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Filter-Aided N-Glycan Separation (FANGS): A Convenient Sample Preparation Method for Mass Spectrometric N-Glycan Profiling
[Image: see text] We have developed a simple method for the release and isolation of glycoprotein N-glycans from whole-cell lysates using less than a million cells, for subsequent implementation with mass spectrometric analysis. Cellular protein extracts prepared using SDS solubilization were sequen...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Chemical
Society
2014
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3971760/ https://www.ncbi.nlm.nih.gov/pubmed/24450425 http://dx.doi.org/10.1021/pr401043r |
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author | Abdul Rahman, Salina Bergström, Ed Watson, Christopher J. Wilson, Katherine M. Ashford, David A. Thomas, Jerry R. Ungar, Daniel Thomas-Oates, Jane E. |
author_facet | Abdul Rahman, Salina Bergström, Ed Watson, Christopher J. Wilson, Katherine M. Ashford, David A. Thomas, Jerry R. Ungar, Daniel Thomas-Oates, Jane E. |
author_sort | Abdul Rahman, Salina |
collection | PubMed |
description | [Image: see text] We have developed a simple method for the release and isolation of glycoprotein N-glycans from whole-cell lysates using less than a million cells, for subsequent implementation with mass spectrometric analysis. Cellular protein extracts prepared using SDS solubilization were sequentially treated in a membrane filter device to ultimately release glycans enzymatically using PNGase F in the volatile buffer ammonium bicarbonate. The released glycans are recovered in the filtrate following centrifugation and typically permethylated prior to mass spectrometric analysis. We call our method “filter-aided N-glycan separation” and have successfully applied it to investigate N-glycan profiles of wild-type and mutant Chinese hamster ovary cells. This method is readily multiplexed and, because of the small numbers of cells needed, is compatible with the analysis of replicate samples to assess the true nature of glycan variability in tissue culture samples. |
format | Online Article Text |
id | pubmed-3971760 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | American Chemical
Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-39717602014-04-01 Filter-Aided N-Glycan Separation (FANGS): A Convenient Sample Preparation Method for Mass Spectrometric N-Glycan Profiling Abdul Rahman, Salina Bergström, Ed Watson, Christopher J. Wilson, Katherine M. Ashford, David A. Thomas, Jerry R. Ungar, Daniel Thomas-Oates, Jane E. J Proteome Res [Image: see text] We have developed a simple method for the release and isolation of glycoprotein N-glycans from whole-cell lysates using less than a million cells, for subsequent implementation with mass spectrometric analysis. Cellular protein extracts prepared using SDS solubilization were sequentially treated in a membrane filter device to ultimately release glycans enzymatically using PNGase F in the volatile buffer ammonium bicarbonate. The released glycans are recovered in the filtrate following centrifugation and typically permethylated prior to mass spectrometric analysis. We call our method “filter-aided N-glycan separation” and have successfully applied it to investigate N-glycan profiles of wild-type and mutant Chinese hamster ovary cells. This method is readily multiplexed and, because of the small numbers of cells needed, is compatible with the analysis of replicate samples to assess the true nature of glycan variability in tissue culture samples. American Chemical Society 2014-01-22 2014-03-07 /pmc/articles/PMC3971760/ /pubmed/24450425 http://dx.doi.org/10.1021/pr401043r Text en Copyright © 2014 American Chemical Society Terms of Use CC-BY (http://pubs.acs.org/page/policy/authorchoice_ccby_termsofuse.html) |
spellingShingle | Abdul Rahman, Salina Bergström, Ed Watson, Christopher J. Wilson, Katherine M. Ashford, David A. Thomas, Jerry R. Ungar, Daniel Thomas-Oates, Jane E. Filter-Aided N-Glycan Separation (FANGS): A Convenient Sample Preparation Method for Mass Spectrometric N-Glycan Profiling |
title | Filter-Aided N-Glycan Separation
(FANGS): A Convenient Sample Preparation Method for Mass Spectrometric N-Glycan Profiling |
title_full | Filter-Aided N-Glycan Separation
(FANGS): A Convenient Sample Preparation Method for Mass Spectrometric N-Glycan Profiling |
title_fullStr | Filter-Aided N-Glycan Separation
(FANGS): A Convenient Sample Preparation Method for Mass Spectrometric N-Glycan Profiling |
title_full_unstemmed | Filter-Aided N-Glycan Separation
(FANGS): A Convenient Sample Preparation Method for Mass Spectrometric N-Glycan Profiling |
title_short | Filter-Aided N-Glycan Separation
(FANGS): A Convenient Sample Preparation Method for Mass Spectrometric N-Glycan Profiling |
title_sort | filter-aided n-glycan separation
(fangs): a convenient sample preparation method for mass spectrometric n-glycan profiling |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3971760/ https://www.ncbi.nlm.nih.gov/pubmed/24450425 http://dx.doi.org/10.1021/pr401043r |
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