Site-specific characterisation of densely O-glycosylated mucin-type peptides using electron transfer dissociation ESI-MS/MS
Site-specific characterisation of mucin-type O-linked glycosylation is an analytical challenge due to glycan heterogeneity, lack of glycosylation site consensus sequence and high density of occupied glycosylation sites. Here, we report the use of electron transfer dissociation (ETD) for the site-spe...
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2011
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ftunivnewengland:oai:rune.une.edu.au:1959.11/20672 2023-08-27T04:04:09+02:00 Site-specific characterisation of densely O-glycosylated mucin-type peptides using electron transfer dissociation ESI-MS/MS Thaysen-Andersen, Morten Wilkinson, Brendan School of Science and Technology orcid:0000-0003-1866-6540 Payne, Richard J Packer, Nicolle H 2011 https://hdl.handle.net/1959.11/20672 en eng Wiley-VCH Verlag GmbH & Co KGaA 10.1002/elps.201100294 https://hdl.handle.net/1959.11/20672 une:20865 Proteins and Peptides Characterisation of Biological Macromolecules Macromolecular and Materials Chemistry Journal Article 2011 ftunivnewengland 2023-08-10T19:42:55Z Site-specific characterisation of mucin-type O-linked glycosylation is an analytical challenge due to glycan heterogeneity, lack of glycosylation site consensus sequence and high density of occupied glycosylation sites. Here, we report the use of electron transfer dissociation (ETD) for the site-specific characterisation of densely glycosylated mucin type O-linked glycopeptides using ESI-IT-MS/MS. Synthetic glycopeptides from the human mucin-1 (MUC-1) tandem repeat region containing a range of O-linked, tumour associated carbohydrate antigens, namely Tn, T and sialyl T, with different glycosylation site occupancies and an increasing number of tandem repeats were studied. In addition, a glycopeptide from the anti-freeze glycoprotein of Antarctic and Arctic notothenoids, bearing four O-linked, per-acetylated T antigens was characterised. ETD MS/MS of infused or capillary LC-separated glycopeptides provided broad peptide sequence coverage (c/z·-type fragment ions) with intact glycans still attached to the Ser/Thr residues. Thus, the glycosylation sites were unambiguously determined, while simultaneously obtaining information about the attached glycan mass and peptide identity. Highly sialylated O-glycopeptides showed less efficient peptide fragmentation, but some sequence and glycosylation site information was still obtained. This study demonstrates the capabilities of ETD MS/MS for site-specific characterisation of mucin-type glyco peptides containing high-density O-linked glycan clusters, using accessible and relative low-resolution/low-mass accuracy IT MS instrumentation. Article in Journal/Newspaper Antarc* Antarctic Arctic Research UNE - University of New England at Armidale, NSW Australia Antarctic Arctic |
institution |
Open Polar |
collection |
Research UNE - University of New England at Armidale, NSW Australia |
op_collection_id |
ftunivnewengland |
language |
English |
topic |
Proteins and Peptides Characterisation of Biological Macromolecules Macromolecular and Materials Chemistry |
spellingShingle |
Proteins and Peptides Characterisation of Biological Macromolecules Macromolecular and Materials Chemistry Thaysen-Andersen, Morten Wilkinson, Brendan School of Science and Technology orcid:0000-0003-1866-6540 Payne, Richard J Packer, Nicolle H Site-specific characterisation of densely O-glycosylated mucin-type peptides using electron transfer dissociation ESI-MS/MS |
topic_facet |
Proteins and Peptides Characterisation of Biological Macromolecules Macromolecular and Materials Chemistry |
description |
Site-specific characterisation of mucin-type O-linked glycosylation is an analytical challenge due to glycan heterogeneity, lack of glycosylation site consensus sequence and high density of occupied glycosylation sites. Here, we report the use of electron transfer dissociation (ETD) for the site-specific characterisation of densely glycosylated mucin type O-linked glycopeptides using ESI-IT-MS/MS. Synthetic glycopeptides from the human mucin-1 (MUC-1) tandem repeat region containing a range of O-linked, tumour associated carbohydrate antigens, namely Tn, T and sialyl T, with different glycosylation site occupancies and an increasing number of tandem repeats were studied. In addition, a glycopeptide from the anti-freeze glycoprotein of Antarctic and Arctic notothenoids, bearing four O-linked, per-acetylated T antigens was characterised. ETD MS/MS of infused or capillary LC-separated glycopeptides provided broad peptide sequence coverage (c/z·-type fragment ions) with intact glycans still attached to the Ser/Thr residues. Thus, the glycosylation sites were unambiguously determined, while simultaneously obtaining information about the attached glycan mass and peptide identity. Highly sialylated O-glycopeptides showed less efficient peptide fragmentation, but some sequence and glycosylation site information was still obtained. This study demonstrates the capabilities of ETD MS/MS for site-specific characterisation of mucin-type glyco peptides containing high-density O-linked glycan clusters, using accessible and relative low-resolution/low-mass accuracy IT MS instrumentation. |
format |
Article in Journal/Newspaper |
author |
Thaysen-Andersen, Morten Wilkinson, Brendan School of Science and Technology orcid:0000-0003-1866-6540 Payne, Richard J Packer, Nicolle H |
author_facet |
Thaysen-Andersen, Morten Wilkinson, Brendan School of Science and Technology orcid:0000-0003-1866-6540 Payne, Richard J Packer, Nicolle H |
author_sort |
Thaysen-Andersen, Morten |
title |
Site-specific characterisation of densely O-glycosylated mucin-type peptides using electron transfer dissociation ESI-MS/MS |
title_short |
Site-specific characterisation of densely O-glycosylated mucin-type peptides using electron transfer dissociation ESI-MS/MS |
title_full |
Site-specific characterisation of densely O-glycosylated mucin-type peptides using electron transfer dissociation ESI-MS/MS |
title_fullStr |
Site-specific characterisation of densely O-glycosylated mucin-type peptides using electron transfer dissociation ESI-MS/MS |
title_full_unstemmed |
Site-specific characterisation of densely O-glycosylated mucin-type peptides using electron transfer dissociation ESI-MS/MS |
title_sort |
site-specific characterisation of densely o-glycosylated mucin-type peptides using electron transfer dissociation esi-ms/ms |
publisher |
Wiley-VCH Verlag GmbH & Co KGaA |
publishDate |
2011 |
url |
https://hdl.handle.net/1959.11/20672 |
geographic |
Antarctic Arctic |
geographic_facet |
Antarctic Arctic |
genre |
Antarc* Antarctic Arctic |
genre_facet |
Antarc* Antarctic Arctic |
op_relation |
10.1002/elps.201100294 https://hdl.handle.net/1959.11/20672 une:20865 |
_version_ |
1775349743201812480 |