Document Detail


Quantitative site-specific reactivity profiling of S-nitrosylation in mouse skeletal muscle using cysteinyl peptide enrichment coupled with mass spectrometry.
MedLine Citation:
PMID:  23277143     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
S-nitrosylation, the formation of S-nitrosothiol (SNO), is an important reversible thiol oxidation event that has been increasingly recognized for its role in cell signaling. Although many proteins susceptible to S-nitrosylation have been reported, site-specific identification of physiologically relevant SNO modifications remains an analytical challenge because of the low abundance and labile nature of this modification. Herein we present further improvement and optimization of the recently reported resin-assisted cysteinyl peptide enrichment protocol for SNO identification and its application to mouse skeletal muscle to identify specific cysteine sites sensitive to S-nitrosylation by a quantitative reactivity profiling strategy. Our results indicate that the protein- and peptide-level enrichment protocols provide comparable specificity and coverage of SNO-peptide identifications. S-nitrosylation reactivity profiling was performed by quantitatively comparing the site-specific SNO modification levels in samples treated with S-nitrosoglutathione, an NO donor, at two different concentrations (i.e., 10 and 100 μM). The reactivity profiling experiments led to the identification of 488 SNO-modified sites from 197 proteins with specificity of ∼95% at the unique peptide level, i.e., ∼95% of enriched peptides contain cysteine residues as the originally SNO-modified sites. Among these sites, 281 from 145 proteins were considered more sensitive to S-nitrosylation based on the ratios of observed SNO levels between the two treatments. These SNO-sensitive sites are more likely to be physiologically relevant. Many of the SNO-sensitive proteins are localized in mitochondria, contractile fiber, and actin cytoskeleton, suggesting the susceptibility of these subcellular compartments to redox regulation. Moreover, these observed SNO-sensitive proteins are primarily involved in metabolic pathways, including the tricarboxylic acid cycle, glycolysis/gluconeogenesis, glutathione metabolism, and fatty acid metabolism, suggesting the importance of redox regulation in muscle metabolism and insulin action.
Authors:
Dian Su; Anil K Shukla; Baowei Chen; Jong-Seo Kim; Ernesto Nakayasu; Yi Qu; Uma Aryal; Karl Weitz; Therese R W Clauss; Matthew E Monroe; David G Camp; Diana J Bigelow; Richard D Smith; Rohit N Kulkarni; Wei-Jun Qian
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, U.S. Gov't, Non-P.H.S.     Date:  2012-12-28
Journal Detail:
Title:  Free radical biology & medicine     Volume:  57     ISSN:  1873-4596     ISO Abbreviation:  Free Radic. Biol. Med.     Publication Date:  2013 Apr 
Date Detail:
Created Date:  2013-03-07     Completed Date:  2013-08-23     Revised Date:  2014-04-02    
Medline Journal Info:
Nlm Unique ID:  8709159     Medline TA:  Free Radic Biol Med     Country:  United States    
Other Details:
Languages:  eng     Pagination:  68-78     Citation Subset:  IM    
Copyright Information:
Copyright © 2012 Elsevier Inc. All rights reserved.
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MeSH Terms
Descriptor/Qualifier:
Actin Cytoskeleton / metabolism
Animals
Contractile Proteins / metabolism
Cysteine / chemistry
Male
Mass Spectrometry
Mice
Mice, Inbred C57BL
Mitochondria / metabolism
Muscle, Skeletal / metabolism*
Oxidation-Reduction
Peptides / chemistry,  metabolism*
Proteomics
S-Nitrosoglutathione / chemistry,  metabolism,  pharmacology
S-Nitrosothiols / metabolism*
Signal Transduction
Grant Support
ID/Acronym/Agency:
DP2 OD006668/OD/NIH HHS; DP2OD006668/OD/NIH HHS; P41 GM103493/GM/NIGMS NIH HHS; P41 RR018522/RR/NCRR NIH HHS; P41GM103493/GM/NIGMS NIH HHS; R01 DK074795/DK/NIDDK NIH HHS; R01DK074795/DK/NIDDK NIH HHS
Chemical
Reg. No./Substance:
0/Contractile Proteins; 0/Peptides; 0/S-Nitrosothiols; 57564-91-7/S-Nitrosoglutathione; K848JZ4886/Cysteine
Comments/Corrections

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