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Proteome-wide detection of S-nitrosylation targets and motifs using bioorthogonal cleavable-linker-based enrichment and switch technique.

Nature communications (2019-05-18)
Ruzanna Mnatsakanyan, Stavroula Markoutsa, Kim Walbrunn, Andreas Roos, Steven H L Verhelst, René P Zahedi
ABSTRACT

Cysteine modifications emerge as important players in cellular signaling and homeostasis. Here, we present a chemical proteomics strategy for quantitative analysis of reversibly modified Cysteines using bioorthogonal cleavable-linker and switch technique (Cys-BOOST). Compared to iodoTMT for total Cysteine analysis, Cys-BOOST shows a threefold higher sensitivity and considerably higher specificity and precision. Analyzing S-nitrosylation (SNO) in S-nitrosoglutathione (GSNO)-treated and non-treated HeLa extracts Cys-BOOST identifies 8,304 SNO sites on 3,632 proteins covering a wide dynamic range of the proteome. Consensus motifs of SNO sites with differential GSNO reactivity confirm the relevance of both acid-base catalysis and local hydrophobicity for NO targeting to particular Cysteines. Applying Cys-BOOST to SH-SY5Y cells, we identify 2,151 SNO sites under basal conditions and reveal significantly changed SNO levels as response to early nitrosative stress, involving neuro(axono)genesis, glutamatergic synaptic transmission, protein folding/translation, and DNA replication. Our work suggests SNO as a global regulator of protein function akin to phosphorylation and ubiquitination.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Tris(3-hydroxypropyltriazolylmethyl)amine, 95%
Sigma-Aldrich
Tetraethylammonium borohydride, technical, ≥95% (T)