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Proteomic analysis of S-nitrosylation and denitrosylation by resin-assisted capture

2009; Nature Portfolio; Volume: 27; Issue: 6 Linguagem: Inglês

10.1038/nbt.1545

1546-1696

Michael T. Forrester, J. Will Thompson, Matthew W. Foster, Leonardo Nogueira, M. Arthur Moseley, Jonathan S. Stamler,

Redox biology and oxidative stress

Forrester et al. adapt the biotin switch technique for studying reversible protein modification by nitric oxide, by using resin-assisted capture of S-nitrosothiols. This enables more sensitive detection of larger S-nitrosylated proteins and can be coupled with quantitative mass spectrometry to study the kinetics of denitrosylation. 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.