Nitric Oxide

Alicia Izquierdo-Álvarez, Daniel Tello, J. Daniel Cabrera-García, Antonio Martínez-Ruiz, Izquierdo-Álvarez, Alicia, Tello, Daniel, Cabrera-García, J. Daniel, Martínez-Ruiz, Antonio

S-nitrosylation and other reversible oxidative posttranslational modifications of proteins are part of the nonclassical mechanisms of nitric oxide signaling. The biotin switch technique for specifically labeling S-nitrosylated proteins opened the way to proteomic identification of these modifications. Since then, several variations and adaptations of the original method have been applied.We describe here the protocols of several techniques that can be used for the proteomic identification of these modifications, as well as for the detailed characterization of the modification of individual proteins. The fluorescence switch technique allows the proteomic identification of S-nitrosylated proteins based on their fluorescent labeling coupled to electrophoretic separation, as well as the comparison of the overall modification in different samples. The redox fluorescence switch is an adaptation to detect all the proteins reversibly oxidized in cysteine residues. We also describe the protocols of complementary techniques that allow comparing the extent of modification of individual proteins in several conditions by biotin switch, and the identification of modified residues by differential labeling adapted for mass spectrometry identification.