**1. Introduction**

Glycosylation is one of the most common post-translational modifications (PTM). There are two main types of glycosylation: (i) *O*-linked glycosylation, in which glycans are covalently linked to the hydroxyl oxygen of serine (S) or threonine (T) residues [1,2], and (ii) *N*-linked glycosylation, where glycans are attached to asparagine (N) residues within the N-X-S/T sequon [3–5]. In *N*-linked glycosylation, the initial glycan moiety, Glc3Man9GlcNAc2, is transferred to the nascent polypeptide chain co-translationally in the ER, and then the initial glycan is processed in the ER and Golgi apparatus resulting in either a high-mannose, hybrid, or complex type N-glycan (Figure S1). Because protein glycosylation is not template driven, it is inherently heterogeneous, with several factors contributing to the final glycan structure, such as protein structure [6,7], enzyme protein levels [8], Golgi transport mechanism [9], and secretory protein load [10]. Overall, this process yields heterogeneously glycosylated proteins, such as IgG, which has 32 possible glycans for its one *N*-linked glycosylation site at N297 [11].

For many glycoproteins, the glycans are critical to the protein's structure, stability, and function [12–14]. For example, monoclonal antibodies (mAbs) that lack core fucose in the Fc region (remote to the antigen binding site) lead to an increase in antibodydependent cell-mediated cytotoxicity [15–18]. IgG sialylation has been linked to antiinflammatory activity [19]. The loss of some HIV-1 gp120 glycans leads to an increase in protein degradation and a decrease in binding to the host cell receptor [20–22]. In Hepatitis C virus envelope 2 protein, the loss of either N2 or N4 glycan results in total loss of HCV infectivity [23]. These are just a few glycoproteins where the location and type of glycan

**Citation:** Hargett, A.A.; Marcella, A.M.; Yu, H.; Li, C.; Orwenyo, J.; Battistel, M.D.; Wang, L.-X.; Freedberg, D.I. Glycosylation States on Intact Proteins Determined by NMR Spectroscopy. *Molecules* **2021**, *26*, 4308. https://doi.org/10.3390/ molecules26144308

Academic Editor: Robert Brinson

Received: 9 June 2021 Accepted: 6 July 2021 Published: 16 July 2021

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are critical to protein function. Thus, developing tools to characterize intact glycoproteins will aid in the understanding of optimal glycosylation for a given function, especially in protein therapeutics.

To improve our understanding of structure/function and to ensure proper glycosylation of protein therapeutics, the glycans must be fully characterized. Typically, mass spectrometry (MS)-based methods are combined with other methods, such as glycan enrichment, affinity separation, enzymatic digestion, liquid chromatography (LC) and/or gas chromatography (GC), to determine protein glycosylation [24–26]. However, the stereochemistry of a glycan, including the type of glycosidic linkage, are challenging to determine by MS, because it is difficult to distinguish between isobaric species like glucose (Glc), galactose (Gal), and mannose (Man). To overcome these limitations, a direct, robust and simple NMR spectroscopy method was recently proposed for the detection and identification of protein glycoforms by denaturing the glycoprotein in urea [24]. This method provides a significant advantage by indirectly detecting modifications on intact proteins without sophisticated sample preparation or isotopic labeling. Moreover, the method is not limited by the protein's molecular weight due to the more favorable nuclear relaxation properties of denatured proteins. RNAse A and RNase B have identical amino acid sequences, but RNase B is glycosylated. In this report, we show that the glycans in intact folded RNase B can be characterized by NMR spectroscopy.

As a proof of concept, we chose RNase B glycoprotein as a model system because it is characterized by the following key properties: it is a ~15 kDa glycoprotein, a size that is amenable to NMR and enables the study of native glycosylation; it contains a single glycosylation site at N34 yet, it exists as five glycosylated variants (Man5-9GlcNAc2) and therefore, RNase B permits the study of the potential microheterogeneity in a single glycosylation (at N34) [27]; finally, previous studies of RNase B can be used to cross-validate our findings. In a study of commercial RNase B, the oligosaccharides were released and isolated, and the relative molar portions of Man5 to Man9 were determined to be 57, 31, 4, 7, and 1%, respectively [28].

In pioneering work, Brown showed that differential T2s can be used to distinguish between fluids with different viscosities [29]. Herein, we build on this idea, using 1H-13C HSQC-TOCSY [30,31], with varied mixing times on natural abundance samples for fast detection and analysis of glycoprotein microheterogeneity, without complicated sample preparation. The mixing time efficiently relaxes away protein resonances and, although this phenomenon is not unexpected, it hasn't been investigated in detail [32]. In this report, we show that using a T2 filter in small glycoproteins reduces the spectral complexity that arises from the protein peaks yet captures the glycosylation microheterogeneity by retaining glycan peaks.
