Assessment of S-nitrosothiols on diaminofluorescein gels

Abstract S-Nitrosylation is the modification of a cysteine thiol on a protein or peptide by a nitric oxide (NO) group. Increasing evidence suggests that S-nitrosylation of critical cysteine residues regulates protein function and cell signaling. However, progress in the field has been hampered by a lack of accurate and easy methods for detecting S-nitrosylation and other labile NO-based modifications in samples. We have developed a rapid method for analyzing protein and peptide S-nitrosothiols on gels using the fluorescent probes 4,5-diaminofluorescein (DAF-2) and 3-amino,4-aminomethyl-2′7′-difluorescein (DAF-FM). Low micromolar levels of S-nitrosylated bovine serum albumin (BSA), but not control BSA, are detected on the gels. In addition, NO-based modifications of proteins and peptides on nonsulfur groups (e.g., carbon, oxygen, nitrogen) are detected on DAF gels. Analysis of intracellular proteins on DAF gels indicated that the NO donor compound S-nitrosoglutathione S-nitrosylates significantly more proteins in mitochondrial lysates than in cytoplasmic lysates. In summary, the use of DAF gels is an easy method to analyze in vitro protein and peptide S-nitrosylation. The assay is also the first gel-based method to identify not only S-nitrosothiols but also other labile NO-based modifications of proteins and peptides.