The levels of HNE and HNE-protein adducts are elevated in cells and tissues exposed to oxidative stress, and HNE can regulate redox-responsive signaling pathways by still poorly understood mechanisms2,8,9. (PTM) of proteins1. Many PTMs are enzyme-catalyzed, but others reflect the direct (non-enzymatic) oxidative or electrophilic modification of nucleophilic residues, such as cysteine, by reactive small-molecules that are often the products of oxidative stress2,3. Chronic inflammation or hypoxia, for instance, induces the peroxidation of polyunsaturated lipids to generate a broad range of electrophilic products2. These lipid-derived electrophiles (LDEs) can change DNA and proteins to promote cytotoxicity and have been implicated in the pathogenesis of many diseases4. A growing body of studies also suggests that, at lesser and more physiological concentrations, LDEs serve as endogenous messengers that modulate the response of signaling pathways5,6. 4-Hydroxynonenal (HNE), for instance, is a major product generated when free radicals initiate the non-enzymatic fragmentation of lipids in biological membranes2,7. The levels of HNE and HNE-protein adducts are elevated in cells and tissues exposed to oxidative stress, and HNE can regulate redox-responsive signaling pathways by still poorly understood mechanisms2,8,9. 15-deoxy-12,14-prostaglandin J2 (15d-PGJ2) is usually another LDE produced by a set of enzymes that metabolize arachidonic acid10. 15d-PGJ2 exhibits anti-inflammatory and cytoprotective properties and has therefore been designated as a pro-resolving transmission10. A third example is the LDE 2-trans-hexadecenal (2-HD), which is a product of sphingolipid metabolism and has recently been shown to function as a protein-modifying cofactor that promotes mitochondrial pathways for apoptosis11. Understanding the protein targets of LDEs is critical for elucidating their cellular functions and mechanisms of action. Here, chemoproteomic methods have proven particularly useful for inventorying a large number of proteins that react with LDEs in cells and tissues2,1217; however, quantifying the potency and specificity of these reactions to identify the most sensitive sites in the proteome to electrophilic modification has proven challenging. Here, we Capromorelin Tartrate describe a competitive activity-based protein profiling Capromorelin Tartrate (ABPP) method for quantifying the reactivity of electrophilic compounds against 1000+ cysteines in parallel in the human proteome. Using this approach, we identify select units of proteins that are preferentially altered by HNE and 15d-PGJ2. We show that one of these proteins, ZAK kinase, is usually labeled by HNE on a conserved, active site-proximal cysteine residue, which inhibits the enzyme and suppresses the activation of JNK pathways by oxidative stress in malignancy cells. == Results == == Quantitative proteomic profiling of Capromorelin Tartrate LDE-cysteine reactions == Among the 20 protein-coding amino acids, cysteine is unique owing to its high nucleophilicity, which renders its sensitivity to modification by endogenous and exogenous electrophiles and oxidants3. Cysteine reactions with electrophilic metabolites have been characterized for purified proteins18,19, and, on a global level in cells and tissues using mass spectrometry-based chemoproteomic2,1217and imaging methods20. These studies, along with analytical, quantum mechanical, and kinetic work21,22, have, for the most part, confirmed MYH9 the preferential reactivity that Michael acceptor electrophiles like HNE show for cysteine over other nucleophilic amino acids (e.g., lysine, histidine) in proteomes. We were interested in building on these past findings to determine whether individual cysteines in the proteome display differences in their reactivity with LDEs, and, if so, whether Capromorelin Tartrate potential hot-spots for electrophile modification might constitute important nodes in signaling pathways of redox sensing and response. We previously explained a chemoproteomic method termed isoTOP-ABPP (isotopic Tandem Orthogonal Proteolysis-ABPP) and its use to quantify the intrinsic reactivity of cysteine residues in cell and tissue proteomes23. Here, we envisioned that isoTOP-ABPP could be advanced to discover and quantify reactions between cysteines and electrophilic small-molecules in proteomes. In this competitive version of isoTOP-ABPP (Fig. 1a, b), a proteome is usually treated with an electrophile (experimental sample) or DMSO (control sample). Both proteomic samples are then labeled with an alkynylated iodoacetamide (IA) probe (Fig. 1b) and conjugated by copper-catalyzed azide-alkyne cycloaddition (CuAAC or click24) chemistry to light and heavy azide-biotin tags, respectively, each made up of a Tobacco Etch Capromorelin Tartrate Virus (TEV) cleavage sequence. The light and heavy samples are then mixed and subjected to our explained isoTOP-ABPP protocol for peptide enrichment, cleavage, and identification, where IA-modified cysteines are recognized and their extent of labeling quantified based on MS2 and MS1 profiles, respectively23. Electrophile-sensitive cysteines.