Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
SHR Neuro Krebs Kardio Lipid Stoffw Microb
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
Doi: 10.1161/CIRCRESAHA.115.307336
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- Co-Autor*innen der Med Uni Graz
- Rainer Peter
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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