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Enhancing Accuracy in Molecular Weight Determination of Highly Heterogeneously Glycosylated Proteins by Native Tandem Mass Spectrometry
[Image: see text] The determination of molecular weights (MWs) of heavily glycosylated proteins is seriously hampered by the physicochemical characteristics and heterogeneity of the attached carbohydrates. Glycosylation impacts protein migration during sodium dodecyl sulfate (SDS)-polyacrylamide gel...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American
Chemical
Society
2017
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5415875/ https://www.ncbi.nlm.nih.gov/pubmed/28383250 http://dx.doi.org/10.1021/acs.analchem.6b05129 |
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author | Wang, Guanbo de Jong, Rob N. van den Bremer, Ewald T. J. Parren, Paul W. H. I. Heck, Albert J. R. |
author_facet | Wang, Guanbo de Jong, Rob N. van den Bremer, Ewald T. J. Parren, Paul W. H. I. Heck, Albert J. R. |
author_sort | Wang, Guanbo |
collection | PubMed |
description | [Image: see text] The determination of molecular weights (MWs) of heavily glycosylated proteins is seriously hampered by the physicochemical characteristics and heterogeneity of the attached carbohydrates. Glycosylation impacts protein migration during sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and size-exclusion chromatography (SEC) analysis. Standard electrospray ionization (ESI)-mass spectrometry does not provide a direct solution as this approach is hindered by extensive interference of ion signals caused by closely spaced charge states of broadly distributed glycoforms. Here, we introduce a native tandem MS-based approach, enabling charge-state resolution and charge assignment of protein ions including those that escape mass analysis under standard MS conditions. Using this method, we determined the MW of two model glycoproteins, the extra-cellular domains of the highly and heterogeneously glycosylated proteins CD38 and epidermal growth factor receptor (EGFR), as well as the overall MW and binding stoichiometries of these proteins in complex with a specific antibody. |
format | Online Article Text |
id | pubmed-5415875 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | American
Chemical
Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-54158752017-05-05 Enhancing Accuracy in Molecular Weight Determination of Highly Heterogeneously Glycosylated Proteins by Native Tandem Mass Spectrometry Wang, Guanbo de Jong, Rob N. van den Bremer, Ewald T. J. Parren, Paul W. H. I. Heck, Albert J. R. Anal Chem [Image: see text] The determination of molecular weights (MWs) of heavily glycosylated proteins is seriously hampered by the physicochemical characteristics and heterogeneity of the attached carbohydrates. Glycosylation impacts protein migration during sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and size-exclusion chromatography (SEC) analysis. Standard electrospray ionization (ESI)-mass spectrometry does not provide a direct solution as this approach is hindered by extensive interference of ion signals caused by closely spaced charge states of broadly distributed glycoforms. Here, we introduce a native tandem MS-based approach, enabling charge-state resolution and charge assignment of protein ions including those that escape mass analysis under standard MS conditions. Using this method, we determined the MW of two model glycoproteins, the extra-cellular domains of the highly and heterogeneously glycosylated proteins CD38 and epidermal growth factor receptor (EGFR), as well as the overall MW and binding stoichiometries of these proteins in complex with a specific antibody. American Chemical Society 2017-04-06 2017-05-02 /pmc/articles/PMC5415875/ /pubmed/28383250 http://dx.doi.org/10.1021/acs.analchem.6b05129 Text en Copyright © 2017 American Chemical Society This is an open access article published under a Creative Commons Non-Commercial No Derivative Works (CC-BY-NC-ND) Attribution License (http://pubs.acs.org/page/policy/authorchoice_ccbyncnd_termsofuse.html) , which permits copying and redistribution of the article, and creation of adaptations, all for non-commercial purposes. |
spellingShingle | Wang, Guanbo de Jong, Rob N. van den Bremer, Ewald T. J. Parren, Paul W. H. I. Heck, Albert J. R. Enhancing Accuracy in Molecular Weight Determination of Highly Heterogeneously Glycosylated Proteins by Native Tandem Mass Spectrometry |
title | Enhancing Accuracy in Molecular Weight Determination
of Highly Heterogeneously Glycosylated Proteins by Native Tandem Mass
Spectrometry |
title_full | Enhancing Accuracy in Molecular Weight Determination
of Highly Heterogeneously Glycosylated Proteins by Native Tandem Mass
Spectrometry |
title_fullStr | Enhancing Accuracy in Molecular Weight Determination
of Highly Heterogeneously Glycosylated Proteins by Native Tandem Mass
Spectrometry |
title_full_unstemmed | Enhancing Accuracy in Molecular Weight Determination
of Highly Heterogeneously Glycosylated Proteins by Native Tandem Mass
Spectrometry |
title_short | Enhancing Accuracy in Molecular Weight Determination
of Highly Heterogeneously Glycosylated Proteins by Native Tandem Mass
Spectrometry |
title_sort | enhancing accuracy in molecular weight determination
of highly heterogeneously glycosylated proteins by native tandem mass
spectrometry |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5415875/ https://www.ncbi.nlm.nih.gov/pubmed/28383250 http://dx.doi.org/10.1021/acs.analchem.6b05129 |
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