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.