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Mapping Yeast N-Glycosites with Isotopically Recoded Glycans

Asparagine-linked glycosylation is a common post-translational modification of proteins; in addition to participating in key macromolecular interactions, N-glycans contribute to protein folding, trafficking, and stability. Despite their importance, few N-glycosites have been experimentally mapped in...

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Autores principales: Breidenbach, Mark A., Palaniappan, Krishnan K., Pitcher, Austin A., Bertozzi, Carolyn R.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The American Society for Biochemistry and Molecular Biology 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3433913/
https://www.ncbi.nlm.nih.gov/pubmed/22261724
http://dx.doi.org/10.1074/mcp.M111.015339
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author Breidenbach, Mark A.
Palaniappan, Krishnan K.
Pitcher, Austin A.
Bertozzi, Carolyn R.
author_facet Breidenbach, Mark A.
Palaniappan, Krishnan K.
Pitcher, Austin A.
Bertozzi, Carolyn R.
author_sort Breidenbach, Mark A.
collection PubMed
description Asparagine-linked glycosylation is a common post-translational modification of proteins; in addition to participating in key macromolecular interactions, N-glycans contribute to protein folding, trafficking, and stability. Despite their importance, few N-glycosites have been experimentally mapped in the Saccharomyces cerevisiae proteome. Factors including glycan heterogeneity, low abundance, and low occupancy can complicate site mapping. Here, we report a novel mass spectrometry-based strategy for detection of N-glycosites in the yeast proteome. Our method imparts N-glycopeptide mass envelopes with a pattern that is computationally distinguishable from background ions. Isotopic recoding is achieved via metabolic incorporation of a defined mixture of N-acetylglucosamine isotopologs into N-glycans. Peptides bearing the recoded envelopes are specifically targeted for fragmentation, facilitating high confidence site mapping. This strategy requires no chemical modification of the N-glycans or stringent sample enrichment. Further, enzymatically simplified N-glycans are preserved on peptides. Using this approach, we identify 133 N-glycosites spanning 58 proteins, nearly doubling the number of experimentally observed N-glycosites in the yeast proteome.
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spelling pubmed-34339132012-09-11 Mapping Yeast N-Glycosites with Isotopically Recoded Glycans Breidenbach, Mark A. Palaniappan, Krishnan K. Pitcher, Austin A. Bertozzi, Carolyn R. Mol Cell Proteomics Research Asparagine-linked glycosylation is a common post-translational modification of proteins; in addition to participating in key macromolecular interactions, N-glycans contribute to protein folding, trafficking, and stability. Despite their importance, few N-glycosites have been experimentally mapped in the Saccharomyces cerevisiae proteome. Factors including glycan heterogeneity, low abundance, and low occupancy can complicate site mapping. Here, we report a novel mass spectrometry-based strategy for detection of N-glycosites in the yeast proteome. Our method imparts N-glycopeptide mass envelopes with a pattern that is computationally distinguishable from background ions. Isotopic recoding is achieved via metabolic incorporation of a defined mixture of N-acetylglucosamine isotopologs into N-glycans. Peptides bearing the recoded envelopes are specifically targeted for fragmentation, facilitating high confidence site mapping. This strategy requires no chemical modification of the N-glycans or stringent sample enrichment. Further, enzymatically simplified N-glycans are preserved on peptides. Using this approach, we identify 133 N-glycosites spanning 58 proteins, nearly doubling the number of experimentally observed N-glycosites in the yeast proteome. The American Society for Biochemistry and Molecular Biology 2012-06 2012-01-19 /pmc/articles/PMC3433913/ /pubmed/22261724 http://dx.doi.org/10.1074/mcp.M111.015339 Text en © 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) applies to Author Choice Articles
spellingShingle Research
Breidenbach, Mark A.
Palaniappan, Krishnan K.
Pitcher, Austin A.
Bertozzi, Carolyn R.
Mapping Yeast N-Glycosites with Isotopically Recoded Glycans
title Mapping Yeast N-Glycosites with Isotopically Recoded Glycans
title_full Mapping Yeast N-Glycosites with Isotopically Recoded Glycans
title_fullStr Mapping Yeast N-Glycosites with Isotopically Recoded Glycans
title_full_unstemmed Mapping Yeast N-Glycosites with Isotopically Recoded Glycans
title_short Mapping Yeast N-Glycosites with Isotopically Recoded Glycans
title_sort mapping yeast n-glycosites with isotopically recoded glycans
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3433913/
https://www.ncbi.nlm.nih.gov/pubmed/22261724
http://dx.doi.org/10.1074/mcp.M111.015339
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