Center for Biologics Evaluation and Review, Laboratory of Bacterial Polysaccharides, Food and Drug Administration (FDA), Silver Spring, MD 20993, USA.
Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA.
Molecules. 2021 Jul 16;26(14):4308. doi: 10.3390/molecules26144308.
Protein glycosylation is important in many organisms for proper protein folding, signaling, cell adhesion, protein-protein interactions, and immune responses. Thus, effectively determining the extent of glycosylation in glycoprotein therapeutics is crucial. Up to now, characterizing protein glycosylation has been carried out mostly by liquid chromatography mass spectrometry (LC-MS), which requires careful sample processing, e.g., glycan removal or protein digestion and glycopeptide enrichment. Herein, we introduce an NMR-based method to better characterize intact glycoproteins in natural abundance. This non-destructive method relies on exploiting differences in nuclear relaxation to suppress the NMR signals of the protein while maintaining glycan signals. Using RNase B Man5 and RNase B Man9, we establish reference spectra that can be used to determine the different glycoforms present in heterogeneously glycosylated commercial RNase B.
蛋白质糖基化对于许多生物体内蛋白质的正确折叠、信号传递、细胞黏附、蛋白质-蛋白质相互作用和免疫反应都很重要。因此，有效地确定糖蛋白治疗药物中的糖基化程度至关重要。到目前为止，蛋白质糖基化的特征主要是通过液相色谱-质谱联用（LC-MS）来进行的，这需要仔细的样品处理，例如糖链的去除或蛋白质的消化和糖肽的富集。在此，我们介绍了一种基于 NMR 的方法，可更好地在自然丰度下对完整糖蛋白进行特征分析。这种非破坏性方法依赖于利用核弛豫的差异来抑制蛋白质的 NMR 信号，同时保持糖链信号。使用 RNase B Man5 和 RNase B Man9，我们建立了参考光谱，可以用来确定异质糖基化的商业 RNase B 中存在的不同糖型。
