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. 2024 Aug 19;4(8):100834.
doi: 10.1016/j.crmeth.2024.100834. Epub 2024 Aug 7.

A chemoenzymatic method for simultaneous profiling N- and O-glycans on glycoproteins using one-pot format

Uriel Ortega-Rodriguez  1 , John Q Bettinger  1 , Guozhang Zou  1 , Vincent M Falkowski  1 , Mari Lehtimaki  1 , Alicia M Matthews  1 , Thomas G Biel  1 , Jordan D Pritts  1 , Wells W Wu  2 , Rong-Fong Shen  2 , Cyrus Agarabi  1 , V Ashutosh Rao  1 , Hang Xie  3 , Tongzhong Ju  4
Affiliations

A chemoenzymatic method for simultaneous profiling N- and O-glycans on glycoproteins using one-pot format

Uriel Ortega-Rodriguez et al. Cell Rep Methods. .

Abstract

Glycosylation is generally characterized and controlled as a critical quality attribute for therapeutic glycoproteins because glycans can impact protein drug-product efficacy, half-life, stability, and safety. Analytical procedures to characterize N-glycans are relatively well established, but the characterization of O-glycans is challenging due to the complex workflows and lack of enzymatic tools. Here, we present a simplified chemoenzymatic method to simultaneously profile N- and O-glycans from the same sample using a one-pot format by mass spectrometry (MS). N-glycans were first released by PNGase F, followed by O-glycopeptide generation by proteinase K, selective N-glycan reduction, and O-glycan release by β-elimination during permethylation of both N- and O-glycans. Glycan structural assignments and determination of N- to O-glycan ratio was obtained from the one-pot mass spectra. The streamlined, one-pot method is a reliable approach that will facilitate advanced characterizations for quality assessments of therapeutic glycoproteins.

Keywords: CP: biotechnology; N-glycans; O-glycans; biotechnology; glycan analysis; glycomics; mass spectrometry; protein drugs.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Qualification of permethylated glycan standards on MALDI-TOF/MS and 2-AB UHPLC-FLR (A) Representative MALDI-TOF spectra of permethylated glycan standards. (B) Representative UHPLC-FLR chromatogram of 2-AB-labeled glycan standards. (C) Comparison of glycan abundances (mean ± SD) determined by MALDI-TOF (blue) or 2-AB UHPLC-FLR (red) to theoretical ratios of mixed glycans (green). (D) Distribution of SD of glycan standards determined by MALDI-TOF (blue) or 2-AB UHPLC-FLR (red); a t test was performed in GraphPad Prism to compare the SD values for glycan abundances. (E) Correlation between theoretical relative abundance (x axis) and measured glycan abundances (y axis) for MALDI-TOF (red) and 2-AB UHPLC-FLR (blue). Data are represented as mean ± SD.
Figure 2
Figure 2
Optimization of the one-pot method for simultaneous analysis of N- and O-glycans of glycoproteins (A) Workflow of one-pot method. (B) Differentiation of permethylated N- and O-glycans by unique reducing ends. (C) One-pot N- and O-glycan profiling of a model glycoprotein fetuin. (D) Summary of glycans identified in six reactions of fetuin plotted by % abundance (mean ± SD). (E) N- to O-glycan ratio of fetuin (top right) and abundance of the total sialylated N- and O-glycan species of fetuin (bottom right). Data are represented as mean ± SD.
Figure 3
Figure 3
Analysis of Fc-fusion glycoproteins by one-pot glycomic method and test of reproducibility with etanercept (A) MALDI-TOF/MS spectra of N- and O-glycans of Fc-fusion proteins: abatacept, etanercept, and aflibercept (n = 3). (B) Summary of unique glycan abundance found in abatacept, etanercept, and aflibercept followed by the N- and O-glycan ratio of each glycoprotein and total % sialylation of N- and O-glycans combined. (C) Summary of the reproducibility of one-pot method using the data from 22 reactions of etanercept lot 1 with the abundance of glycan species identified in etanercept. (D) N- and O-glycan ratio. (E) Summary of abundance of total sialylated and asialylated glycan species; abundance of sialylated N- and O-glycan species is shown in the upper panel. Other glycan determinants include N-glycan fucosylation, total sialylation, and abundance of mannose receptor (MR) and asialoglycoprotein receptor (ASGPR) target glycans, summarized in the lower panel. Data are represented as mean ± SD.
Figure 4
Figure 4
Lot-to-lot comparability with three lots of etanercept (A) Comparison of relative abundance of glycan species detected in three lots of etanercept. (B) Lot-to-lot comparison of etanercept in N- and O-glycan ratio, total and N- and O-glycan specific sialylation and fucosylation status of N-glycans, and abundance of MR and ASGPR target glycans. Data are represented as mean ± SD. Non-parametric analyses; Kruskal-Wallis and Mann-Whitney tests were performed at alpha level of 0.05 to study glycosylation differences between three lots; ∗p < 0.05.
Figure 5
Figure 5
Intermediate precision of one-pot glycomic method performed by two analysts (A) MALDI-TOF/MS scans of 2,5-DHB matrix alone, followed by N- and O-glycan profiles of three preparations of etanercept by analyst 1 (A1) and analyst 2 (A2), respectively. (B) Comparison of N- and O-glycan ratio, sialylation, and fucosylation of glycans measured in etanercept by two analysts demonstrate the intermediate precision of one-pot glycan method. Data are represented as mean ± SD. Variability between two analysts was assessed using non-parametric Mann-Whitney test ∗p < 0.05.
Figure 6
Figure 6
Simultaneous N- and O-glycan profiling of cellular glycoproteins N- and O-glycans from CHO S cell line were prepared with optimized one-pot method and analyzed on MALDI-TOF/MS (n = 6). The representative spectra are shown.
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