Preparation of samples for nuclear magnetic resonance (NMR) characterization of larger proteins requires enrichment with less abundant, NMR-active, isotopes such as ¹³C and ¹⁵N. This is routine for proteins that can be expressed in bacterial culture where low-cost isotopically enriched metabolic substrates can be used. However, it can be expensive for glycosylated proteins expressed in mammalian culture where more costly isotopically enriched amino acids are usually used. We describe a simple, relatively inexpensive procedure in which standard commercial media is supplemented with ¹³C-enriched glucose to achieve labeling of all glycans plus all alanines of the N-terminal domain of the highly glycosylated protein, CEACAM1. We demonstrate an ability to detect partially occupied N-glycan sites, sites less susceptible to processing by an endoglycosidase, and some unexpected truncation of the amino acid sequence. The labeling of both the protein (through alanines) and the glycans in a single culture requiring no additional technical expertise past standard mammalian expression requirements is anticipated to have several applications, including structural and functional screening of the many glycosylated proteins important to human health.

中文翻译：

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使用 13C-葡萄糖对糖蛋白进行核磁共振 (NMR) 的稀疏同位素标记

制备用于较大蛋白质的核磁共振 (NMR) 表征的样品需要用丰度较低的 NMR 活性同位素（如¹³ C 和¹⁵ N）进行富集。这对于可以在细菌培养物中表达的蛋白质来说是常规操作，其中低成本同位素可以使用富集的代谢底物。然而，在哺乳动物培养物中表达的糖基化蛋白质可能很昂贵，其中通常使用更昂贵的同位素富集氨基酸。我们描述了一个简单、相对便宜的程序，其中标准商业媒体补充了¹³富含 C 的葡萄糖可标记高度糖基化蛋白 CEACAM1 的 N 端结构域的所有聚糖和所有丙氨酸。我们展示了检测部分占据的N-聚糖位点、不太容易受到内切糖苷酶处理的位点以及氨基酸序列的一些意外截断的能力。在单一培养物中标记蛋白质（通过丙氨酸）和聚糖，不需要超出标准哺乳动物表达要求的额外技术专长，预计会有多种应用，包括对人类健康很重要的许多糖基化蛋白质的结构和功能筛选。