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In-House Packed Porous Graphitic Carbon Columns for Liquid Chromatography-Mass Spectrometry Analysis of N-Glycans
Protein glycosylation is a common post-translational modification that modulates biological processes such as the immune response and protein trafficking. Altered glycosylation profiles are associated with cancer and inflammatory diseases, as well as impacting the efficacy of therapeutic monoclonal...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8226321/ https://www.ncbi.nlm.nih.gov/pubmed/34178940 http://dx.doi.org/10.3389/fchem.2021.653959 |
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author | Young, Clifford Condina, Mark R. Briggs, Matthew T. Moh, Edward S. X. Kaur, Gurjeet Oehler, Martin K. Hoffmann, Peter |
author_facet | Young, Clifford Condina, Mark R. Briggs, Matthew T. Moh, Edward S. X. Kaur, Gurjeet Oehler, Martin K. Hoffmann, Peter |
author_sort | Young, Clifford |
collection | PubMed |
description | Protein glycosylation is a common post-translational modification that modulates biological processes such as the immune response and protein trafficking. Altered glycosylation profiles are associated with cancer and inflammatory diseases, as well as impacting the efficacy of therapeutic monoclonal antibodies. Consisting of oligosaccharides attached to asparagine residues, enzymatically released N-linked glycans are analytically challenging due to the diversity of isomeric structures that exist. A commonly used technique for quantitative N-glycan analysis is liquid chromatography-mass spectrometry (LC-MS), which performs glycan separation and characterization. Although many reversed and normal stationary phases have been utilized for the separation of N-glycans, porous graphitic carbon (PGC) chromatography has become desirable because of its higher resolving capability, but is difficult to implement in a robust and reproducible manner. Herein, we demonstrate the analytical properties of a 15 cm fused silica capillary (75 µm i.d., 360 µm o.d.) packed in-house with Hypercarb PGC (3 µm) coupled to an Agilent 6550 Q-TOF mass spectrometer for N-glycan analysis in positive ion mode. In repeatability and intermediate precision measurements conducted on released N-glycans from a glycoprotein standard mixture, the majority of N-glycans reported low coefficients of variation with respect to retention times (≤4.2%) and peak areas (≤14.4%). N-glycans released from complex samples were also examined by PGC LC-MS. A total of 120 N-glycan structural and compositional isomers were obtained from formalin-fixed paraffin-embedded ovarian cancer tissue sections. Finally, a comparison between early- and late-stage formalin-fixed paraffin-embedded ovarian cancer tissues revealed qualitative changes in the α2,3- and α2,6-sialic acid linkage of a fucosylated bi-antennary complex N-glycan. Although the α2,3-linkage was predominant in late-stage ovarian cancer, the alternate α2,6-linkage was more prevalent in early-stage ovarian cancer. This study establishes the utility of in-house packed PGC columns for the robust and reproducible LC-MS analysis of N-glycans. |
format | Online Article Text |
id | pubmed-8226321 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-82263212021-06-26 In-House Packed Porous Graphitic Carbon Columns for Liquid Chromatography-Mass Spectrometry Analysis of N-Glycans Young, Clifford Condina, Mark R. Briggs, Matthew T. Moh, Edward S. X. Kaur, Gurjeet Oehler, Martin K. Hoffmann, Peter Front Chem Chemistry Protein glycosylation is a common post-translational modification that modulates biological processes such as the immune response and protein trafficking. Altered glycosylation profiles are associated with cancer and inflammatory diseases, as well as impacting the efficacy of therapeutic monoclonal antibodies. Consisting of oligosaccharides attached to asparagine residues, enzymatically released N-linked glycans are analytically challenging due to the diversity of isomeric structures that exist. A commonly used technique for quantitative N-glycan analysis is liquid chromatography-mass spectrometry (LC-MS), which performs glycan separation and characterization. Although many reversed and normal stationary phases have been utilized for the separation of N-glycans, porous graphitic carbon (PGC) chromatography has become desirable because of its higher resolving capability, but is difficult to implement in a robust and reproducible manner. Herein, we demonstrate the analytical properties of a 15 cm fused silica capillary (75 µm i.d., 360 µm o.d.) packed in-house with Hypercarb PGC (3 µm) coupled to an Agilent 6550 Q-TOF mass spectrometer for N-glycan analysis in positive ion mode. In repeatability and intermediate precision measurements conducted on released N-glycans from a glycoprotein standard mixture, the majority of N-glycans reported low coefficients of variation with respect to retention times (≤4.2%) and peak areas (≤14.4%). N-glycans released from complex samples were also examined by PGC LC-MS. A total of 120 N-glycan structural and compositional isomers were obtained from formalin-fixed paraffin-embedded ovarian cancer tissue sections. Finally, a comparison between early- and late-stage formalin-fixed paraffin-embedded ovarian cancer tissues revealed qualitative changes in the α2,3- and α2,6-sialic acid linkage of a fucosylated bi-antennary complex N-glycan. Although the α2,3-linkage was predominant in late-stage ovarian cancer, the alternate α2,6-linkage was more prevalent in early-stage ovarian cancer. This study establishes the utility of in-house packed PGC columns for the robust and reproducible LC-MS analysis of N-glycans. Frontiers Media S.A. 2021-06-11 /pmc/articles/PMC8226321/ /pubmed/34178940 http://dx.doi.org/10.3389/fchem.2021.653959 Text en Copyright © 2021 Young, Condina, Briggs, Moh, Kaur, Oehler and Hoffmann. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Chemistry Young, Clifford Condina, Mark R. Briggs, Matthew T. Moh, Edward S. X. Kaur, Gurjeet Oehler, Martin K. Hoffmann, Peter In-House Packed Porous Graphitic Carbon Columns for Liquid Chromatography-Mass Spectrometry Analysis of N-Glycans |
title | In-House Packed Porous Graphitic Carbon Columns for Liquid Chromatography-Mass Spectrometry Analysis of N-Glycans |
title_full | In-House Packed Porous Graphitic Carbon Columns for Liquid Chromatography-Mass Spectrometry Analysis of N-Glycans |
title_fullStr | In-House Packed Porous Graphitic Carbon Columns for Liquid Chromatography-Mass Spectrometry Analysis of N-Glycans |
title_full_unstemmed | In-House Packed Porous Graphitic Carbon Columns for Liquid Chromatography-Mass Spectrometry Analysis of N-Glycans |
title_short | In-House Packed Porous Graphitic Carbon Columns for Liquid Chromatography-Mass Spectrometry Analysis of N-Glycans |
title_sort | in-house packed porous graphitic carbon columns for liquid chromatography-mass spectrometry analysis of n-glycans |
topic | Chemistry |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8226321/ https://www.ncbi.nlm.nih.gov/pubmed/34178940 http://dx.doi.org/10.3389/fchem.2021.653959 |
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