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ESNOQ, Proteomic Quantification of Endogenous S-Nitrosation

S-nitrosation is a post-translational protein modification and is one of the most important mechanisms of NO signaling. Endogenous S-nitrosothiol (SNO) quantification is a challenge for detailed functional studies. Here we developed an ESNOQ (Endogenous SNO Quantification) method which combines the...

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Autores principales: Zhou, Xixi, Han, Peiwei, Li, Jiangmei, Zhang, Xu, Huang, Bo, Ruan, Hong-Qiang, Chen, Chang
Formato: Texto
Lenguaje:English
Publicado: Public Library of Science 2010
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2848867/
https://www.ncbi.nlm.nih.gov/pubmed/20368813
http://dx.doi.org/10.1371/journal.pone.0010015
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author Zhou, Xixi
Han, Peiwei
Li, Jiangmei
Zhang, Xu
Huang, Bo
Ruan, Hong-Qiang
Chen, Chang
author_facet Zhou, Xixi
Han, Peiwei
Li, Jiangmei
Zhang, Xu
Huang, Bo
Ruan, Hong-Qiang
Chen, Chang
author_sort Zhou, Xixi
collection PubMed
description S-nitrosation is a post-translational protein modification and is one of the most important mechanisms of NO signaling. Endogenous S-nitrosothiol (SNO) quantification is a challenge for detailed functional studies. Here we developed an ESNOQ (Endogenous SNO Quantification) method which combines the stable isotope labeling by amino acids in cell culture (SILAC) technique with the detergent-free biotin-switch assay and LC-MS/MS. After confirming the accuracy of quantification in this method, we obtained an endogenous S-nitrosation proteome for LPS/IFN-γ induced RAW264.7 cells. 27 S-nitrosated protein targets were confirmed and using our method we were able to obtain quantitative information on the level of S-nitrosation on each modified Cys. With this quantitative information, over 15 more S-nitrosated targets were identified than in previous studies. Based on the quantification results, we found that the S-nitrosation levels of different cysteines varied within one protein, providing direct evidence for differences in the sensitivity of cysteine residues to reactive nitrosative stress and that S-nitrosation is a site-specific modification. Gene ontology clustering shows that S-nitrosation targets in the LPS/IFN-γ induced RAW264.7 cell model were functionally enriched in protein translation and glycolysis, suggesting that S-nitrosation may function by regulating multiple pathways. The ESNOQ method described here thus provides a solution for quantification of multiple endogenous S-nitrosation events, and makes it possible to elucidate the network of relationships between endogenous S-nitrosation targets involved in different cellular processes.
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spelling pubmed-28488672010-04-05 ESNOQ, Proteomic Quantification of Endogenous S-Nitrosation Zhou, Xixi Han, Peiwei Li, Jiangmei Zhang, Xu Huang, Bo Ruan, Hong-Qiang Chen, Chang PLoS One Research Article S-nitrosation is a post-translational protein modification and is one of the most important mechanisms of NO signaling. Endogenous S-nitrosothiol (SNO) quantification is a challenge for detailed functional studies. Here we developed an ESNOQ (Endogenous SNO Quantification) method which combines the stable isotope labeling by amino acids in cell culture (SILAC) technique with the detergent-free biotin-switch assay and LC-MS/MS. After confirming the accuracy of quantification in this method, we obtained an endogenous S-nitrosation proteome for LPS/IFN-γ induced RAW264.7 cells. 27 S-nitrosated protein targets were confirmed and using our method we were able to obtain quantitative information on the level of S-nitrosation on each modified Cys. With this quantitative information, over 15 more S-nitrosated targets were identified than in previous studies. Based on the quantification results, we found that the S-nitrosation levels of different cysteines varied within one protein, providing direct evidence for differences in the sensitivity of cysteine residues to reactive nitrosative stress and that S-nitrosation is a site-specific modification. Gene ontology clustering shows that S-nitrosation targets in the LPS/IFN-γ induced RAW264.7 cell model were functionally enriched in protein translation and glycolysis, suggesting that S-nitrosation may function by regulating multiple pathways. The ESNOQ method described here thus provides a solution for quantification of multiple endogenous S-nitrosation events, and makes it possible to elucidate the network of relationships between endogenous S-nitrosation targets involved in different cellular processes. Public Library of Science 2010-04-02 /pmc/articles/PMC2848867/ /pubmed/20368813 http://dx.doi.org/10.1371/journal.pone.0010015 Text en Zhou et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Zhou, Xixi
Han, Peiwei
Li, Jiangmei
Zhang, Xu
Huang, Bo
Ruan, Hong-Qiang
Chen, Chang
ESNOQ, Proteomic Quantification of Endogenous S-Nitrosation
title ESNOQ, Proteomic Quantification of Endogenous S-Nitrosation
title_full ESNOQ, Proteomic Quantification of Endogenous S-Nitrosation
title_fullStr ESNOQ, Proteomic Quantification of Endogenous S-Nitrosation
title_full_unstemmed ESNOQ, Proteomic Quantification of Endogenous S-Nitrosation
title_short ESNOQ, Proteomic Quantification of Endogenous S-Nitrosation
title_sort esnoq, proteomic quantification of endogenous s-nitrosation
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2848867/
https://www.ncbi.nlm.nih.gov/pubmed/20368813
http://dx.doi.org/10.1371/journal.pone.0010015
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