骤中通过酶解来去除干扰的 OSI 的选择。因此，我们筛选了十种市售酶，以研究它们是否有可能有效地降解最常见的 OSI - 麦芽糊精和葡聚糖。在这些酶中，只有 的葡聚糖酶和 的糖化酶 P 能够在 30 分钟内有效地降解 OSI，而且不会与 - 聚糖发生副反应。最后，我们将 OSI 的简单酶解应用于来自不同标准糖蛋白和各种干细胞裂解物的 - 聚糖样品。
