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

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.

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Publication Details

Journal
Cell Reports Methods
Published
2024-08-01
DOI
https://doi.org/10.1016/j.crmeth.2024.100834
Citations
5
Primary Topic
Glycosylation and Glycoproteins Research
Type
article
Field-Weighted Citation Impact
1.17

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article

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

Wells W. Wu, Vincent M. Falkowski, Uriel Ortega‐Rodriguez, Cyrus Agarabi et al.
5 citations
Cell Reports Methods
Glycosylation and Glycoproteins Research
1.17
article

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

Wells W. Wu, Vincent M. Falkowski, Uriel Ortega‐Rodriguez, Cyrus Agarabi, Jordan D. Pritts, John Q. Bettinger, Alicia M. Matthews, Tongzhong Ju, V. Ashutosh Rao, Mari Lehtimaki, Hang Xie, Thomas Biel, Rong‐Fong Shen, Guozhang Zou
article en
5 citations

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.

Cell Reports MethodsVol. 4(8)
Center for Biologics Evaluation and Research (US), United States Food and Drug Administration (US), Center for Drug Evaluation and Research (US)
U.S. Department of Health and Human Services, Oak Ridge Institute for Science and Education, Office of Women's Health
Openalex Percentile: Top 22%
Glycosylation and Glycoproteins Research
1.17
DOIOpen Access PDFOpenAlex
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