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Oxonium Ion Guided Analysis of Quantitative Proteomics Data Reveals Site-Specific O-Glycosylation of Anterior Gradient Protein 2 (AGR2)

Developments in mass spectrometry (MS)-based analyses of glycoproteins have been important to study changes in glycosylation related to disease. Recently, the characteristic pattern of oxonium ions in glycopeptide fragmentation spectra had been used to assign different sets of glycopeptides. In part...

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Autores principales: Pirro, Martina, Mohammed, Yassene, de Ru, Arnoud H., Janssen, George M. C., Tjokrodirijo, Rayman T. N., Madunić, Katarina, Wuhrer, Manfred, van Veelen, Peter A., Hensbergen, Paul J.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8160981/
https://www.ncbi.nlm.nih.gov/pubmed/34065225
http://dx.doi.org/10.3390/ijms22105369
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author Pirro, Martina
Mohammed, Yassene
de Ru, Arnoud H.
Janssen, George M. C.
Tjokrodirijo, Rayman T. N.
Madunić, Katarina
Wuhrer, Manfred
van Veelen, Peter A.
Hensbergen, Paul J.
author_facet Pirro, Martina
Mohammed, Yassene
de Ru, Arnoud H.
Janssen, George M. C.
Tjokrodirijo, Rayman T. N.
Madunić, Katarina
Wuhrer, Manfred
van Veelen, Peter A.
Hensbergen, Paul J.
author_sort Pirro, Martina
collection PubMed
description Developments in mass spectrometry (MS)-based analyses of glycoproteins have been important to study changes in glycosylation related to disease. Recently, the characteristic pattern of oxonium ions in glycopeptide fragmentation spectra had been used to assign different sets of glycopeptides. In particular, this was helpful to discriminate between O-GalNAc and O-GlcNAc. Here, we thought to investigate how such information can be used to examine quantitative proteomics data. For this purpose, we used tandem mass tag (TMT)-labeled samples from total cell lysates and secreted proteins from three different colorectal cancer cell lines. Following automated glycopeptide assignment (Byonic) and evaluation of the presence and relative intensity of oxonium ions, we observed that, in particular, the ratio of the ions at m/z 144.066 and 138.055, respectively, could be used to discriminate between O-GlcNAcylated and O-GalNAcylated peptides, with concomitant relative quantification between the different cell lines. Among the O-GalNAcylated proteins, we also observed anterior gradient protein 2 (AGR2), a protein which glycosylation site and status was hitherto not well documented. Using a combination of multiple fragmentation methods, we then not only assigned the site of modification, but also showed different glycosylation between intracellular (ER-resident) and secreted AGR2. Overall, our study shows the potential of broad application of the use of the relative intensities of oxonium ions for the confident assignment of glycopeptides, even in complex proteomics datasets.
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spelling pubmed-81609812021-05-29 Oxonium Ion Guided Analysis of Quantitative Proteomics Data Reveals Site-Specific O-Glycosylation of Anterior Gradient Protein 2 (AGR2) Pirro, Martina Mohammed, Yassene de Ru, Arnoud H. Janssen, George M. C. Tjokrodirijo, Rayman T. N. Madunić, Katarina Wuhrer, Manfred van Veelen, Peter A. Hensbergen, Paul J. Int J Mol Sci Article Developments in mass spectrometry (MS)-based analyses of glycoproteins have been important to study changes in glycosylation related to disease. Recently, the characteristic pattern of oxonium ions in glycopeptide fragmentation spectra had been used to assign different sets of glycopeptides. In particular, this was helpful to discriminate between O-GalNAc and O-GlcNAc. Here, we thought to investigate how such information can be used to examine quantitative proteomics data. For this purpose, we used tandem mass tag (TMT)-labeled samples from total cell lysates and secreted proteins from three different colorectal cancer cell lines. Following automated glycopeptide assignment (Byonic) and evaluation of the presence and relative intensity of oxonium ions, we observed that, in particular, the ratio of the ions at m/z 144.066 and 138.055, respectively, could be used to discriminate between O-GlcNAcylated and O-GalNAcylated peptides, with concomitant relative quantification between the different cell lines. Among the O-GalNAcylated proteins, we also observed anterior gradient protein 2 (AGR2), a protein which glycosylation site and status was hitherto not well documented. Using a combination of multiple fragmentation methods, we then not only assigned the site of modification, but also showed different glycosylation between intracellular (ER-resident) and secreted AGR2. Overall, our study shows the potential of broad application of the use of the relative intensities of oxonium ions for the confident assignment of glycopeptides, even in complex proteomics datasets. MDPI 2021-05-20 /pmc/articles/PMC8160981/ /pubmed/34065225 http://dx.doi.org/10.3390/ijms22105369 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Pirro, Martina
Mohammed, Yassene
de Ru, Arnoud H.
Janssen, George M. C.
Tjokrodirijo, Rayman T. N.
Madunić, Katarina
Wuhrer, Manfred
van Veelen, Peter A.
Hensbergen, Paul J.
Oxonium Ion Guided Analysis of Quantitative Proteomics Data Reveals Site-Specific O-Glycosylation of Anterior Gradient Protein 2 (AGR2)
title Oxonium Ion Guided Analysis of Quantitative Proteomics Data Reveals Site-Specific O-Glycosylation of Anterior Gradient Protein 2 (AGR2)
title_full Oxonium Ion Guided Analysis of Quantitative Proteomics Data Reveals Site-Specific O-Glycosylation of Anterior Gradient Protein 2 (AGR2)
title_fullStr Oxonium Ion Guided Analysis of Quantitative Proteomics Data Reveals Site-Specific O-Glycosylation of Anterior Gradient Protein 2 (AGR2)
title_full_unstemmed Oxonium Ion Guided Analysis of Quantitative Proteomics Data Reveals Site-Specific O-Glycosylation of Anterior Gradient Protein 2 (AGR2)
title_short Oxonium Ion Guided Analysis of Quantitative Proteomics Data Reveals Site-Specific O-Glycosylation of Anterior Gradient Protein 2 (AGR2)
title_sort oxonium ion guided analysis of quantitative proteomics data reveals site-specific o-glycosylation of anterior gradient protein 2 (agr2)
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8160981/
https://www.ncbi.nlm.nih.gov/pubmed/34065225
http://dx.doi.org/10.3390/ijms22105369
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