*3.1. Preparation of RNase B with Homogenous Man5 and Man9 N-Glycans*

The synthesis of RNase B Man9 followed our previously reported method [39]. ESI-MS: theoretical mass for RNase B Man9, M = 15,546 Da; found (deconvolution data) (*m*/*z*) 15,547 Da. RNase B Man5 was prepared following a similar chemoenzymatic method. Briefly, the Man5 oxazoline was obtained by α1,2-mannosidease catalyzed hydrolysis of Man9 N-glycan, followed by Endo-A treatment to provide the Man5GlcNAc, which was converted to Man5GlcNAc-oxazoline by treatment with 2-chloro-1,3-dimethylimidazolium chloride (DMC) and triethylamine in water [40]. A solution of Man5GlcNAc-oxazoline (500 μg, 0.49 μmol) and GlcNAc-RNase (500 μg, 0.036 μmol) was incubated with EndoA-N171A (200 μg) in buffer (PBS, 100 mM, pH 7.4, 10 μL) at 30 ◦C for 8 h. The reaction was monitored by analytical HPLC, and the glycoprotein product was isolated by preparative HPLC to give Man5GlcNAc2-RNase as a white foam after lyophilization (418 μg, 78%). ESI-MS: calc'd. for RNase B Man5, M = 14,897 Da; found (deconvolution data) (*m*/*z*) 14,898 Da.

Analytical reverse-phase high-performance liquid chromatography (RP-HPLC) was performed on a Waters 626 HPLC instrument equipped with an YMC-Triart C18 column (5 μm, 4.6 × 250 mm) for reverse phase. The YMC-Triart column was eluted using a linear gradient of acetonitrile (22–29%, *v*/*v*) with water containing 0.1% TFA over 35 min at a flowrate of 0.5 mL/min under UV 280. The LC-ESI-MS was performed on an Exactive™ Plus Orbitrap mass spectrometer (Thermo Scientific) equipped with a C8 column (Poroshell

300SB-C8, 1.0 × 75 mm, 5 μm, Agilent). Mass spectra were analyzed, and deconvolution of MS data was obtained by MagTran.

#### *3.2. NMR of Glycoproteins*

All NMR experiments were performed at 700 MHz 1H Frequency (16.5 T magnet) with a Bruker Avance III HD console and 5 mm triple gradient TCI cryoprobe. RNase B solution concentrations varied from 5-20 mg/mL, depending on sample, in 20 mM phosphate buffer at pH\* 6.0 with added DSS as an internal reference in ~99% D2O. For HSQC (Bruker pulse sequence hsqcetgpsi) and HSQC-TOCSY (Bruker pulse sequence hsqcdietgpsi) experiments, 4096 and 768 total points in 1H and 13C, respectively, were collected. Non-uniform sampling was used in the indirect dimension with 30–50% of the points collected based on the schedules from the Wagner group [41]. Data were collected with spectral windows of 7002.801 Hz (10 ppm) and 10,563.504 Hz (60 ppm), with carrier frequencies of 4.7 ppm and 60 ppm in 1H and 13C, respectively, and were reconstructed using SMILE [42]. The anomeric 13C peaks appear at ~40 ppm, due to folding in most of the spectra collected. Data were processed with zero-filling to 2x the total points collected in 1H and 13C. A square cosine bell window function was applied in both dimensions. For linewidth measurements, data were collected with traditional sampling and 2048 × 1024 total points in 1H and 13C, respectively. Spectral widths of 10 ppm (7002.8 Hz) in 1H and 100 ppm (17,605.8 Hz) in 13C were used with the 13C carrier set at 60 ppm (10,562.9 Hz). Data were processed in the same way as described above.

For 1H-15N experiments, samples of RNase B and RNase A were dissolved in 20 mM phosphate buffer pH 6.5 in 2.5% D2O at ~15 mg/mL (~1 mM). 1H-15N experiments were collected using an HSQC pulse sequence with a flip back pulse and WATERGATE [43] element for water suppression. Acquisition times of 41 and 26 ms for 1H and 15N respectively were used with spectral resolution of 12 and 19 Hz/pt. High-quality data were collected over ~10 h of experiment time; two 10 h spectra were added to increase signal to noise.

**ESI-MS of RNase B**: 2.5 μL of a 0.5 mg/mL solution of RNAse B was injected into a C18 trap column and eluted using a gradient from 0 to 40% acetonitrile in acetate buffer, pH 4.5, at a flow rate of 0.5 μL/min. Data were collected on a Waters Synapt G2 HDMS system with a nanoAcquity LC system. Data containing the entire charge state envelope were deconvoluted using the Masslynx software yielding the mass of the singly charged species.

**MALDI-TOF MS of RNase B**: 0.5 mg/mL RNase B was mixed 1:1 with dihydroxybenzoic acid matrix solution (10 mg/mL in 50:50 acetonitrile:water with 0.1 % TFA). The mixture was then spotted on a stainless steel MALDI target and allowed to air dry. Samples were analyzed using a Bruker Autoflex Speed instrument with a voltage of 13 keV, 4000 shots per spectrum and delay time of 800 ns. Samples were all shot in positive ion mode with singly, doubly, and triply charged states observed.

#### **4. Conclusions**

Multiple RNase B samples were tested using a standard set of HSQC and HSQC-TOCSY pulse sequences with varying mixing times. The size of RNase B offers a protein with favorable T2 relaxation times when compared to even larger proteins and even more beneficial is the expected mobility of the N-glycan. The RNase B N-glycan was not observed in the crystal structure suggesting an unrestrained conformation which allowed us to exploit the relaxation differences between the protein and glycan. This difference in protein and carbohydrate relaxation times provides the opportunity to analyze the two components of the spectra independently on increasingly complex samples. We will use these experiments to fine-tune the conditions under which NMR spectra of polysaccharide conjugate vaccines can be better analyzed.

**Supplementary Materials:** The following are available online, Figure S1: Schematic of N-glycans using symbol nomenclature; Figure S2: Overlay of 1H-15N HSQC of RNase A and RNase B; Figure S3: Anomeric region of the 1H-13C HSQC of uniformly glycosylated RNase B Man5 and RNase B-Man9; Figure S4: Plots of the average T2 relaxation for proteins and glycans; Figure S5: Intensity regions used in Table 1 for the 1H-13C HSQC and the HSQC-TOCSY for RNase B Man5. Table S1: Linewidths and estimated T2 relaxation values for protein Cα's and glycan anomeric ring 1H-13C correlations.

**Author Contributions:** A.A.H.—ran experiments and contributed to manuscript writing; A.M.M. ran experiments and contributed to manuscript writing; H.Y.—ran experiments; C.L.—prepared glycosylated RNase B Man5 and RNase B-Man9; J.O.—prepared glycosylated RNase B Man5 and RNase B-Man9; M.D.B.—Trained and assisted in NMR data acquisition and processing; L.-X.W.—manuscript preparation, supervision and funding; D.I.F.—Project design and implementation, manuscript preparation, supervision and funding. All authors have read and agreed to the published version of the manuscript.

**Funding:** NIH Grant R01GM080374 to L.X.W.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

**Sample Availability:** Not applicable.
