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Improvement of the glycoproteomic toolbox with the discovery of a unique C-terminal cleavage specificity of flavastacin for N-glycosylated asparagine

To determine all potential N-glycosylation sites of a glycoprotein, one central aspect of every bottom-up N-glycoproteomic strategy is to generate suitable N-glycopeptides that can be detected and analyzed by mass spectrometry. Specific proteases, such as trypsin, bear the potential of generating N-...

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Autores principales: Pralow, Alexander, Hoffmann, Marcus, Nguyen-Khuong, Terry, Rapp, Erdmann, Reichl, Udo
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5595805/
https://www.ncbi.nlm.nih.gov/pubmed/28900186
http://dx.doi.org/10.1038/s41598-017-11668-1
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author Pralow, Alexander
Hoffmann, Marcus
Nguyen-Khuong, Terry
Rapp, Erdmann
Reichl, Udo
author_facet Pralow, Alexander
Hoffmann, Marcus
Nguyen-Khuong, Terry
Rapp, Erdmann
Reichl, Udo
author_sort Pralow, Alexander
collection PubMed
description To determine all potential N-glycosylation sites of a glycoprotein, one central aspect of every bottom-up N-glycoproteomic strategy is to generate suitable N-glycopeptides that can be detected and analyzed by mass spectrometry. Specific proteases, such as trypsin, bear the potential of generating N-glycopeptides that either carry more than one N-glycosylation site or are too long to be readily analyzed by mass spectrometry– both due to the lack of tryptic cleavage sites near the N-glycosylation site. Here, we present a newly identified cleavage specificity of flavastacin, a protease from Flavobacterium menigosepticum, which - up to now - was only reported to cleave peptide bonds N-terminal to aspartic acid residues. In contrast to literature, we could not confirm this N-terminal specificity of flavastacin for aspartic acid. However, for the first time, we show a unique cleavage specificity of flavastacin towards the C-terminus of N-glycosylated asparagine residues. Implemented in an N-glycoproteomic workflow the use of flavastacin can thus not only render data analysis much easier, it can also significantly increase the confidence of MS-based N-glycoproteomic analyses. We demonstrate this newly discovered specificity of flavastacin by in-depth LC-MS(/MS) analysis of complex-type glycosylated human lactotransferrin and bovine serum albumin peptides and N-glycopeptides that were generated by trypsin and flavastacin digestion. Following to this work, further elucidation of the efficiency, specificity and mode of action of flavastacin is needed, but we believe that our discovery has great potential to facilitate and improve the characterization of N-glycoproteomes.
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spelling pubmed-55958052017-09-14 Improvement of the glycoproteomic toolbox with the discovery of a unique C-terminal cleavage specificity of flavastacin for N-glycosylated asparagine Pralow, Alexander Hoffmann, Marcus Nguyen-Khuong, Terry Rapp, Erdmann Reichl, Udo Sci Rep Article To determine all potential N-glycosylation sites of a glycoprotein, one central aspect of every bottom-up N-glycoproteomic strategy is to generate suitable N-glycopeptides that can be detected and analyzed by mass spectrometry. Specific proteases, such as trypsin, bear the potential of generating N-glycopeptides that either carry more than one N-glycosylation site or are too long to be readily analyzed by mass spectrometry– both due to the lack of tryptic cleavage sites near the N-glycosylation site. Here, we present a newly identified cleavage specificity of flavastacin, a protease from Flavobacterium menigosepticum, which - up to now - was only reported to cleave peptide bonds N-terminal to aspartic acid residues. In contrast to literature, we could not confirm this N-terminal specificity of flavastacin for aspartic acid. However, for the first time, we show a unique cleavage specificity of flavastacin towards the C-terminus of N-glycosylated asparagine residues. Implemented in an N-glycoproteomic workflow the use of flavastacin can thus not only render data analysis much easier, it can also significantly increase the confidence of MS-based N-glycoproteomic analyses. We demonstrate this newly discovered specificity of flavastacin by in-depth LC-MS(/MS) analysis of complex-type glycosylated human lactotransferrin and bovine serum albumin peptides and N-glycopeptides that were generated by trypsin and flavastacin digestion. Following to this work, further elucidation of the efficiency, specificity and mode of action of flavastacin is needed, but we believe that our discovery has great potential to facilitate and improve the characterization of N-glycoproteomes. Nature Publishing Group UK 2017-09-12 /pmc/articles/PMC5595805/ /pubmed/28900186 http://dx.doi.org/10.1038/s41598-017-11668-1 Text en © The Author(s) 2017 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Pralow, Alexander
Hoffmann, Marcus
Nguyen-Khuong, Terry
Rapp, Erdmann
Reichl, Udo
Improvement of the glycoproteomic toolbox with the discovery of a unique C-terminal cleavage specificity of flavastacin for N-glycosylated asparagine
title Improvement of the glycoproteomic toolbox with the discovery of a unique C-terminal cleavage specificity of flavastacin for N-glycosylated asparagine
title_full Improvement of the glycoproteomic toolbox with the discovery of a unique C-terminal cleavage specificity of flavastacin for N-glycosylated asparagine
title_fullStr Improvement of the glycoproteomic toolbox with the discovery of a unique C-terminal cleavage specificity of flavastacin for N-glycosylated asparagine
title_full_unstemmed Improvement of the glycoproteomic toolbox with the discovery of a unique C-terminal cleavage specificity of flavastacin for N-glycosylated asparagine
title_short Improvement of the glycoproteomic toolbox with the discovery of a unique C-terminal cleavage specificity of flavastacin for N-glycosylated asparagine
title_sort improvement of the glycoproteomic toolbox with the discovery of a unique c-terminal cleavage specificity of flavastacin for n-glycosylated asparagine
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5595805/
https://www.ncbi.nlm.nih.gov/pubmed/28900186
http://dx.doi.org/10.1038/s41598-017-11668-1
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