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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Main Authors: Xixi Zhou, Peiwei Han, Jiangmei Li, Xu Zhang, Bo Huang, Hong-Qiang Ruan, Chang Chen
Format: Article
Language:English
Published: Public Library of Science (PLoS) 2010-01-01
Series:PLoS ONE
Online Access:http://europepmc.org/articles/PMC2848867?pdf=render
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spelling doaj-e2fc5b8eab114117a37bc27c881f97322020-11-25T01:30:27ZengPublic Library of Science (PLoS)PLoS ONE1932-62032010-01-0154e1001510.1371/journal.pone.0010015ESNOQ, proteomic quantification of endogenous S-nitrosation.Xixi ZhouPeiwei HanJiangmei LiXu ZhangBo HuangHong-Qiang RuanChang ChenS-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-gamma 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-gamma 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.http://europepmc.org/articles/PMC2848867?pdf=render
collection DOAJ
language English
format Article
sources DOAJ
author Xixi Zhou
Peiwei Han
Jiangmei Li
Xu Zhang
Bo Huang
Hong-Qiang Ruan
Chang Chen
spellingShingle Xixi Zhou
Peiwei Han
Jiangmei Li
Xu Zhang
Bo Huang
Hong-Qiang Ruan
Chang Chen
ESNOQ, proteomic quantification of endogenous S-nitrosation.
PLoS ONE
author_facet Xixi Zhou
Peiwei Han
Jiangmei Li
Xu Zhang
Bo Huang
Hong-Qiang Ruan
Chang Chen
author_sort Xixi Zhou
title ESNOQ, proteomic quantification of endogenous S-nitrosation.
title_short 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_sort esnoq, proteomic quantification of endogenous s-nitrosation.
publisher Public Library of Science (PLoS)
series PLoS ONE
issn 1932-6203
publishDate 2010-01-01
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-gamma 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-gamma 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.
url http://europepmc.org/articles/PMC2848867?pdf=render
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