*Article* **Glycosylation States on Intact Proteins Determined by NMR Spectroscopy**

**Audra A. Hargett 1, Aaron M. Marcella 1, Huifeng Yu 1, Chao Li 2, Jared Orwenyo 2, Marcos D. Battistel 1, Lai-Xi Wang <sup>2</sup> and Darón I. Freedberg 1,\***


**Abstract:** 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.

**Keywords:** glycosylated proteins; heteronuclear NMR; HSQC-TOCSY; natural abundance; T2 filter; glycoprotein
