title: Quantitative chemoproteomics for site-specific analysis of protein alkylation by 4-hydroxy-2-nonenal in cells.
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.
authors: Department of Biochemistry, Vanderbilt University School of Medicine , 465 21st Avenue South, U1213 MRB III, Nashville, Tennessee 37232, United States.
authors: Jing
authors: J
authors: Yang
authors: Keri A
authors: KA
authors: Tallman
authors: Ned A
authors: NA
authors: Porter
authors: Daniel C
authors: DC
authors: Liebler
mesh_terms: D000447:Aldehydes / Q000737:chemistry*; D000478:Alkylation; D002853:Chromatography, Liquid / Q000379:methods*; D015179:Colorectal Neoplasms / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000737:chemistry; D006801:Humans; D009363:Neoplasm Proteins / Q000032:analysis* / Q000737:chemistry*; D011499:Protein Processing, Post-Translational; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014407:Tumor Cells, Cultured
chemical_list: D000447:Aldehydes; D003432:Cross-Linking Reagents; D009363:Neoplasm Proteins; C027576:4-hydroxy-2-nonenal
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