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

• Published in: Nature communications 2019-05, Vol.10 (1), p.2195-2195, Article 2195
• Main Authors: Mnatsakanyan, Ruzanna, Markoutsa, Stavroula, Walbrunn, Kim, Roos, Andreas, Verhelst, Steven H. L., Zahedi, René P.
• Format: Article
• Language: English
• Subjects: 631/1647/296; 631/337/458/2386; 631/45/612/1248; 631/92/475; 82/58; Catalysis; Chromatography, High Pressure Liquid - instrumentation; Chromatography, High Pressure Liquid - methods; Cysteine; Cysteine - analysis; Cysteine - metabolism; DNA biosynthesis; Glutamatergic transmission; HeLa Cells; Homeostasis; Humanities and Social Sciences; Humans; Hydrophobicity; multidisciplinary; Nitrosation - physiology; Organic chemistry; Phosphorylation; Protein folding; Protein Processing, Post-Translational - physiology; Proteins; Proteome - analysis; Proteome - metabolism; Proteomics; Proteomics - instrumentation; Proteomics - methods; Quantitative analysis; S-Nitrosoglutathione - chemistry; S-Nitrosoglutathione - metabolism; Science; Science (multidisciplinary); Sensitivity analysis; Synaptic transmission; Tandem Mass Spectrometry - instrumentation; Tandem Mass Spectrometry - methods; Target detection; Ubiquitination
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-019-10182-4

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. Reversible cysteine modifications play important roles in cellular redox signaling. Here, the authors develop a chemical proteomics strategy that enables the quantitative analysis of endogenous cysteine nitrosylation sites and their dynamic regulation under nitrosative stress conditions.