Glycosylation States on Intact Proteins Determined by NMR Spectroscopy

• Published in: Molecules (Basel, Switzerland) Vol. 26; no. 14; p. 4308
• Main Authors: Hargett, Audra A., Marcella, Aaron M., Yu, Huifeng, Li, Chao, Orwenyo, Jared, Battistel, Marcos D., Wang, Lai-Xi, Freedberg, Darón I.
• Format: Journal Article
• Language: English
• Published: Basel MDPI AG 16-07-2021; MDPI
• Subjects: Amino acids; Binding sites; Cell adhesion; Chromatography; Glycan; Glycopeptides; glycoprotein; Glycoproteins; glycosylated proteins; Glycosylation; heteronuclear NMR; HSQC-TOCSY; Immune response; Liquid chromatography; Magnetic resonance spectroscopy; Mass spectrometry; Mass spectroscopy; Medical research; natural abundance; NMR; NMR spectroscopy; Nondestructive testing; Nuclear magnetic resonance; Nuclear relaxation; Polypeptides; Protein folding; Protein interaction; Proteins; Ribonuclease B; Scientific imaging; T2 filter
• ISSN: 1420-3049; 1420-3049
• 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.