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Top-Down 193-nm Ultraviolet Photodissociation Mass Spectrometry for Simultaneous Determination of Polyubiquitin Chain Length and Topology
[Image: see text] Protein ubiquitin modifications present a vexing analytical challenge, because of the dynamic changes in the site of modification on the substrate, the number of ubiquitin moieties attached, and the diversity of linkage patterns in which they are attached. Presented here is a metho...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American
Chemical
Society
2015
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4318624/ https://www.ncbi.nlm.nih.gov/pubmed/25559986 http://dx.doi.org/10.1021/ac5038363 |
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author | Cannon, Joe R. Martinez-Fonts, Kirby Robotham, Scott A. Matouschek, Andreas Brodbelt, Jennifer S. |
author_facet | Cannon, Joe R. Martinez-Fonts, Kirby Robotham, Scott A. Matouschek, Andreas Brodbelt, Jennifer S. |
author_sort | Cannon, Joe R. |
collection | PubMed |
description | [Image: see text] Protein ubiquitin modifications present a vexing analytical challenge, because of the dynamic changes in the site of modification on the substrate, the number of ubiquitin moieties attached, and the diversity of linkage patterns in which they are attached. Presented here is a method to confidently assign size and linkage type of polyubiquitin modifications. The method combines intact mass measurement to determine the number of ubiquitin moieties in the chain with backbone fragmentation by 193-nm ultraviolet photodissociation (UVPD) to determine the linkage pattern. UVPD fragmentation of proteins leads to reproducible backbone cleavage at almost every inter-residue position, and in polyubiquitin chains, the N-terminally derived fragments from each constituent monomer are identical, up to the site of conjugation. The N-terminal ubiquitin fragment ions are superimposed to create a diagnostic pattern that allows easy recognition of the dominant chain linkages. The method is demonstrated by achieving almost-complete fragmentation of monoubiquitin and then, subsequently, fragmentation of dimeric, tetrameric, and longer Lys48- and Lys63-linked ubiquitin chains. The utility of the method for the analysis of mixed linkage chains is confirmed for mixtures of Lys48 and Lys63 tetramers with known relative concentrations and for an in vitro-formulated ubiquitin chain attached to a substrate protein. |
format | Online Article Text |
id | pubmed-4318624 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | American
Chemical
Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-43186242016-01-05 Top-Down 193-nm Ultraviolet Photodissociation Mass Spectrometry for Simultaneous Determination of Polyubiquitin Chain Length and Topology Cannon, Joe R. Martinez-Fonts, Kirby Robotham, Scott A. Matouschek, Andreas Brodbelt, Jennifer S. Anal Chem [Image: see text] Protein ubiquitin modifications present a vexing analytical challenge, because of the dynamic changes in the site of modification on the substrate, the number of ubiquitin moieties attached, and the diversity of linkage patterns in which they are attached. Presented here is a method to confidently assign size and linkage type of polyubiquitin modifications. The method combines intact mass measurement to determine the number of ubiquitin moieties in the chain with backbone fragmentation by 193-nm ultraviolet photodissociation (UVPD) to determine the linkage pattern. UVPD fragmentation of proteins leads to reproducible backbone cleavage at almost every inter-residue position, and in polyubiquitin chains, the N-terminally derived fragments from each constituent monomer are identical, up to the site of conjugation. The N-terminal ubiquitin fragment ions are superimposed to create a diagnostic pattern that allows easy recognition of the dominant chain linkages. The method is demonstrated by achieving almost-complete fragmentation of monoubiquitin and then, subsequently, fragmentation of dimeric, tetrameric, and longer Lys48- and Lys63-linked ubiquitin chains. The utility of the method for the analysis of mixed linkage chains is confirmed for mixtures of Lys48 and Lys63 tetramers with known relative concentrations and for an in vitro-formulated ubiquitin chain attached to a substrate protein. American Chemical Society 2015-01-05 2015-02-03 /pmc/articles/PMC4318624/ /pubmed/25559986 http://dx.doi.org/10.1021/ac5038363 Text en Copyright © 2015 American Chemical Society This is an open access article published under an ACS AuthorChoice License (http://pubs.acs.org/page/policy/authorchoice_termsofuse.html) , which permits copying and redistribution of the article or any adaptations for non-commercial purposes. |
spellingShingle | Cannon, Joe R. Martinez-Fonts, Kirby Robotham, Scott A. Matouschek, Andreas Brodbelt, Jennifer S. Top-Down 193-nm Ultraviolet Photodissociation Mass Spectrometry for Simultaneous Determination of Polyubiquitin Chain Length and Topology |
title | Top-Down 193-nm Ultraviolet Photodissociation Mass
Spectrometry for Simultaneous Determination of Polyubiquitin Chain
Length and Topology |
title_full | Top-Down 193-nm Ultraviolet Photodissociation Mass
Spectrometry for Simultaneous Determination of Polyubiquitin Chain
Length and Topology |
title_fullStr | Top-Down 193-nm Ultraviolet Photodissociation Mass
Spectrometry for Simultaneous Determination of Polyubiquitin Chain
Length and Topology |
title_full_unstemmed | Top-Down 193-nm Ultraviolet Photodissociation Mass
Spectrometry for Simultaneous Determination of Polyubiquitin Chain
Length and Topology |
title_short | Top-Down 193-nm Ultraviolet Photodissociation Mass
Spectrometry for Simultaneous Determination of Polyubiquitin Chain
Length and Topology |
title_sort | top-down 193-nm ultraviolet photodissociation mass
spectrometry for simultaneous determination of polyubiquitin chain
length and topology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4318624/ https://www.ncbi.nlm.nih.gov/pubmed/25559986 http://dx.doi.org/10.1021/ac5038363 |
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