Quantification of mitochondrial S-nitrosylation by CysTMT⁶ switch assay.

Abstract

S-nitrosylation (SNO) is an important oxidative posttranslational modification in the regulation of cardiac mitochondria. SNO modification of several mitochondrial proteins has been associated with cardiac preconditioning and improved cell survival following ischemia/reperfusion injury. Due to their labile nature, SNO modifications are challenging to study using traditional biochemical techniques; particularly, the identification of individual modified cysteine residues. Here, we describe the details of the cysTMT(6) switch assay, a variation of the classic biotin switch protocol. The cysTMT(6) reagent provides a simplified and powerful approach to SNO detection by combining unambiguous identification of the modified cysteine residue and relative quantification of up to six samples by mass spectrometry analysis.

• Authors: Christopher I Murray, Hea Seung Chung, Helge Uhrigshardt, Jennifer E Van Eyk
• Journal: Methods in molecular biology (Clifton, N.J.) (Methods Mol Biol) Vol. 1005 Pg. 169-79 ( 2013) ISSN: 1940-6029 [Electronic] United States
• PMID: 23606257 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)

Chemical References

• Mitochondrial Proteins
• Nitric Oxide
• S-Nitrosoglutathione
• Biotin
• Cysteine

Topics

• Animals
• Biological Assay
• Biotin (chemistry)
• Cysteine (chemistry, metabolism)
• Mitochondria, Heart (drug effects, metabolism)
• Mitochondrial Proteins (chemistry, metabolism)
• Myocardium (metabolism)
• Nitric Oxide (biosynthesis)
• Oxidation-Reduction
• Protein Processing, Post-Translational
• Rats
• S-Nitrosoglutathione (pharmacology)