Generation and detection of S-nitrosothiols.
Nitric oxide (NO) plays a pivotal role in cellular signaling in many different organisms as the result of the modification of protein activities/functions by protein S-nitrosylation. This NO-dependent posttranslational modification is based on the attachment of NO to the sulfur moiety of cysteine residues. However, the instability of S-nitrosothiols makes it difficult to analyze this type of protein modification in vitro as well as in vivo. Jeffrey and colleagues developed a method--named the biotin switch method--that allows the detection and purification of S-nitrosylated proteins. The principle behind this technology is the substitution of the NO group by a biotin linker in a three-step procedure. First, the all free thiol groups are blocked with a thiol-reactive agent, followed by selective reduction of the S-nitrosylated cysteine residues using ascorbate. In the final step, the reduced thiol groups are labeled with a biotin linker, so that the previously S-nitrosylated cysteine residues are finally biotinylated. Afterwards, the biotinylated proteins can be detected with anti-biotin antibodies or can be purified by affinity chromatography on neutravidin agarose. In this chapter, we give a detailed description of the biotin switch method, which can be used for proteomics approach to identify candidates for protein S-nitrosylation as well as to analyse S-nitrosylation of selected proteins.
Lindermayr
Christian
C
Institute of Biochemical Plant Pathology, GSF - National Research Center for Environmental and Health, Munich/Neuherberg, Germany.
Sell
Simone
S
Durner
Jörg
J
D017360:Arabidopsis / Q000166:cytology / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000032:analysis / Q000737:chemistry; D001360:Avidin / Q000378:metabolism; D019921:Biotinylation; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D008967:Molecular Biology / Q000379:methods*; D015538:Nitrosation; D017433:Protein Structure, Secondary; D026403:S-Nitrosothiols / Q000032:analysis*
D029681:Arabidopsis Proteins; D026403:S-Nitrosothiols; C093483:neutravidin; D001360:Avidin