Abstract
Protein alkylation by 4-hydroxy-2-nonenal (HNE), an endogenous lipid derived electrophile, contributes to stress signaling and cellular toxicity. Although previous work has identified protein targets for HNE alkylation, the sequence specificity of alkylation and dynamics in a cellular context remain largely unexplored. We developed a new quantitative chemoproteomic platform, which uses isotopically tagged, photocleavable azido-biotin reagents to selectively capture and quantify the cellular targets labeled by the alkynyl analogue of HNE (aHNE). Our analyses site-specifically identified and quantified 398 aHNE protein alkylation events (386 cysteine sites and 12 histidine sites) in intact cells. This data set expands by at least an order of magnitude the number of such modification sites previously reported. Although adducts formed by Michael addition are thought to be largely irreversible, we found that most aHNE modifications are lost rapidly in situ. Moreover, aHNE adduct turnover occurs only in intact cells and loss rates are site-selective. This quantitative chemoproteomics platform provides a versatile general approach to map bioorthogonal-chemically engineered post-translational modifications and their cellular dynamics in a site-specific and unbiased manner.
Highlights
The covalent modification of proteins by endogenous lipid derived electrophiles (LDEs) triggers cytotoxic and adaptive responses associated with oxidative stress.[1,2] Of the dozens of known LDEs, 4-hydroxy-2-nonenal (HNE) is the most studied, owing to its high reactivity and evidence that it activates diverse pathways governing cellular signaling and stress.[3,4] Understanding how HNE and other LDEs modify cellular proteomes can offer new insights into mechanisms of chemical toxicity, inflammation, and disease
We recently described a chemoproteomics method for sitespecific mapping of protein S-sulfenylation in cells,[13] in which S-sulfenyl residues are tagged with the alkynyl-dimedone probe DYn-2, biotinylated by Click chemistry with a UVcleavable azido-biotin (Az-UV-biotin),[14] which permits efficient streptavidin capture and photorelease of tagged, S-sulfenyl peptides
We have adopted key features of our recently published chemoproteomic method for sitespecific mapping of protein S-sulfenylation in cells,[13] including site labeling with an alkynyl probe, bioorthogonal conjugation with Az-UV-biotin, and high resolution LC−Mass spectrometry (MS)/MS
Summary
Editors' Highlight mediated covalent modification in cells, with respect to adduct stability and turnover?. Metabolic incorporation of cells under different conditions with an alkyne tagged probe, (2) digesting cell lysates into peptides with trypsin, (3) conjugating the alkyne tagged peptides with light Az-UV-biotin or heavy Az-UV-biotin via CuI-catalyzed azide−alkyne cycloaddition reaction (Click chemistry),[21] (4) enrichment of biotin-tagged tryptic peptides by streptavidin capture and photorelease, (5) liquid chromatography−tandem mass spectrometry (LC−MS/MS)-based shotgun proteomics and informatics analyses for peptide identification and quantification. After tryptic digestion of cell lysates, aHNE-modified peptides from two identical proteome samples were conjugated with the light and heavy Az-UV-biotin reagents, respectively, and mixed at a 1:1 ratio. We enriched alkylated peptides from the lysate and intact cell recovery experiments, digested the proteins and quantified adducted peptides by light/heavy AzUV-biotin labeling and LC−MS/MS (Table S3 in the Supporting Information). The results suggest that aHNE adduct instability is mediated by factors present in intact, metabolically competent cells and is not due to simple chemical instability
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