Comparison of N-glycopeptide to released N-glycan abundances and the influence of glycopeptide mass and charge state on N-linked glycosylation of IgG antibodies

Published

March 7, 2024

Author(s)

Concepcion Remoroza, Meghan Burke Harris, Tytus Mak, Sergey Sheetlin, Yuri Mirokhin, Zachary Goecker, Brian T. Cooper, Mark Lowenthal, Xiaoyu (Sara) Yang, Guanghui Wang, Dmitrii V. Tchekhovskoi, Stephen E. Stein

Abstract

We report the comparison of mass-spectral-based abundances of tryptic glycopeptides to fluorescence abundances of released labeled glycans and the effects of mass and charge state and in-source fragmentation on glycopeptide abundances. The primary glycoforms derived from Rituximab, NISTmAb, Evolocumab, and Infliximab were high-mannose and biantennary complex galactosylated and fucosylated N-glycans. Except for Evolocumab, in-source ions derived from the loss of HexNAc or HexNAc-Hex sugars are prominent for other therapeutic IgGs. After excluding in-source fragmentation of glycopeptide ions from the results, a linear correlation was observed between fluorescently labeled N-glycan and glycopeptide abundance over a dynamic range 500. Different charge states of human IgG-derived glycopeptides containing a wider variety of abundant attached glycans were also investigated to examine the effects of charge state on ion abundances. These revealed a linear dependence of glycopeptide abundance on the mass of the glycan, with higher charge states favoring higher-mass glycans. Findings indicate that the mass-spectrometry-based bottom-up approach can provide results as accurate as those of glycan release studies while revealing the origin of each attached glycan. These site-specific relative abundances are conveniently displayed and compared using previously described Glycopeptide Abundance Distribution Spectra 'GADS' representations. Mass spectrometry data are available at the MAssIVE repository (MSV000093562).

Citation

ACS Journal of Proteome Research

Pub Type

Journals

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https://doi.org/10.1021/acs.jproteome.3c00904

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Keywords

Therapeutic monoclonal antibodies, in-source fragments, charge-state effects, Mass spectrometry, fluorescently labeled N-glycans, HILIC-FLD timsTOF, N-glycopeptides/glycoproteomics

Citation

Remoroza, C. , Burke Harris, M. , Mak, T. , Sheetlin, S. , Mirokhin, Y. , Goecker, Z. , Cooper, B. , Lowenthal, M. , Yang, X. , Wang, G. , Tchekhovskoi, D. and Stein, S. (2024), Comparison of N-glycopeptide to released N-glycan abundances and the influence of glycopeptide mass and charge state on N-linked glycosylation of IgG antibodies, ACS Journal of Proteome Research, [online], https://doi.org/10.1021/acs.jproteome.3c00904, https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=956442 (Accessed October 14, 2025)