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Expanding the Chemical Cross-Linking Toolbox by the Use of Multiple Proteases and Enrichment by Size Exclusion Chromatography

Chemical cross-linking in combination with mass spectrometric analysis offers the potential to obtain low-resolution structural information from proteins and protein complexes. Identification of peptides connected by a cross-link provides direct evidence for the physical interaction of amino acid si...

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Autores principales: Leitner, Alexander, Reischl, Roland, Walzthoeni, Thomas, Herzog, Franz, Bohn, Stefan, Förster, Friedrich, Aebersold, Ruedi
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/PMC3316732/
https://www.ncbi.nlm.nih.gov/pubmed/22286754
http://dx.doi.org/10.1074/mcp.M111.014126
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author Leitner, Alexander
Reischl, Roland
Walzthoeni, Thomas
Herzog, Franz
Bohn, Stefan
Förster, Friedrich
Aebersold, Ruedi
author_facet Leitner, Alexander
Reischl, Roland
Walzthoeni, Thomas
Herzog, Franz
Bohn, Stefan
Förster, Friedrich
Aebersold, Ruedi
author_sort Leitner, Alexander
collection PubMed
description Chemical cross-linking in combination with mass spectrometric analysis offers the potential to obtain low-resolution structural information from proteins and protein complexes. Identification of peptides connected by a cross-link provides direct evidence for the physical interaction of amino acid side chains, information that can be used for computational modeling purposes. Despite impressive advances that were made in recent years, the number of experimentally observed cross-links still falls below the number of possible contacts of cross-linkable side chains within the span of the cross-linker. Here, we propose two complementary experimental strategies to expand cross-linking data sets. First, enrichment of cross-linked peptides by size exclusion chromatography selects cross-linked peptides based on their higher molecular mass, thereby depleting the majority of unmodified peptides present in proteolytic digests of cross-linked samples. Second, we demonstrate that the use of proteases in addition to trypsin, such as Asp-N, can additionally boost the number of observable cross-linking sites. The benefits of both SEC enrichment and multiprotease digests are demonstrated on a set of model proteins and the improved workflow is applied to the characterization of the 20S proteasome from rabbit and Schizosaccharomyces pombe.
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spelling pubmed-33167322012-04-10 Expanding the Chemical Cross-Linking Toolbox by the Use of Multiple Proteases and Enrichment by Size Exclusion Chromatography Leitner, Alexander Reischl, Roland Walzthoeni, Thomas Herzog, Franz Bohn, Stefan Förster, Friedrich Aebersold, Ruedi Mol Cell Proteomics Special Issue: Prospects in Space and Time Chemical cross-linking in combination with mass spectrometric analysis offers the potential to obtain low-resolution structural information from proteins and protein complexes. Identification of peptides connected by a cross-link provides direct evidence for the physical interaction of amino acid side chains, information that can be used for computational modeling purposes. Despite impressive advances that were made in recent years, the number of experimentally observed cross-links still falls below the number of possible contacts of cross-linkable side chains within the span of the cross-linker. Here, we propose two complementary experimental strategies to expand cross-linking data sets. First, enrichment of cross-linked peptides by size exclusion chromatography selects cross-linked peptides based on their higher molecular mass, thereby depleting the majority of unmodified peptides present in proteolytic digests of cross-linked samples. Second, we demonstrate that the use of proteases in addition to trypsin, such as Asp-N, can additionally boost the number of observable cross-linking sites. The benefits of both SEC enrichment and multiprotease digests are demonstrated on a set of model proteins and the improved workflow is applied to the characterization of the 20S proteasome from rabbit and Schizosaccharomyces pombe. The American Society for Biochemistry and Molecular Biology 2012-03 2012-01-27 /pmc/articles/PMC3316732/ /pubmed/22286754 http://dx.doi.org/10.1074/mcp.M111.014126 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 Special Issue: Prospects in Space and Time
Leitner, Alexander
Reischl, Roland
Walzthoeni, Thomas
Herzog, Franz
Bohn, Stefan
Förster, Friedrich
Aebersold, Ruedi
Expanding the Chemical Cross-Linking Toolbox by the Use of Multiple Proteases and Enrichment by Size Exclusion Chromatography
title Expanding the Chemical Cross-Linking Toolbox by the Use of Multiple Proteases and Enrichment by Size Exclusion Chromatography
title_full Expanding the Chemical Cross-Linking Toolbox by the Use of Multiple Proteases and Enrichment by Size Exclusion Chromatography
title_fullStr Expanding the Chemical Cross-Linking Toolbox by the Use of Multiple Proteases and Enrichment by Size Exclusion Chromatography
title_full_unstemmed Expanding the Chemical Cross-Linking Toolbox by the Use of Multiple Proteases and Enrichment by Size Exclusion Chromatography
title_short Expanding the Chemical Cross-Linking Toolbox by the Use of Multiple Proteases and Enrichment by Size Exclusion Chromatography
title_sort expanding the chemical cross-linking toolbox by the use of multiple proteases and enrichment by size exclusion chromatography
topic Special Issue: Prospects in Space and Time
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3316732/
https://www.ncbi.nlm.nih.gov/pubmed/22286754
http://dx.doi.org/10.1074/mcp.M111.014126
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