Characterization and application of the biotin-switch assay for the identification of S-nitrosated proteins. Free Radic Biol Med 2005 Apr 01;38(7):874-81
Date
03/08/2005Pubmed ID
15749383DOI
10.1016/j.freeradbiomed.2004.12.012Scopus ID
2-s2.0-14644423288 (requires institutional sign-in at Scopus site) 104 CitationsAbstract
S-Nitrosation of protein cysteinyl residues has been suggested to be an important nitric oxide-dependent posttranslational modification. The so-called biotin-switch method has been developed to identify S-nitrosated proteins. This method relies on the selective reduction of S-nitrosothiols by ascorbate. In this study we have assessed the ability of ascorbate to reduce S-nitrosothiols and show that ascorbate is a very inefficient reducing agent. We show that higher concentrations of ascorbate and longer incubation times can significantly improve immunological detection of S-nitrosothiols. We have compared immunological detection of S-nitrosothiols with the level of intracellular S-nitrosothiols measured by tri-iodide chemiluminescence and show that the biotin-switch method is capable of detecting only high (nmol/mg protein) levels of intracellular S-nitrosothiols obtained after exposing cells to S-nitrosocysteine, but not the low levels observed during physiological nitric oxide formation. Preliminary proteomic analysis of protein S-nitrosothiols has identified elongation factor 2, heat shock protein 90 beta, and a 65-kDa macrophage protein homologous to human L-plastin as major nitrosation targets at high intracellular nitrosation levels in the murine macrophage-derived RAW 264.7 cell line. While the biotin-switch method may be a useful tool to aid in the positive identification of protein S-nitrosothiols, it cannot match the sensitivity of chemiluminescence-based methods and its use in proteomic studies likely suffers from selective detection of more easily reducible S-nitrosothiols.
Author List
Zhang Y, Keszler A, Broniowska KA, Hogg NAuthors
Neil Hogg PhD Associate Dean, Professor in the Biophysics department at Medical College of WisconsinAgnes Keszler PhD Research Scientist I in the Biophysics department at Medical College of Wisconsin
MESH terms used to index this publication - Major topics in bold
AnimalsAscorbic Acid
Biotin
Cells, Cultured
Cysteine
Mice
Nitrosation
Oxidation-Reduction
Proteins
Proteomics
S-Nitrosothiols