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Gewählte Publikation:

SHR Neuro Krebs Kardio Lipid

Chung, HS; Murray, CI; Venkatraman, V; Crowgey, EL; Rainer, PP; Cole, RN; Bomgarden, RD; Rogers, JC; Balkan, W; Hare, JM; Kass, DA; Van Eyk, JE.
Dual Labeling Biotin Switch Assay to Reduce Bias Derived From Different Cysteine Subpopulations: A Method to Maximize S-Nitrosylation Detection.
Circ Res. 2015; 117(10):846-857 [OPEN ACCESS]
Web of Science PubMed PUBMED Central FullText FullText_MUG

 

Autor/innen der Med Uni Graz:
Rainer Peter
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Number of Figures: 7
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Abstract:
S-nitrosylation (SNO), an oxidative post-translational modification of cysteine residues, responds to changes in the cardiac redox-environment. Classic biotin-switch assay and its derivatives are the most common methods used for detecting SNO. In this approach, the labile SNO group is selectively replaced with a single stable tag. To date, a variety of thiol-reactive tags have been introduced. However, these methods have not produced a consistent data set, which suggests an incomplete capture by a single tag and potentially the presence of different cysteine subpopulations. To investigate potential labeling bias in the existing methods with a single tag to detect SNO, explore if there are distinct cysteine subpopulations, and then, develop a strategy to maximize the coverage of SNO proteome. We obtained SNO-modified cysteine data sets for wild-type and S-nitrosoglutathione reductase knockout mouse hearts (S-nitrosoglutathione reductase is a negative regulator of S-nitrosoglutathione production) and nitric oxide-induced human embryonic kidney cell using 2 labeling reagents: the cysteine-reactive pyridyldithiol and iodoacetyl based tandem mass tags. Comparison revealed that <30% of the SNO-modified residues were detected by both tags, whereas the remaining SNO sites were only labeled by 1 reagent. Characterization of the 2 distinct subpopulations of SNO residues indicated that pyridyldithiol reagent preferentially labels cysteine residues that are more basic and hydrophobic. On the basis of this observation, we proposed a parallel dual-labeling strategy followed by an optimized proteomics workflow. This enabled the profiling of 493 SNO sites in S-nitrosoglutathione reductase knockout hearts. Using a protocol comprising 2 tags for dual-labeling maximizes overall detection of SNO by reducing the previously unrecognized labeling bias derived from different cysteine subpopulations. © 2015 American Heart Association, Inc.
Find related publications in this database (using NLM MeSH Indexing)
Alcohol Dehydrogenase - deficiency
Alcohol Dehydrogenase - genetics
Animals -
Biotin - metabolism
Cysteine - metabolism
Female -
HEK293 Cells -
Humans -
Male -
Mice, Inbred C57BL -
Mice, Knockout -
Molecular Probes -
Myocardium - metabolism
Nitrosation -
Nitroso Compounds - metabolism
Protein Processing, Post-Translational -
Proteomics - methods
Reproducibility of Results -
Tandem Mass Spectrometry -

Find related publications in this database (Keywords)
nitric oxide
oxidation-reduction
proteomics
S-nitrosothiols
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