Biotin Switch Assay for S-Nitrosylation

Changes in S-nitrosylation levels were analyzed with the Biotin switch assay as described by Jaffrey and Snyder [88 (link)]. Briefly, samples of 200 μg protein were treated with S-methyl methanethiosulfonate (MMTS, Sigma Aldrich, USA, Cat. #64,306) for 1 h at 50 °C in the dark to block cysteine-free thiols, and then, proteins were precipitated with ice-cold acetone for 24 h at − 20 °C. The S-nitrosylated cysteine residues were reduced with 2.5 mM sodium ascorbate (Sigma Aldrich, USA, Cat. #A7506) and 4 mM N-[6-(Biotinamido) hexyl]-3′-(2′-pyridyldithio) propionamide (HPDP-biotin, Thermo Scientific, Cat. #21,341) was used to label the reduced thiols with biotin. Samples were incubated for 1 h with agarose-conjugated streptavidin beads (Thermo Scientific, Cat. #20,353) and centrifuged to pull down HPDP-biotinylated proteins, which were finally separated by SDS-PAGE to be detected with specific antibodies.