Characterization and application of the biotin-switch assay for the identification of S-nitrosated proteins.
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.
Zhang
Yanhong
Y
Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.
Keszler
Agnes
A
Broniowska
Katarzyna A
KA
Hogg
Neil
N
D000818:Animals; D001205:Ascorbic Acid / Q000737:chemistry*; D001710:Biotin / Q000032:analysis* / Q000737:chemistry; D002478:Cells, Cultured; D003545:Cysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D051379:Mice; D015538:Nitrosation; D010084:Oxidation-Reduction; D011506:Proteins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D040901:Proteomics; D026403:S-Nitrosothiols / Q000032:analysis* / Q000494:pharmacology
D011506:Proteins; D026403:S-Nitrosothiols; D001710:Biotin; C021315:S-nitrosocysteine; D003545:Cysteine; D001205:Ascorbic Acid