Figure 1

Development of NitroDIGE to detect SNO-proteins. (A) Schematic showing the NitroDIGE method for labeling of S-nitrosylated cysteine thiols. A hypothetical protein is indicated with cysteines in the free thiol, disulfide, or nitrosothiol conformation. Free thiols are first blocked with MMTS. Ascorbate selectively releases NO from S-nitrosylated cysteine thiols. The fluorescent thiol-reactive CyDye™ (Cy3 or Cy5) reacts with NO-released thiols to form stable fluorescent complexes. (B) The specificity and sensitivity of NitroDIGE. (a) Cell lysates (20 μg) were treated with 200 μM SNOC, and SNO-proteins were analyzed by NitroDIGE; 5 μg of proteins from each NitroDIGE-labeled sample were separated by SDS-PAGE and Cy5 fluorescence signals were collected. Control omitting SNOC, MMTS, ascorbate, or Cy5 demonstrated that CyDye™ specifically labeled SNO-proteins. (b) In the BST, 250 μg of SNO-proteins were labeled with Biotin-HPDP, pulled down by avidin-agarose, and visualized by silver staining. (C) BV-2 cell lysates were exposed to different doses of SNOC as a test of sensitivity. The NitroDIGE method detected SNO-proteins in cell lysates treated with as low as 10 μM SNOC.