Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, and ‡Department of Plant Pathology, Nanjing Agricultural University , Nanjing, Jiangsu 210095, People's Republic of China.
J Agric Food Chem. 2015 Dec 9;63(48):10550-5. doi: 10.1021/acs.jafc.5b03633. Epub 2015 Nov 23.
The quantification of potentially allergenic carbohydrate motifs of plant and insect glycoproteins is increasingly important in biotechnological and agricultural applications as a result of the use of insect cell-based expression systems and transgenic plants. The need to analyze N-glycan moieties in a highly parallel manner inspired us to develop a quick N-glycan analysis method based on a recently discovered bacterial protein N-glycanase (PNGase H(+)). In contrast to the traditionally used PNGase A, which is isolated from almond seeds and only releases N-glycans from proteolytically derived glycopeptides, the herein implemented PNGase H(+) allows for the release of N-glycans directly from the glycoprotein samples. Because PNGase H(+) is highly active under acidic conditions, the consecutive fluorescence labeling step using 2-aminobenzamide (2AB) can be directly performed in the same mixture used for the enzymatic deglycosylation step. All sample handling and incubation steps can be performed in less than 4 h and are compatible with microwell-plate sampling, without the need for tedious centrifugation, precipitation, or sample-transfer steps. The versatility of this methodology was evaluated by analyzing glycoproteins derived from various plant sources using ultra-performance liquid chromatography (UPLC) analysis and further demonstrated through the activity analysis of four PNGase H(+) mutant variants.
由于昆虫细胞表达系统和转基因植物的使用，植物和昆虫糖蛋白中潜在过敏原碳水化合物序列的定量分析在生物技术和农业应用中变得越来越重要。需要以高度平行的方式分析 N-糖链部分，这激发我们开发了一种基于最近发现的细菌蛋白 N-聚糖酶 (PNGase H(+)) 的快速 N-糖链分析方法。与传统上从杏仁种子中分离出来的仅从蛋白水解衍生的糖肽中释放 N-聚糖的 PNGase A 不同，本文中使用的 PNGase H(+) 允许直接从糖蛋白样品中释放 N-聚糖。由于 PNGase H(+) 在酸性条件下具有很高的活性，因此可以直接在用于酶解糖基化步骤的相同混合物中进行随后的荧光标记步骤，使用 2-氨基苯甲酰胺 (2AB)。所有样品处理和孵育步骤可以在不到 4 小时内完成，并与微孔板采样兼容，无需繁琐的离心、沉淀或样品转移步骤。该方法的多功能性通过使用超高效液相色谱 (UPLC) 分析来自各种植物来源的糖蛋白进行了评估，并通过对四个 PNGase H(+) 突变变体的活性分析进一步证明。
