*2.1. 1H-15N HSQC of RNase A and RNase B*

1H-15N HSQC spectra of unlabeled RNase A and RNase B were collected at 700 MHz, 37 ◦C in 5 h with all expected signals, consistent with previous results [33]. Peaks were assigned based on 1H-15N chemical shifts deposited in the Biological Magnetic Resonance Data Bank (BMRB) for RNase A. Like mapping protein ligand binding sites by comparing apo and bound forms' 1H-15N chemical shift changes, protein backbone amino acid chemical shifts can be affected by PTMs. Backbone resonance assignments of RNase A/B provide useful data that were used to identify the effect of glycosylation on the polypeptide chain. Upon glycosylation, the backbone 1H-15N chemical shift perturbation in RNase B compared to RNase A is confined to the region around the glycosylation site (±4 amino acids, Figure S2). Minimal changes were observed for most of the glycoprotein's NMR signals. However, measurable differences were observed at N34 (glycosylation site). Specifically, T36 shifts 0.064 ppm in 1H, and S32, N34, and K37 change by 0.014 ppm in 1H. In 15N, S32, N34, T36, and K37 change by 0.28, 0.93, 0.63, and 0.28 ppm, respectively. Interestingly, R33 is absent in the RNase B spectrum, and L35 s chemical shift is unchanged. Thus, in RNase

B only polar or charged residues proximal to the glycosylation site exhibit a change in chemical shift. It may also be that both charged residues and N34, the glycosylated residue, are exposed, thus when N34 is glycosylated, other exposed residues are affected. While these 1H-15N spectra suggest that PTMs effect the protein and the location of attachment, they do not provide accurate information regarding the precise identity of the modification.
