*2.2. 1H-13C HSQC of RNase B Man5 and RNase B Man9*

In contrast to natural abundance 1H-15N HSQC, natural abundance 1H-13C HSQC spectra are higher in sensitivity and can provide substantially more information regarding protein glycosylation. The larger number of 13C atoms in an amino acid than 15N atoms increases spectral complexity; nevertheless, the uniqueness of 13C chemical shift ranges and NMR experiments can be used to differentiate between protein and glycan subspectra.

Glycan anomeric 1H-13C correlations occur in a unique spectral region which does not overlap with most protein signals [24,34], since both nuclei are typically deshielded in 1H (4.3–5.8 ppm) and 13C (98–106 ppm). In the case of pure glycans or single glycoforms, the number of anomeric peaks can be used to determine the number of saccharide residues in a given glycan. 1H-13C HSQC spectra were taken of two engineered RNase B glycoproteins (Figures 1 and 2), each uniformly glycosylated at N34 with either Man5GlcNAc2 (Man5) or Man9GlcNAc2 (Man9). Intact electrospray ionization mass spectrometry (ESI-MS) showed that each glycoprotein contained one predominant mass after charge state deconvolution (14898 Da, RNase B Man5; 15546 Da, RNase B Man9, Figure 3). Figure S1 provides schematics of different types of glycan and their linkages. For RNase B Man5, seven anomeric peaks were unambiguously observed (Figure S3a). GlcNAc2, Man3, Man4, Man4 , ManA, and ManB have 13C chemical shifts between 100 and 144 ppm and 1H chemical shifts between 4.6 and 5.2 ppm. The GlcNAc1 anomeric peak is shifted significantly, in 13C, to 78.4 ppm as it is amide linked to the protein [24]. Notably, there are no overlapping protein chemical shifts in this region.

**Figure 1.** Comparison of 1H-13C HSQC and HSQC-TOCSY spectra of RNase B Man5 and Man9. (**a**) HSQC spectrum of 0.3 mM RNase B Man5 contains peaks for both protein (black circles) and glycans (red circles). RNase B Man5 anomeric protons (upper red circle) are folded in the 13C dimension and range from 98–103 ppm. (**b**) An HSQC-TOCSY with a 90 ms mixing time of RNase B Man5 takes advantage of the longer glycan T2, so that the glycans peaks are retained while the protein peaks are greatly reduced. Similarly, an HSQC of 0.6 mM RNase B Man9 (**c**) shows peaks for both protein and glycan, while in the HSQC-TOCSY experiment (**d**) mostly glycan peaks are retained.

**Figure 2.** Overlay of a 2D 1H-13C HSQC (blue) and 1H-13C HSQC-TOCSY (red) of 0.3 mM RNase B Man5 at pH 6, 37 oC (blue) and 1H-13C HSQC-TOCSY (red) (**a**). Chemical shifts of 13C anomeric signals are folded and range from 98 to 104 ppm, except GlcNAc1 which is more shielded with a 13C chemical shift of 78 ppm. (**b**) Glycan ring signals with lines drawn to show the 90 ms TOCSY correlations for each of the monosaccharides except GlcNac1 (GN1) and GlcNAc2 (GN2).

In a 1H-13C HSQC of RNase B Man9, anomeric correlations are observed in a similar spectral region as RNase B Man5 (Figure S3b). However, because RNase B Man9 contains additional Man residues D1-D3, with α1-2 linkages, their signals overlap and were resolved with Lorentz-to-Gauss processing for line narrowing [35]. RNase B Man9 provides an additional challenge because the NMR signals for the C2-C6 positions on each glycan significantly overlap. Although the spectra were collected at 34 Hz/pt 13C resolution, it is still insufficient to resolve the individual signals within the ring. Nevertheless, the unique fingerprint in the anomeric region is the ideal method for distinguishing glycoforms. The most evident signals are those belonging to ManA (+D3), ManC (+D1), ManB (+D2) and Man4 (+C). These three signals are unique to Man9, as Man5 does not contain a 'C' residue and ManA and Man4 are more deshielded in the 1H dimension when linked to the D mannoses. A similar strategy was used to characterize the glycoprofile of FcεRiα [36]. These researchers were able to assess the different glycoforms using HSQC spectra of uniformly 15N/13C-labeled glycoproteins under both folded and denatured sample conditions at lower concentrations than we report. They also report assessing the relative abundances of

each glycoform using the anomeric region only. Thus, both their methods and those we report here can be used to assess glycoforms.

**Figure 3.** Deconvoluted ESI-MS spectra of (**a**) RNase B Man5, (**b**) RNase B Man9, (**c**) vendor 1 RNase B, and (**d**) vendor 2 RNase B. RNase B Man5 and RNase B Man9 are singly glycosylated. Both vendor 1 and vendor 2 contain a population of high-mannose glycan with predominantly GlcNAc2Man5. The vendor 2 sample did have a significant population of RNase A that was not present in Vendor 1's sample.

#### *2.3. Relaxation Selection for Glycan Regions of Spectrum*

To enrich glycan regions of the spectrum for peak assignment and reduce ambiguity observed in ring regions of the spectrum, 1H-13C HSQC-TOCSY experiments were used [30,31]. TOCSY mixing times were optimized by a simple linewidth analysis. Data collected at a sufficient resolution to obtain reliable linewidths in both 1H and 13C dimensions can be used to estimate the upper limits of the T2s, using the relation (*T*<sup>2</sup> ≈ *π* ∗ Δ*ν* <sup>1</sup> 2 −<sup>1</sup> , where Δν<sup>1</sup> 2 is the linewidth at half height [37]. Table S1 shows a list of peaks corresponding mostly to either the ring or anomeric region of the N-glycan or 1H, 13Cα peaks from the protein. Based on linewidths, the range of 13C transverse relaxation times for the protein specific regions is 9–13 ms with an average of 11.6 ms, whereas in the glycan regions the T2 range is 12 to 17 ms and an average of 14.4 ms. This yields an approximate difference in relaxation time of 25% between the glycan and protein components, limiting the amount of relaxation effect to exploit. In contrast to the 13C relaxation times, 1H relaxation times displayed a greater disparity between the protein and glycan resonances. The proteinspecific relaxation times in 1H were between 10 and 35 ms, with an average of 16.4 ms. The glycan relaxation times in 1H ranged from 14 to 45 ms and averaged 29 ms. This provides a nearly twofold (80%) difference in relaxation times which is easier and more effective to exploit. The average T2 determined from this analysis was then used to plot transverse magnetization loss over time (Figure S4). This allows for quantitatively selecting mixing times to maximize the intensity difference between the protein and glycan peaks. Because

the relaxation rate difference is nearly twofold, it allows most of the protein signals to relax while maintaining enough glycan signal so as not to increase experiment time.

Signal-to-noise ratios (SNR) in protein dominant regions (2a/2b) and glycan dominant regions (1a/1b) were assessed in an HSQC and HSQC-TOCSY of RNase B Man5 (Table 1, Figure S5). The glycan regions maintain 43.3% of their signal intensity in the HSQC-TOCSY (90 ms mixing time), compared to the HSQC, where in the protein regions only an average of 11.8% of the initial intensity remains. This 3.7-fold difference agrees with the estimated signal loss calculated using the relaxation times (3.2-fold) and significantly simplifies the spectra while also providing the benefit of intra-ring correlations of coupled 1Hs through the TOCSY (Figures 1 and 2). Interestingly, signal loss is observed for glycan residues GlcNAc1 and GlcNac2 which are spatially close to the protein and have a T2 closer to that of the protein Cα. Other NMR experiments such as the HSQC-ROESY have a similar effect on protein signal attenuation.

**Table 1.** Peak volume between glycan dominant (1a/1b) and protein dominant (2a/2b) spectral regions.


*2.4. Analysis of Commercial RNase B Samples*

Using the uniformly glycosylated RNase glycoproteins as references, two commercially available RNase B samples were evaluated. RNase B from vendor 1 was reported to be 80% pure, and RNase B from vendor 2 was reported to be 50% pure. All RNase B samples were analyzed for glycosylation heterogeneity and purity using ESI-MS. Mass spectra of intact RNase B were collected and a charge envelope consisting of +8 to +15 charged ions were observed for each of the samples. The charge state envelope was deconvoluted [38] using the Waters MassLynx MS software and the glycosylation pattern was determined for each of the RNase B samples (Figure 3). The commercial RNase B from vendor 1 contained predominantly GlcNAc2Man5 at N34 (exp = 14,898 Da, calc = 14,897 Da), with a small percentage of GlcNAc2Man6-9. Similarly, commercial RNase B from vendor 2 was mostly glycosylated with GlcNAc2Man5; however, this sample also contained RNase A (exp = 13,682 Da, calc = 13,681 Da). To have similar amounts of RNase B for the NMR analysis in both vendors, the percent of RNase A was accounted for when determining RNase B sample concentration for vendor 2. Overall, the distribution of N-glycans in RNase B is similar between the two manufacturers which should lead to nearly identical samples in the NMR experiments.

Initial 1H-13C HSQC analysis of the commercial RNase B revealed a contaminating peak present in vendor 1's sample (Figure S6, blue) that was not observed in the ESI-MS analysis (data not shown). It is possible that this glycoside-like molecule is a methyl mannoside that was either not completely removed after lectin affinity chromatography or was used to stabilize RNase B. Due to similarities in chemical shift between this contaminant and the RNase B glycan and its high SNR in the HSQC-TOCSY, vendor 1 RNase B was dialyzed using a 1 kDa MWCO membrane to remove the contaminant (Figure S6, red). After the dialysis, there were only minor differences between the vendor RNase B samples (Figure 4). Specifically, RNase B from vendor 2 contained peaks from 2.5–3.0 ppm 1H and 30–40 ppm 13C that are not present in vendor 1 s RNase B. The chemical shifts that correspond to the glycan anomeric (4.5–5.5 ppm 1H and 95–105 ppm 13C) and ring regions (3.3–4.3 ppm 1H and 60–80 ppm 13C) are the same in both vendor RNase B spectra.

**Figure 4.** 2D 1H-13C HSQC of (**a**) 1.4 mM vendor 1 RNase B and (**b**) 1.0 mM vendor 2 RNase B. Glycan anomeric region and ring region are the same between the two vendors; however, the vendor 2 spectrum contains additional peaks from 2.5 to 3.0 ppm in 1H and 30 to 40 ppm in 13C that are not present in the RNase B vendor 1 spectrum. (**c**) Vendor 1 glycan anomeric signals used for quantitative analysis (inset: schematic of Man9 glycan).

The 1H-13C HSQC and 1H-13C HSQC-TOCSY spectra from the vendor samples, which contained a heterogenous population of glycans (Man5-9GlcNAc2), was compared to the uniformly glycosylated reference RNase B spectra. Figure 4 shows the anomeric region of the commercially available RNase B from vendor 1 with transferred assignments from literature values [33]. In addition to the peaks observed in the RNase B Man5 reference spectrum, there are additional peaks corresponding to Man 4, A, B, and C as each of these positions can be further modified by an α1-2 linked mannose residue. Man D1, D2, and D3 chemical shifts overlap Man A, C, and 4 and cannot be assigned at the current spectral resolution. Thus, the ratios of the entire glycan population cannot be qualitatively

estimated using these signals. Nevertheless, the SNR of some of the glycan anomeric signals can be used for quantification, as we show below.

#### *2.5. Quantitative Analysis of Commercial RNase B Glycoforms*

To normalize the results for quantitative analysis, all experiments performed were collected on a 700 MHz (1H) magnet equipped with cryoprobe, which provided increased sensitivity. This is especially useful in experiments carried out at natural abundance, as performed in the present study. One HSQC was run with nearly identical experimental conditions for the three samples analyzed (RNase B Man5, RNase B Man9, and commercial RNase B (vendor 1)), protein concentrations were between 18–22 mg/mL. The temperature was set to 25 ◦C for Man5 and commercial RNase B and 37 ◦C for Man9 RNase B. In all cases, the lowest SNR was observed for GlcNAc1 and GlcNAc2 anomeric signals. The SNRs of GlcNAc1 were standardized to account for differences in protein concentration between samples, as all experiments were performed with the same number of scans and t1 points. RNase B Man9 GlcNAc1 had a SNR of 19:1, RNase B Man5 GlcNAc1 had a s/n of 16:1 and commercial RNase B 16:1.

Quantitative ratios of each glycoform present in the commercial RNase B are difficult to obtain due to differential T2 relaxation. For example, Man C and Man 3 cannot be compared, since Man 3 is far more restricted and would be expected to have more efficient relaxation, leading to a decrease in peak intensity that would reduce accuracy in assessing relative abundance of glycoforms. Therefore, to better estimate the relative glycoforms abundance, only residues with similar T2s can be compared such as Man B and Man C. The ratio of Man B: Man C correlates to glycoforms GlcNAc2Man5 and GlcNAc2Man6. This ratio was determined to be 1.8:1 by peak height comparison, which is in line with the reported estimate of 1.84:1 [28]. Another ratio that should be close to 1:1 is that of ManC (+D1):ManB (+D2) as there is more GlcNAc2Man8 than GlcNAc2Man7 according to the MS analysis leading to equal amounts of the two terminal mannose residues present in GlcNAc2Man8. In this sample the ratio was 1.04:1 in line with the expectation.
