Oral Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Proteome-wide detection of cysteine nitrosylation targets and motifs using bioorthogonal cleavable-linker-based enrichment and switch technique (Cys-BOOST) (#227)

Ruzanna Mnatsakanyan 1 , Stavroula Markoutsa 1 , Kim Walbrunn 1 , Andreas Roos 1 2 , Steven H. L. Verhelst 1 3 , René P. Zahedi 1 4 5
  1. Leibniz-Institut für Analytische Wissenschaften – ISAS - e.V., Dortmund, Germany
  2. Department of Neuropediatrics, University Hospital Essen, Essen, Germany
  3. Laboratory of Chemical Biology, KU Leuven, Leuven, Belgium
  4. Segal Cancer Proteomics Centre, Montreal, QC, Canada
  5. Gerald Bronfman Department of Oncology, Jewish General Hospital, Montreal, Canada

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.

Using iodoTMT-based enrichment we quantified 9,966 Cys peptides and 3,446 background peptides (74% specificity), compared to 25,019 Cys peptides and only 581 background peptides for Cys-BOOST (98% specificity). We assessed the technical reproducibility of both workflows through individual processing of technical replicates and obtained relative standard deviations of 36 % (iodoTMT) vs 9 % (Cys-BOOST). We compared the average scaled TMT reporter intensities (sum of all TMT reporter intensities of Cys containing PSMs/number of Cys containing PSMs) of all Cys containing PSMs quantified by either Cys-BOOST or iodoTMT. The average scaled TMT intensity observed for Cys-BOOST was around 4 times higher, despite the higher number of Cys peptides detected with Cys-BOOST which may come along with the identification of many low abundant peptides. These results indicate a considerably higher recovery, which comes along with more precise quantification.

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.

  1. Proteome-wide detection of S-nitrosylation targets and motifs using bioorthogonal cleavable-linker-based enrichment and switch technique. Mnatsakanyan R, Markoutsa S, Walbrunn K, Roos A, Verhelst SHL, Zahedi RP. Nat Commun. 2019 May 16;10(1):2195. doi: 10.1038/s41467-019-10182-4.