Proteomic analysis of S-nitrosylation and denitrosylation by resin-assisted capture.
We have modified the biotin switch assay for protein S-nitrosothiols (SNOs), using resin-assisted capture (SNO-RAC). Compared with existing methodologies, SNO-RAC requires fewer steps, detects high-mass S-nitrosylated proteins more efficiently, and facilitates identification and quantification of S-nitrosylated sites by mass spectrometry. When combined with iTRAQ labeling, SNO-RAC revealed that intracellular proteins may undergo rapid denitrosylation on a global scale. This methodology is readily adapted to analyzing diverse cysteine-based protein modifications, including S-acylation.
Forrester
Michael T
MT
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina, USA.
Thompson
J Will
JW
Foster
Matthew W
MW
Nogueira
Leonardo
L
Moseley
M Arthur
MA
Stamler
Jonathan S
JS
D001710:Biotin / Q000378:metabolism; D002460:Cell Line; D003545:Cysteine / Q000031:analogs & derivatives / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D026403:S-Nitrosothiols / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013438:Sulfhydryl Compounds / Q000378:metabolism*
D020543:Proteome; D026403:S-Nitrosothiols; D013438:Sulfhydryl Compounds; D001710:Biotin; C021315:S-nitrosocysteine; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D003545:Cysteine