Glycosylation States on Intact Proteins Determined by NMR Spectroscopy

Audra A. Hargett; Aaron M. Marcella; Huifeng Yu; Chao Li; Jared Orwenyo; Marcos D. Battistel; Lai-Xi Wang; Darón I. Freedberg

doi:10.3390/molecules26144308

Molecules (Jul 2021)

Glycosylation States on Intact Proteins Determined by NMR Spectroscopy

Audra A. Hargett,
Aaron M. Marcella,
Huifeng Yu,
Chao Li,
Jared Orwenyo,
Marcos D. Battistel,
Lai-Xi Wang,
Darón I. Freedberg

Affiliations

Audra A. Hargett: Center for Biologics Evaluation and Review, Laboratory of Bacterial Polysaccharides, Food and Drug Administration (FDA), Silver Spring, MD 20993, USA
Aaron M. Marcella: Center for Biologics Evaluation and Review, Laboratory of Bacterial Polysaccharides, Food and Drug Administration (FDA), Silver Spring, MD 20993, USA
Huifeng Yu: Center for Biologics Evaluation and Review, Laboratory of Bacterial Polysaccharides, Food and Drug Administration (FDA), Silver Spring, MD 20993, USA
Chao Li: Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA
Jared Orwenyo: Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA
Marcos D. Battistel: Center for Biologics Evaluation and Review, Laboratory of Bacterial Polysaccharides, Food and Drug Administration (FDA), Silver Spring, MD 20993, USA
Lai-Xi Wang: Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA
Darón I. Freedberg: Center for Biologics Evaluation and Review, Laboratory of Bacterial Polysaccharides, Food and Drug Administration (FDA), Silver Spring, MD 20993, USA

DOI: https://doi.org/10.3390/molecules26144308
Journal volume & issue: Vol. 26, no. 14
p. 4308

Abstract

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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.

Published in Molecules

ISSN: 1420-3049 (Online)
Publisher: MDPI AG
Country of publisher: Switzerland
LCC subjects: Science: Chemistry: Organic chemistry
Website: http://www.mdpi.com/journal/molecules

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