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Quantitative Chemoproteomics for Site-Specific Analysis of Protein Alkylation by 4-Hydroxy-2-Nonenal in Cells
[Image: see text] Protein alkylation by 4-hydroxy-2-nonenal (HNE), an endogenous lipid derived electrophile, contributes to stress signaling and cellular toxicity. Although previous work has identified protein targets for HNE alkylation, the sequence specificity of alkylation and dynamics in a cellu...
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/PMC4350606/ https://www.ncbi.nlm.nih.gov/pubmed/25654326 http://dx.doi.org/10.1021/ac504685y |
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author | Yang, Jing Tallman, Keri A. Porter, Ned A. Liebler, Daniel C. |
author_facet | Yang, Jing Tallman, Keri A. Porter, Ned A. Liebler, Daniel C. |
author_sort | Yang, Jing |
collection | PubMed |
description | [Image: see text] Protein alkylation by 4-hydroxy-2-nonenal (HNE), an endogenous lipid derived electrophile, contributes to stress signaling and cellular toxicity. Although previous work has identified protein targets for HNE alkylation, the sequence specificity of alkylation and dynamics in a cellular context remain largely unexplored. We developed a new quantitative chemoproteomic platform, which uses isotopically tagged, photocleavable azido-biotin reagents to selectively capture and quantify the cellular targets labeled by the alkynyl analogue of HNE (aHNE). Our analyses site-specifically identified and quantified 398 aHNE protein alkylation events (386 cysteine sites and 12 histidine sites) in intact cells. This data set expands by at least an order of magnitude the number of such modification sites previously reported. Although adducts formed by Michael addition are thought to be largely irreversible, we found that most aHNE modifications are lost rapidly in situ. Moreover, aHNE adduct turnover occurs only in intact cells and loss rates are site-selective. This quantitative chemoproteomics platform provides a versatile general approach to map bioorthogonal-chemically engineered post-translational modifications and their cellular dynamics in a site-specific and unbiased manner. |
format | Online Article Text |
id | pubmed-4350606 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | American
Chemical
Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-43506062016-02-05 Quantitative Chemoproteomics for Site-Specific Analysis of Protein Alkylation by 4-Hydroxy-2-Nonenal in Cells Yang, Jing Tallman, Keri A. Porter, Ned A. Liebler, Daniel C. Anal Chem [Image: see text] Protein alkylation by 4-hydroxy-2-nonenal (HNE), an endogenous lipid derived electrophile, contributes to stress signaling and cellular toxicity. Although previous work has identified protein targets for HNE alkylation, the sequence specificity of alkylation and dynamics in a cellular context remain largely unexplored. We developed a new quantitative chemoproteomic platform, which uses isotopically tagged, photocleavable azido-biotin reagents to selectively capture and quantify the cellular targets labeled by the alkynyl analogue of HNE (aHNE). Our analyses site-specifically identified and quantified 398 aHNE protein alkylation events (386 cysteine sites and 12 histidine sites) in intact cells. This data set expands by at least an order of magnitude the number of such modification sites previously reported. Although adducts formed by Michael addition are thought to be largely irreversible, we found that most aHNE modifications are lost rapidly in situ. Moreover, aHNE adduct turnover occurs only in intact cells and loss rates are site-selective. This quantitative chemoproteomics platform provides a versatile general approach to map bioorthogonal-chemically engineered post-translational modifications and their cellular dynamics in a site-specific and unbiased manner. American Chemical Society 2015-02-05 2015-03-03 /pmc/articles/PMC4350606/ /pubmed/25654326 http://dx.doi.org/10.1021/ac504685y Text en Copyright © 2015 American Chemical Society This is an open access article published under a Creative Commons Attribution (CC-BY) License (http://pubs.acs.org/page/policy/authorchoice_ccby_termsofuse.html) , which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. |
spellingShingle | Yang, Jing Tallman, Keri A. Porter, Ned A. Liebler, Daniel C. Quantitative Chemoproteomics for Site-Specific Analysis of Protein Alkylation by 4-Hydroxy-2-Nonenal in Cells |
title | Quantitative Chemoproteomics for Site-Specific Analysis
of Protein Alkylation by 4-Hydroxy-2-Nonenal in Cells |
title_full | Quantitative Chemoproteomics for Site-Specific Analysis
of Protein Alkylation by 4-Hydroxy-2-Nonenal in Cells |
title_fullStr | Quantitative Chemoproteomics for Site-Specific Analysis
of Protein Alkylation by 4-Hydroxy-2-Nonenal in Cells |
title_full_unstemmed | Quantitative Chemoproteomics for Site-Specific Analysis
of Protein Alkylation by 4-Hydroxy-2-Nonenal in Cells |
title_short | Quantitative Chemoproteomics for Site-Specific Analysis
of Protein Alkylation by 4-Hydroxy-2-Nonenal in Cells |
title_sort | quantitative chemoproteomics for site-specific analysis
of protein alkylation by 4-hydroxy-2-nonenal in cells |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4350606/ https://www.ncbi.nlm.nih.gov/pubmed/25654326 http://dx.doi.org/10.1021/ac504685y |
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