Identification and quantification of S-nitrosylation by cysteine reactive tandem mass tag switch assay - PubMed (original) (raw)

In vivo detection of S-nitrosylation by cysTMT6 switch assay. A, Representative silver stained gel of SNO-modifications detected using the biotin switch assay on HPAEC in culture treated with SNO-Cys (50, 200 μ

m

) or control conditions (untreated/DTT, 1 m

m l

-NAME, untreated or 200 μ

m

Cys) (n = 3). Staining indicates SNO-modification with 50 μ

m

SNO-Cys treatment, increasing with 200 μ

m

. B, Experimental schema of the in vivo cysTMT6 switch assay to identify and map SNO-modifications in cultured HPAECs induced by 50 and 200 μ

m

SNO-Cys treatment or control conditions (n = 3). Samples were labeled with cysTMT6 reagent in the following order; untreated/DTT (126 Da), 1 m

m l

-NAME (127 Da), untreated (128 Da), 200 μ

m

Cys (129 Da), 50 μ

m

SNO-Cys (130 Da), and 200 μ

m

SNO-Cys (131 Da). C–E, Examples of average normalized reporter ion intensities for cysteine residues identified to be SNO-modified in vivo (n = 4, 2, and 1 observations, respectively. * = p < 0.05). Error bars indicate S.E. A total of 25 sites were indentified, 22 of which corresponded to sites identified in the in vitro data set (

supplemental Tables S5

for detailed MS data). F, In vivo SNO-modifications were matched to the corresponding in vitro site GSNO-reactivity (m) (gray line) and plotted. Greater SNO stimuli (200 μ

m

SNO-cys, green line, 22 sites) was found to modify more insensitive sites than lower doses (50 μ

m

, red line, 9 sites). For ease of interpretation, the 200 μ

m

site responses were offset below the in vitro data plot. Black arrow heads indicate the relative position of thiol-reactivities determined for the modified sites presented in C–E. (See

supplemental Fig. S6

).