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Chemoproteomics Reveals Chemical Diversity and Dynamics of 4-Oxo-2-nonenal Modifications in Cells*

  • Author Footnotes
    ‡‡ These authors contributed equally to this work.
    Rui Sun
    Footnotes
    ‡‡ These authors contributed equally to this work.
    Affiliations
    From the State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Drug Discovery for Metabolic Disease, Center for New Drug Safety Evaluation and Research, China Pharmaceutical University, Nanjing 211198, China;

    State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences, Beijing Institute of Radiation Medicine, Beijing 102206, China;
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  • Author Footnotes
    ‡‡ These authors contributed equally to this work.
    Ling Fu
    Footnotes
    ‡‡ These authors contributed equally to this work.
    Affiliations
    State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences, Beijing Institute of Radiation Medicine, Beijing 102206, China;
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  • Keke Liu
    Affiliations
    State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences, Beijing Institute of Radiation Medicine, Beijing 102206, China;
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  • Caiping Tian
    Affiliations
    State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences, Beijing Institute of Radiation Medicine, Beijing 102206, China;
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  • Author Footnotes
    §§ These authors are co-corresponding authors.
    Yong Yang
    Footnotes
    §§ These authors are co-corresponding authors.
    Affiliations
    From the State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Drug Discovery for Metabolic Disease, Center for New Drug Safety Evaluation and Research, China Pharmaceutical University, Nanjing 211198, China;
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  • Keri A. Tallman
    Affiliations
    Department of Chemistry, Vanderbilt University, Nashville, Tennessee 37232;
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  • Ned A. Porter
    Affiliations
    Department of Chemistry, Vanderbilt University, Nashville, Tennessee 37232;
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  • Author Footnotes
    §§ These authors are co-corresponding authors.
    Daniel C. Liebler
    Footnotes
    §§ These authors are co-corresponding authors.
    Affiliations
    Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232
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  • Jing Yang
    Correspondence
    To whom correspondence should be addressed:State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences, Beijing Institute of Radiation Medicine, Beijing 102206, China. Tel.:86-10-61777114
    Affiliations
    State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences, Beijing Institute of Radiation Medicine, Beijing 102206, China;
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  • Author Footnotes
    * This work was supported in part by the National Key R&D Program of China (No. 2016YFA0501303), the National Natural Science Foundation of China (No. 31500666 and No. 81573395), the Beijing Natural Science Foundation (No.5162009), and Beijing Nova Program (No. Z171100001117014) to J.Y. and the National Institutes of Health (U24CA159988) to D.C.L.
    This article contains supplemental material.
    ‡‡ These authors contributed equally to this work.
    §§ These authors are co-corresponding authors.
Open AccessPublished:August 16, 2017DOI:https://doi.org/10.1074/mcp.RA117.000116
      4-Oxo-2-nonenal (ONE) derived from lipid peroxidation modifies nucleophiles and transduces redox signaling by its reactions with proteins. However, the molecular interactions between ONE and complex proteomes and their dynamics in situ remain largely unknown. Here we describe a quantitative chemoproteomic analysis of protein adduction by ONE in cells, in which the cellular target profile of ONE is mimicked by its alkynyl surrogate. The analyses reveal four types of ONE-derived modifications in cells, including ketoamide and Schiff-base adducts to lysine, Michael adducts to cysteine, and a novel pyrrole adduct to cysteine. ONE-derived adducts co-localize and exhibit crosstalk with many histone marks and redox sensitive sites. All four types of modifications derived from ONE can be reversed site-specifically in cells. Taken together, our study provides much-needed mechanistic insights into the cellular signaling and potential toxicities associated with this important lipid derived electrophile.
      Reactive oxygen species generated from biological processes or environmental insults can result in damage to biomacromolecules including proteins and DNA (
      • Ezraty B.
      • Gennaris A.
      • Barras F.
      • Collet J.F.
      Oxidative stress, protein damage and repair in bacteria.
      ,
      • Sies H.
      • Berndt C.
      • Jones D.P.
      Oxidative Stress.
      ). The polyunsaturated fatty acyl chains found in biological membranes and lipoproteins are particularly susceptible to reactive oxygen species, leading to free radical chain autoxidation and the formation of a variety of unsaturated lipid hydroperoxides and their electrophilic decomposition products, such as 4-hydroxy-2-nonenal (HNE)
      The abbreviations used are: HNE, 4-hydroxy-2-nonenal; ACN, acetonitrile; BCA, bicinchoninic acid; DMEM, Dulbecco's modified eagle's medium; DTT, dithiothreitol; FDR, false discovery rate; HCD, high energy collisional dissociation; LC-MS/MS, liquid chromatography-tandem mass spectrometry; LDE, lipid derived electrophile; IAM, iodoacetamide; IR, ionizing radiation; MeOH, methanol; ONE, 4-oxo-2-nonenal; PBS, phosphate buffered saline; PTM, post-translational modification; PVDF, polyvinylidene difluoride; PSM, peptide-spectrum match; RT, room temperature; RSA, residue solvent accessibility; SCX, strong cation exchange; S/N, signal to noise; TBST, tris-buffered saline plus 0.05% Tween-20 (v/v); TBTA, tris[(1-benzyl-1H-1,2,3-triazol-4-yl) methyl] amine.
      1The abbreviations used are: HNE, 4-hydroxy-2-nonenal; ACN, acetonitrile; BCA, bicinchoninic acid; DMEM, Dulbecco's modified eagle's medium; DTT, dithiothreitol; FDR, false discovery rate; HCD, high energy collisional dissociation; LC-MS/MS, liquid chromatography-tandem mass spectrometry; LDE, lipid derived electrophile; IAM, iodoacetamide; IR, ionizing radiation; MeOH, methanol; ONE, 4-oxo-2-nonenal; PBS, phosphate buffered saline; PTM, post-translational modification; PVDF, polyvinylidene difluoride; PSM, peptide-spectrum match; RT, room temperature; RSA, residue solvent accessibility; SCX, strong cation exchange; S/N, signal to noise; TBST, tris-buffered saline plus 0.05% Tween-20 (v/v); TBTA, tris[(1-benzyl-1H-1,2,3-triazol-4-yl) methyl] amine.
      and 4-oxo-2-nonenal (ONE) (
      • Sayre L.M.
      • Lin D.
      • Yuan Q.
      • Zhu X.
      • Tang X.
      Protein adducts generated from products of lipid oxidation: focus on HNE and one.
      ). These lipid derived electrophiles (LDE) can react with nucleophiles on proteins, including cysteine, lysine, and histidine (
      • Schopfer F.J.
      • Cipollina C.
      • Freeman B.A.
      Formation and signaling actions of electrophilic lipids.
      ). Chemical modification induced by the lipid derived electrophiles (LDEs) has emerged an important mechanism for cells to regulate redox signaling and drive cytotoxic responses (
      • Rudolph T.K.
      • Freeman B.A.
      Transduction of redox signaling by electrophile-protein reactions.
      ). Dysregulation triggered by these LDE-protein interactions is associated with inflammation, diabetes, neurodegenerative disorders, and cardiovascular diseases (
      • Uchida K.
      HNE as an inducer of COX-2.
      ,
      • Cohen G.
      • Riahi Y.
      • Sunda V.
      • Deplano S.
      • Chatgilialoglu C.
      • Ferreri C.
      • Kaiser N.
      • Sasson S.
      Signaling properties of 4-hydroxyalkenals formed by lipid peroxidation in diabetes.
      ,
      • Di Domenico F.
      • Tramutola A.
      • Butterfield D.A.
      Role of 4-hydroxy-2-nonenal (HNE) in the pathogenesis of alzheimer disease and other selected age-related neurodegenerative disorders.
      ,
      • Koenitzer J.R.
      • Freeman B.A.
      Redox signaling in inflammation: interactions of endogenous electrophiles and mitochondria in cardiovascular disease.
      ).
      Identifying the protein targets of LDEs is critical for better understanding of their functional impact on specific signaling pathways and cellular functions. Recent advances in proteomics have improved the detection of LDE-induced protein modifications and greatly expanded the global inventories of targeted proteins and/or sites of LDEs both in vitro and in situ, especially for HNE (
      • Yang J.
      • Tallman K.A.
      • Porter N.A.
      • Liebler D.C.
      Quantitative chemoproteomics for site-specific analysis of protein alkylation by 4-hydroxy-2-nonenal in cells.
      ,
      • Codreanu S.G.
      • Ullery J.C.
      • Zhu J.
      • Tallman K.A.
      • Beavers W.N.
      • Porter N.A.
      • Marnett L.J.
      • Zhang B.
      • Liebler D.C.
      Alkylation damage by lipid electrophiles targets functional protein systems.
      ,
      • Wang C.
      • Weerapana E.
      • Blewett M.M.
      • Cravatt B.F.
      A chemoproteomic platform to quantitatively map targets of lipid-derived electrophiles.
      ,
      • Yuan W.
      • Zhang Y.
      • Xiong Y.
      • Tao T.
      • Wang Y.
      • Yao J.
      • Zhang L.
      • Yan G.
      • Bao H.
      • Lu H.
      Highly selective and large scale mass spectrometric analysis of 4-Hydroxynonenal modification via fluorous derivatization and fluorous solid-phase extraction.
      ,
      • Chen Y.
      • Cong Y.
      • Quan B.
      • Lan T.
      • Chu X.
      • Ye Z.
      • Hou X.
      • Wang C.
      Chemoproteomic profiling of targets of lipid-derived electrophiles by bioorthogonal aminooxy probe.
      ). Although ONE and HNE share a nearly identical chemical structure (supplemental Scheme S1), ONE is more reactive and cytotoxic than HNE in neuronal cells (
      • Lin D.
      • Lee H.G.
      • Liu Q.
      • Perry G.
      • Smith M.A.
      • Sayre L.M.
      4-Oxo-2-nonenal is both more neurotoxic and more protein reactive than 4-hydroxy-2-nonenal.
      ). Unlike HNE, which preferentially reacts with proteomic cysteines (
      • Yang J.
      • Tallman K.A.
      • Porter N.A.
      • Liebler D.C.
      Quantitative chemoproteomics for site-specific analysis of protein alkylation by 4-hydroxy-2-nonenal in cells.
      ,
      • Wang C.
      • Weerapana E.
      • Blewett M.M.
      • Cravatt B.F.
      A chemoproteomic platform to quantitatively map targets of lipid-derived electrophiles.
      ), ONE displays a broader range of adduction chemistry because of differences in its stereoelectronic properties (
      • Sayre L.M.
      • Lin D.
      • Yuan Q.
      • Zhu X.
      • Tang X.
      Protein adducts generated from products of lipid oxidation: focus on HNE and one.
      ). For instance, ONE exhibits more potent chemical reactivity with lysine residues, as compared with HNE. Of interest, Galligan et al. recently showed that ONE forms stable ketoamide adducts with several lysine residues on histones and blocks nucleosome assembly, thereby suggesting a potential link between oxidative stress and epigenetic effects (
      • Galligan J.J.
      • Rose K.L.
      • Beavers W.N.
      • Hill S.
      • Tallman K.A.
      • Tansey W.P.
      • Marnett L.J.
      Stable histone adduction by 4-oxo-2-nonenal: a potential link between oxidative stress and epigenetics.
      ). In addition, ONE renders more likely intra- or intermolecular cross-linking of its targets, which has been implicated in many diseases associated with protein aggregation. For example, ONE facilitates the formation of more stable α-synuclein oligomers than those induced by HNE (
      • Nasstrom T.
      • Fagerqvist T.
      • Barbu M.
      • Karlsson M.
      • Nikolajeff F.
      • Kasrayan A.
      • Ekberg M.
      • Lannfelt L.
      • Ingelsson M.
      • Bergstrom J.
      The lipid peroxidation products 4-oxo-2-nonenal and 4-hydroxy-2-nonenal promote the formation of alpha-synuclein oligomers with distinct biochemical, morphological, and functional properties.
      ). More recently, Marnett and coworkers showed that ONE, rather than HNE, forms cross-links and alters the activities of pyruvate kinase M2 and peptidylprolyl cis/trans isomerase A1 in cells (
      • Camarillo J.M.
      • Ullery J.C.
      • Rose K.L.
      • Marnett L.J.
      Electrophilic modification of PKM2 by 4-hydroxynonenal and 4-oxononenal results in protein cross-linking and kinase inhibition.
      ,
      • Aluise C.D.
      • Camarillo J.M.
      • Shimozu Y.
      • Galligan J.J.
      • Rose K.L.
      • Tallman K.A.
      • Marnett L.J.
      Site-specific, intramolecular cross-linking of Pin1 active site residues by the lipid electrophile 4-oxo-2-nonenal.
      ). Despite these interesting findings, the molecular interactions between ONE and complex proteomes and their dynamics remain uncertain with respect to the following issues. First, the full nature of in situ adduction chemistry of ONE is still unknown, although the chemical reactivity of ONE with nucleophilic residues has been analyzed in chemical model systems (
      • Sayre L.M.
      • Lin D.
      • Yuan Q.
      • Zhu X.
      • Tang X.
      Protein adducts generated from products of lipid oxidation: focus on HNE and one.
      ,
      • Zhang W.H.
      • Liu J.
      • Xu G.
      • Yuan Q.
      • Sayre L.M.
      Model studies on protein side chain modification by 4-oxo-2-nonenal.
      ,
      • Doorn J.A.
      • Petersen D.R.
      Covalent modification of amino acid nucleophiles by the lipid peroxidation products 4-hydroxy-2-nonenal and 4-oxo-2-nonenal.
      ). Second, the site-specific target profile and selectivity of ONE across native proteomes are still unexplored. Third, it is unclear whether ONE-derived adductions are reversible in cells, though two recent studies have shown that one of these modifications on histones can be removed by deacylase Sirt2 (
      • Cui Y.
      • Li X.
      • Lin J.
      • Hao Q.
      • Li X.D.
      Histone ketoamide adduction by 4-oxo-2-nonenal is a reversible posttranslational modification regulated by Sirt2.
      ,
      • Jin J.
      • He B.
      • Zhang X.
      • Lin H.
      • Wang Y.
      SIRT2 Reverses 4-oxononanoyl lysine modification on histones.
      ).
      Here we present the first global survey of ONE adduct chemistry, targeting sites, and dynamics in intact cells using a generalized quantitative chemoproteomic platform (
      • Yang J.
      • Tallman K.A.
      • Porter N.A.
      • Liebler D.C.
      Quantitative chemoproteomics for site-specific analysis of protein alkylation by 4-hydroxy-2-nonenal in cells.
      ), in which the cellular target profile of ONE is mimicked by its alkynyl surrogate (aONE, Fig. 1). This analysis not only greatly expand the inventory of ONE-adducts in cells but also identify a novel pyrrole adduct to cysteine. Biochemical analyses further show that these ONE-derived adducts co-localize and exhibit crosstalk with many histone marks and redox sensitive sites. Moreover, quantitative analyses reveal that all four types of modifications derived from ONE are reversible in cells in a site-specific manner, which may be controlled by Sirt2-mediated deacylation and other unknown mechanisms.
      Figure thumbnail gr1
      Fig. 1Workflow for quantitative chemoproteomic analysis of dynamic aONE-derived protein adducts in cells.

      DISCUSSION

      Among all known LDEs, HNE is the most extensively studied, and hundreds of proteins have been identified as the targets of this electrophile by either traditional analytical approaches or state-of-the-art chemoproteomics (
      • Yang J.
      • Tallman K.A.
      • Porter N.A.
      • Liebler D.C.
      Quantitative chemoproteomics for site-specific analysis of protein alkylation by 4-hydroxy-2-nonenal in cells.
      ,
      • Codreanu S.G.
      • Ullery J.C.
      • Zhu J.
      • Tallman K.A.
      • Beavers W.N.
      • Porter N.A.
      • Marnett L.J.
      • Zhang B.
      • Liebler D.C.
      Alkylation damage by lipid electrophiles targets functional protein systems.
      ,
      • Wang C.
      • Weerapana E.
      • Blewett M.M.
      • Cravatt B.F.
      A chemoproteomic platform to quantitatively map targets of lipid-derived electrophiles.
      ,
      • Yuan W.
      • Zhang Y.
      • Xiong Y.
      • Tao T.
      • Wang Y.
      • Yao J.
      • Zhang L.
      • Yan G.
      • Bao H.
      • Lu H.
      Highly selective and large scale mass spectrometric analysis of 4-Hydroxynonenal modification via fluorous derivatization and fluorous solid-phase extraction.
      ,
      • Chen Y.
      • Cong Y.
      • Quan B.
      • Lan T.
      • Chu X.
      • Ye Z.
      • Hou X.
      • Wang C.
      Chemoproteomic profiling of targets of lipid-derived electrophiles by bioorthogonal aminooxy probe.
      ,
      • Carini M.
      • Aldini G.
      • Facino R.M.
      Mass spectrometry for detection of 4-hydroxy-trans-2-nonenal (HNE) adducts with peptides and proteins.
      ,
      • Sousa B.C.
      • Pitt A.R.
      • Spickett C.M.
      Chemistry and analysis of HNE and other prominent carbonyl-containing lipid oxidation compounds.
      ). ONE, the 4-keto analog of HNE (supplemental Scheme S1), exhibits much higher intrinsic reactivity and cytotoxicity than those of HNE and has drawn much attention since its discovery approximately two decades ago (
      • Lee S.H.
      • Blair I.A.
      Characterization of 4-oxo-2-nonenal as a novel product of lipid peroxidation.
      ). However, study of ONE has lagged far behind study of HNE. The potential target profile of ONE-derived adductions is far more complicated than that of HNE because of the distinct stereoelectronic properties of ONE. A recent proteomic analysis confirmed that HNE and ONE display significant differences in cellular protein targets and sites of reactivity (
      • Codreanu S.G.
      • Ullery J.C.
      • Zhu J.
      • Tallman K.A.
      • Beavers W.N.
      • Porter N.A.
      • Marnett L.J.
      • Zhang B.
      • Liebler D.C.
      Alkylation damage by lipid electrophiles targets functional protein systems.
      ). However, a site-specific target profile of ONE in cells has never been reported.
      In this study, we present the first proteome-wide survey of ONE adduct chemistry, target preference, and site-specific selectivity in intact cells using a chemoproteomic approach. Our analysis reveals four types of ONE-derived modifications on 234 nucleophilic sites of 183 proteins in RKO and HeLa cells. Notably, we map ONE-derived lysine adductions (ketoamide or Schiff-base) onto many histone marks. We further demonstrate anti-synergistic crosstalk of ONE adduction with monoubiquitination of H2BK120, a key histone mark, which promotes IR-induced DNA damage (Fig. 2). We also uncovered potential crosstalk between ONE adduction and sulfinic acid formation of PARK7/DJ-1 C106 (supplemental Fig. S4). Considering the chemical diversity of ONE-derived adductions, the future exploration of crosstalk within the same protein or across a protein-protein network is warranted.
      We also have identified and validated novel pyrrole adducts to cysteine residue, which are major products of ONE-proteome reactions. However, it is important to note that we do not yet know whether this novel modification exists in blood or tissues in vivo. This question merits future investigation. Moreover, the functional impact of ONE-derived adductions on individual protein targets or the global thiol proteome remains to be determined. Regardless, we foresee that our data set, together with proximity-directed chemical approaches described recently by Aye and coworkers (
      • Fang X.
      • Fu Y.
      • Long M.J.
      • Haegele J.A.
      • Ge E.J.
      • Parvez S.
      • Aye Y.
      Temporally controlled targeting of 4-hydroxynonenal to specific proteins in living cells.
      ,
      • Parvez S.
      • Fu Y.
      • Li J.
      • Long M.J.
      • Lin H.Y.
      • Lee D.K.
      • Hu G.S.
      • Aye Y.
      Substoichiometric hydroxynonenylation of a single protein recapitulates whole-cell-stimulated antioxidant response.
      ,
      • Long M.J.
      • Poganik J.R.
      • Aye Y.
      On-Demand Targeting: Investigating Biology with Proximity-Directed Chemistry.
      ,
      • Long M.J.
      • Parvez S.
      • Zhao Y.
      • Surya S.L.
      • Wang Y.
      • Zhang S.
      • Aye Y.
      Akt3 is a privileged first responder in isozyme-specific electrophile response.
      ,
      • Parvez S.
      • Long M.J.
      • Lin H.Y.
      • Zhao Y.
      • Haegele J.A.
      • Pham V.N.
      • Lee D.K.
      • Aye Y.
      T-REX on-demand redox targeting in live cells.
      ), will greatly facilitate future exploration of the complex networks affected by this type of electrophilic stress.
      Furthermore, our analysis and emerging evidence demonstrate that both ONE and HNE adduction reactions are highly dynamic cellular events (
      • Yang J.
      • Tallman K.A.
      • Porter N.A.
      • Liebler D.C.
      Quantitative chemoproteomics for site-specific analysis of protein alkylation by 4-hydroxy-2-nonenal in cells.
      ,
      • Randall M.J.
      • Hristova M.
      • van der Vliet A.
      Protein alkylation by the alpha, beta-unsaturated aldehyde acrolein. A reversible mechanism of electrophile signaling?.
      ), although ONE-derived adducts exhibit relatively higher stability in biological systems. Interestingly, two independent groups have recently shown that ONE-derived lysine ketoamide adductions of histone proteins can be reversed by Sirt2-mediated deacylation (
      • Cui Y.
      • Li X.
      • Lin J.
      • Hao Q.
      • Li X.D.
      Histone ketoamide adduction by 4-oxo-2-nonenal is a reversible posttranslational modification regulated by Sirt2.
      ,
      • Jin J.
      • He B.
      • Zhang X.
      • Lin H.
      • Wang Y.
      SIRT2 Reverses 4-oxononanoyl lysine modification on histones.
      ). Our analysis not only confirms this previous finding, but also reveals a broader role of Sirt2 in regulation of ONE-derived lysine adductions of nonhistones. In addition, our results suggest that the cellular turnover of ONE-derived adduction may also be controlled by yet unidentified mechanisms, especially for those adducts on cysteine residues. Our findings, therefore, provide much-needed mechanistic insights into the potential toxicities associated with ONE as well as the self-protection machinery against electrophilic stress in human cells.
      It is important to note that endogenous LDE generation in specific cell and tissue locations and over a time course that tracks the onset of inflammation or toxicity. Thus, our experimental approach, in which we exogenously apply aONE (50 μm, 2 h) to cells, may not adequately reflect cellular microenvironmental factors that modulate native ONE formation and reactivity. Beavers et al. recently developed several metabolically competent surrogates including ω-alkynyl linoleic acid and ω-alkynyl arachidonic acid for tracking the fate and protein targets of LDEs derived from these polyunsaturated fatty acids in cells under physiological oxidative stress (
      • Beavers W.N.
      • Serwa R.
      • Shimozu Y.
      • Tallman K.A.
      • Vaught M.
      • Dalvie E.D.
      • Marnett L.J.
      • Porter N.A.
      omega-Alkynyl lipid surrogates for polyunsaturated fatty acids: free radical and enzymatic oxidations.
      ,
      • Beavers W.N.
      • Rose K.L.
      • Galligan J.J.
      • Mitchener M.M.
      • Rouzer C.A.
      • Tallman K.A.
      • Lamberson C.R.
      • Wang X.
      • Hill S.
      • Ivanova P.T.
      • Brown H.A.
      • Zhang B.
      • Porter N.A.
      • Marnett L.J.
      Protein modification by endogenously generated lipid electrophiles: mitochondria as the source and target.
      ). However, site-specific mapping of endogenous LDE-adducts generated from radical-mediated peroxidation of these alkynyl ω-lipid surrogates remains an unmet analytical challenge. We expect that the chemoproteomic workflow outlined here, which includes detection of unanticipated adducts by blind PTM search and isotope signature-based identification and quantification, can be applied to globally and site-specifically map and quantify known and unexpected chemical modifications derived from endogenously generated LDEs and to estimate their stoichiometry in situ.

      Data Availability

      The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (
      • Vizcaino J.A.
      • Csordas A.
      • Del-Toro N.
      • Dianes J.A.
      • Griss J.
      • Lavidas I.
      • Mayer G.
      • Perez-Riverol Y.
      • Reisinger F.
      • Ternent T.
      • Xu Q.W.
      • Wang R.
      • Hermjakob H.
      2016 update of the PRIDE database and its related tools.
      ) partner repository with the data set identifier PXD007149 and 10.6019/PXD007149.

      Acknowledgments

      We thank Quan Zhou and Dr. Wenchuan Leng from mass spectrometry facility of the National Center for Protein Science (Beijing) for expert technical assistance.

      REFERENCES

        • Ezraty B.
        • Gennaris A.
        • Barras F.
        • Collet J.F.
        Oxidative stress, protein damage and repair in bacteria.
        Nat. Rev. Microbiol. 2017; 15: 385-396
        • Sies H.
        • Berndt C.
        • Jones D.P.
        Oxidative Stress.
        Annu. Rev. Biochem. 2017; 86: 715-748
        • Sayre L.M.
        • Lin D.
        • Yuan Q.
        • Zhu X.
        • Tang X.
        Protein adducts generated from products of lipid oxidation: focus on HNE and one.
        Drug Metab. Rev. 2006; 38: 651-675
        • Schopfer F.J.
        • Cipollina C.
        • Freeman B.A.
        Formation and signaling actions of electrophilic lipids.
        Chem. Rev. 2011; 111: 5997-6021
        • Rudolph T.K.
        • Freeman B.A.
        Transduction of redox signaling by electrophile-protein reactions.
        Sci. Signal. 2009; 2: re7
        • Uchida K.
        HNE as an inducer of COX-2.
        Free Radic. Biol. Med. 2017; 111: 169-172
        • Cohen G.
        • Riahi Y.
        • Sunda V.
        • Deplano S.
        • Chatgilialoglu C.
        • Ferreri C.
        • Kaiser N.
        • Sasson S.
        Signaling properties of 4-hydroxyalkenals formed by lipid peroxidation in diabetes.
        Free Radic. Biol. Med. 2013; 65: 978-987
        • Di Domenico F.
        • Tramutola A.
        • Butterfield D.A.
        Role of 4-hydroxy-2-nonenal (HNE) in the pathogenesis of alzheimer disease and other selected age-related neurodegenerative disorders.
        Free Radic. Biol. Med. 2017; 111: 253-261
        • Koenitzer J.R.
        • Freeman B.A.
        Redox signaling in inflammation: interactions of endogenous electrophiles and mitochondria in cardiovascular disease.
        Ann. N.Y. Acad. Sci. 2010; 1203: 45-52
        • Yang J.
        • Tallman K.A.
        • Porter N.A.
        • Liebler D.C.
        Quantitative chemoproteomics for site-specific analysis of protein alkylation by 4-hydroxy-2-nonenal in cells.
        Anal. Chem. 2015; 87: 2535-2541
        • Codreanu S.G.
        • Ullery J.C.
        • Zhu J.
        • Tallman K.A.
        • Beavers W.N.
        • Porter N.A.
        • Marnett L.J.
        • Zhang B.
        • Liebler D.C.
        Alkylation damage by lipid electrophiles targets functional protein systems.
        Mol. Cell. Proteomics. 2014; 13: 849-859
        • Wang C.
        • Weerapana E.
        • Blewett M.M.
        • Cravatt B.F.
        A chemoproteomic platform to quantitatively map targets of lipid-derived electrophiles.
        Nat. Methods. 2014; 11: 79-85
        • Yuan W.
        • Zhang Y.
        • Xiong Y.
        • Tao T.
        • Wang Y.
        • Yao J.
        • Zhang L.
        • Yan G.
        • Bao H.
        • Lu H.
        Highly selective and large scale mass spectrometric analysis of 4-Hydroxynonenal modification via fluorous derivatization and fluorous solid-phase extraction.
        Anal. Chem. 2017; 89: 3093-3100
        • Chen Y.
        • Cong Y.
        • Quan B.
        • Lan T.
        • Chu X.
        • Ye Z.
        • Hou X.
        • Wang C.
        Chemoproteomic profiling of targets of lipid-derived electrophiles by bioorthogonal aminooxy probe.
        Redox Biol. 2017; 12: 712-718
        • Lin D.
        • Lee H.G.
        • Liu Q.
        • Perry G.
        • Smith M.A.
        • Sayre L.M.
        4-Oxo-2-nonenal is both more neurotoxic and more protein reactive than 4-hydroxy-2-nonenal.
        Chem. Res. Toxicol. 2005; 18: 1219-1231
        • Galligan J.J.
        • Rose K.L.
        • Beavers W.N.
        • Hill S.
        • Tallman K.A.
        • Tansey W.P.
        • Marnett L.J.
        Stable histone adduction by 4-oxo-2-nonenal: a potential link between oxidative stress and epigenetics.
        J. Am. Chem. Soc. 2014; 136: 11864-11866
        • Nasstrom T.
        • Fagerqvist T.
        • Barbu M.
        • Karlsson M.
        • Nikolajeff F.
        • Kasrayan A.
        • Ekberg M.
        • Lannfelt L.
        • Ingelsson M.
        • Bergstrom J.
        The lipid peroxidation products 4-oxo-2-nonenal and 4-hydroxy-2-nonenal promote the formation of alpha-synuclein oligomers with distinct biochemical, morphological, and functional properties.
        Free Radic. Biol. Med. 2011; 50: 428-437
        • Camarillo J.M.
        • Ullery J.C.
        • Rose K.L.
        • Marnett L.J.
        Electrophilic modification of PKM2 by 4-hydroxynonenal and 4-oxononenal results in protein cross-linking and kinase inhibition.
        Chem. Res. Toxicol. 2017; 30: 635-641
        • Aluise C.D.
        • Camarillo J.M.
        • Shimozu Y.
        • Galligan J.J.
        • Rose K.L.
        • Tallman K.A.
        • Marnett L.J.
        Site-specific, intramolecular cross-linking of Pin1 active site residues by the lipid electrophile 4-oxo-2-nonenal.
        Chem. Res. Toxicol. 2015; 28: 817-827
        • Zhang W.H.
        • Liu J.
        • Xu G.
        • Yuan Q.
        • Sayre L.M.
        Model studies on protein side chain modification by 4-oxo-2-nonenal.
        Chem. Res. Toxicol. 2003; 16: 512-523
        • Doorn J.A.
        • Petersen D.R.
        Covalent modification of amino acid nucleophiles by the lipid peroxidation products 4-hydroxy-2-nonenal and 4-oxo-2-nonenal.
        Chem. Res. Toxicol. 2002; 15: 1445-1450
        • Cui Y.
        • Li X.
        • Lin J.
        • Hao Q.
        • Li X.D.
        Histone ketoamide adduction by 4-oxo-2-nonenal is a reversible posttranslational modification regulated by Sirt2.
        ACS Chem. Biol. 2017; 12: 47-51
        • Jin J.
        • He B.
        • Zhang X.
        • Lin H.
        • Wang Y.
        SIRT2 Reverses 4-oxononanoyl lysine modification on histones.
        J. Am. Chem. Soc. 2016; 138: 12304-12307
        • Yang J.
        • Gupta V.
        • Tallman K.A.
        • Porter N.A.
        • Carroll K.S.
        • Liebler D.C.
        Global, in situ, site-specific analysis of protein S-sulfenylation.
        Nat. Protoc. 2015; 10: 1022-1037
        • Yang J.
        • Gupta V.
        • Carroll K.S.
        • Liebler D.C.
        Site-specific mapping and quantification of protein S-sulphenylation in cells.
        Nat. Commun. 2014; 5: 4776
        • Dasari S.
        • Chambers M.C.
        • Codreanu S.G.
        • Liebler D.C.
        • Collins B.C.
        • Pennington S.R.
        • Gallagher W.M.
        • Tabb D.L.
        Sequence tagging reveals unexpected modifications in toxicoproteomics.
        Chem. Res. Toxicol. 2011; 24: 204-216
        • Dasari S.
        • Chambers M.C.
        • Slebos R.J.
        • Zimmerman L.J.
        • Ham A.J.
        • Tabb D.L.
        TagRecon: high-throughput mutation identification through sequence tagging.
        J. Proteome Res. 2010; 9: 1716-1726
        • Kim S.
        • Pevzner P.A.
        MS-GF+ makes progress towards a universal database search tool for proteomics.
        Nat. Commun. 2014; 5: 5277
        • Huang da W.
        • Sherman B.T.
        • Lempicki R.A.
        Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.
        Nat. Protoc. 2009; 4: 44-57
        • Zhu X.
        • Sayre L.M.
        Long-lived 4-oxo-2-enal-derived apparent lysine michael adducts are actually the isomeric 4-ketoamides.
        Chem. Res. Toxicol. 2007; 20: 165-170
        • Oe T.
        • Arora J.S.
        • Lee S.H.
        • Blair I.A.
        A novel lipid hydroperoxide-derived cyclic covalent modification to histone H4.
        J. Biol. Chem. 2003; 278: 42098-42105
        • Sun Z.W.
        • Allis C.D.
        Ubiquitination of histone H2B regulates H3 methylation and gene silencing in yeast.
        Nature. 2002; 418: 104-108
        • Sadeghi L.
        • Siggens L.
        • Svensson J.P.
        • Ekwall K.
        Centromeric histone H2B monoubiquitination promotes noncoding transcription and chromatin integrity.
        Nat. Struct. Mol. Biol. 2014; 21: 236-243
        • Trujillo K.M.
        • Osley M.A.
        A role for H2B ubiquitylation in DNA replication.
        Mol. Cell. 2012; 48: 734-746
        • Karpiuk O.
        • Najafova Z.
        • Kramer F.
        • Hennion M.
        • Galonska C.
        • Konig A.
        • Snaidero N.
        • Vogel T.
        • Shchebet A.
        • Begus-Nahrmann Y.
        • Kassem M.
        • Simons M.
        • Shcherbata H.
        • Beissbarth T.
        • Johnsen S.A.
        The histone H2B monoubiquitination regulatory pathway is required for differentiation of multipotent stem cells.
        Mol. Cell. 2012; 46: 705-713
        • Fuchs G.
        • Shema E.
        • Vesterman R.
        • Kotler E.
        • Wolchinsky Z.
        • Wilder S.
        • Golomb L.
        • Pribluda A.
        • Zhang F.
        • Haj-Yahya M.
        • Feldmesser E.
        • Brik A.
        • Yu X.
        • Hanna J.
        • Aberdam D.
        • Domany E.
        • Oren M.
        RNF20 and USP44 regulate stem cell differentiation by modulating H2B monoubiquitylation.
        Mol. Cell. 2012; 46: 662-673
        • Moyal L.
        • Lerenthal Y.
        • Gana-Weisz M.
        • Mass G.
        • So S.
        • Wang S.Y.
        • Eppink B.
        • Chung Y.M.
        • Shalev G.
        • Shema E.
        • Shkedy D.
        • Smorodinsky N.I.
        • van Vliet N.
        • Kuster B.
        • Mann M.
        • Ciechanover A.
        • Dahm-Daphi J.
        • Kanaar R.
        • Hu M.C.
        • Chen D.J.
        • Oren M.
        • Shiloh Y.
        Requirement of ATM-dependent monoubiquitylation of histone H2B for timely repair of DNA double-strand breaks.
        Mol. Cell. 2011; 41: 529-542
        • Nakamura K.
        • Kato A.
        • Kobayashi J.
        • Yanagihara H.
        • Sakamoto S.
        • Oliveira D.V.
        • Shimada M.
        • Tauchi H.
        • Suzuki H.
        • Tashiro S.
        • Zou L.
        • Komatsu K.
        Regulation of homologous recombination by RNF20-dependent H2B ubiquitination.
        Mol. Cell. 2011; 41: 515-528
        • Bennaars-Eiden A.
        • Higgins L.
        • Hertzel A.V.
        • Kapphahn R.J.
        • Ferrington D.A.
        • Bernlohr D.A.
        Covalent modification of epithelial fatty acid-binding protein by 4-hydroxynonenal in vitro and in vivo. Evidence for a role in antioxidant biology.
        J. Biol. Chem. 2002; 277: 50693-50702
        • Doulias P.T.
        • Greene J.L.
        • Greco T.M.
        • Tenopoulou M.
        • Seeholzer S.H.
        • Dunbrack R.L.
        • Ischiropoulos H.
        Structural profiling of endogenous S-nitrosocysteine residues reveals unique features that accommodate diverse mechanisms for protein S-nitrosylation.
        Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 16958-16963
        • Taira T.
        • Saito Y.
        • Niki T.
        • Iguchi-Ariga S.M.
        • Takahashi K.
        • Ariga H.
        DJ-1 has a role in antioxidative stress to prevent cell death.
        EMBO Rep. 2004; 5: 213-218
        • Klamt F.
        • Zdanov S.
        • Levine R.L.
        • Pariser A.
        • Zhang Y.
        • Zhang B.
        • Yu L.R.
        • Veenstra T.D.
        • Shacter E.
        Oxidant-induced apoptosis is mediated by oxidation of the actin-regulatory protein cofilin.
        Nat. Cell Biol. 2009; 11: 1241-1246
        • Cameron J.M.
        • Gabrielsen M.
        • Chim Y.H.
        • Munro J.
        • McGhee E.J.
        • Sumpton D.
        • Eaton P.
        • Anderson K.I.
        • Yin H.
        • Olson M.F.
        Polarized cell motility induces hydrogen peroxide to inhibit cofilin via cysteine oxidation.
        Curr. Biol. 2015; 25: 1520-1525
        • Barglow K.T.
        • Knutson C.G.
        • Wishnok J.S.
        • Tannenbaum S.R.
        • Marletta M.A.
        Site-specific and redox-controlled S-nitrosation of thioredoxin.
        Proc. Natl. Acad. Sci. U.S.A. 2011; 108: E600-E606
        • Conway M.E.
        • Poole L.B.
        • Hutson S.M.
        Roles for cysteine residues in the regulatory CXXC motif of human mitochondrial branched chain aminotransferase enzyme.
        Biochemistry. 2004; 43: 7356-7364
        • Stradal T.B.
        • Troxler H.
        • Heizmann C.W.
        • Gimona M.
        Mapping the zinc ligands of S100A2 by site-directed mutagenesis.
        J. Biol. Chem. 2000; 275: 13219-13227
        • She P.
        • Reid T.M.
        • Bronson S.K.
        • Vary T.C.
        • Hajnal A.
        • Lynch C.J.
        • Hutson S.M.
        Disruption of BCATm in mice leads to increased energy expenditure associated with the activation of a futile protein turnover cycle.
        Cell Metab. 2007; 6: 181-194
        • Vogel C.
        • Silva G.M.
        • Marcotte E.M.
        Protein expression regulation under oxidative stress.
        Mol. Cell. Proteomics. 2011; 10 (M111 009217)
        • Xu S.N.
        • Wang T.S.
        • Li X.
        • Wang Y.P.
        SIRT2 activates G6PD to enhance NADPH production and promote leukaemia cell proliferation.
        Sci. Rep. 2016; 6: 32734
        • Fiskus W.
        • Coothankandaswamy V.
        • Chen J.
        • Ma H.
        • Ha K.
        • Saenz D.T.
        • Krieger S.S.
        • Mill C.P.
        • Sun B.
        • Huang P.
        • Mumm J.S.
        • Melnick A.M.
        • Bhalla K.N.
        SIRT2 deacetylates and inhibits the peroxidase activity of peroxiredoxin-1 to sensitize breast cancer cells to oxidant stress-inducing agents.
        Cancer Res. 2016; 76: 5467-5478
        • Szabo-Taylor K.
        • Ryan B.
        • Osteikoetxea X.
        • Szabo T.G.
        • Sodar B.
        • Holub M.
        • Nemeth A.
        • Paloczi K.
        • Pallinger E.
        • Winyard P.
        • Buzas E.I.
        Oxidative and other posttranslational modifications in extracellular vesicle biology.
        Semin. Cell Dev. Biol. 2015; 40: 8-16
        • Anavi S.
        • Ni Z.
        • Tirosh O.
        • Fedorova M.
        Steatosis-induced proteins adducts with lipid peroxidation products and nuclear electrophilic stress in hepatocytes.
        Redox Biol. 2015; 4: 158-168
        • Gentile F.
        • Pizzimenti S.
        • Arcaro A.
        • Pettazzoni P.
        • Minelli R.
        • D'Angelo D.
        • Mamone G.
        • Ferranti P.
        • Toaldo C.
        • Cetrangolo G.
        • Formisano S.
        • Dianzani M.U.
        • Uchida K.
        • Dianzani C.
        • Barrera G.
        Exposure of HL-60 human leukaemic cells to 4-hydroxynonenal promotes the formation of adduct (s) with alpha-enolase devoid of plasminogen binding activity.
        Biochem. J. 2009; 422: 285-294
        • Carini M.
        • Aldini G.
        • Facino R.M.
        Mass spectrometry for detection of 4-hydroxy-trans-2-nonenal (HNE) adducts with peptides and proteins.
        Mass Spectrom. Rev. 2004; 23: 281-305
        • Sousa B.C.
        • Pitt A.R.
        • Spickett C.M.
        Chemistry and analysis of HNE and other prominent carbonyl-containing lipid oxidation compounds.
        Free Radic. Biol. Med. 2017; 111: 294-308
        • Lee S.H.
        • Blair I.A.
        Characterization of 4-oxo-2-nonenal as a novel product of lipid peroxidation.
        Chem. Res. Toxicol. 2000; 13: 698-702
        • Fang X.
        • Fu Y.
        • Long M.J.
        • Haegele J.A.
        • Ge E.J.
        • Parvez S.
        • Aye Y.
        Temporally controlled targeting of 4-hydroxynonenal to specific proteins in living cells.
        J. Am. Chem. Soc. 2013; 135: 14496-14499
        • Parvez S.
        • Fu Y.
        • Li J.
        • Long M.J.
        • Lin H.Y.
        • Lee D.K.
        • Hu G.S.
        • Aye Y.
        Substoichiometric hydroxynonenylation of a single protein recapitulates whole-cell-stimulated antioxidant response.
        J. Am. Chem. Soc. 2015; 137: 10-13
        • Long M.J.
        • Poganik J.R.
        • Aye Y.
        On-Demand Targeting: Investigating Biology with Proximity-Directed Chemistry.
        J. Am. Chem. Soc. 2016; 138: 3610-3622
        • Long M.J.
        • Parvez S.
        • Zhao Y.
        • Surya S.L.
        • Wang Y.
        • Zhang S.
        • Aye Y.
        Akt3 is a privileged first responder in isozyme-specific electrophile response.
        Nat. Chem. Biol. 2017; 13: 333-338
        • Parvez S.
        • Long M.J.
        • Lin H.Y.
        • Zhao Y.
        • Haegele J.A.
        • Pham V.N.
        • Lee D.K.
        • Aye Y.
        T-REX on-demand redox targeting in live cells.
        Nat. Protoc. 2016; 11: 2328-2356
        • Randall M.J.
        • Hristova M.
        • van der Vliet A.
        Protein alkylation by the alpha, beta-unsaturated aldehyde acrolein. A reversible mechanism of electrophile signaling?.
        FEBS Lett. 2013; 587: 3808-3814
        • Beavers W.N.
        • Serwa R.
        • Shimozu Y.
        • Tallman K.A.
        • Vaught M.
        • Dalvie E.D.
        • Marnett L.J.
        • Porter N.A.
        omega-Alkynyl lipid surrogates for polyunsaturated fatty acids: free radical and enzymatic oxidations.
        J. Am. Chem. Soc. 2014; 136: 11529-11539
        • Beavers W.N.
        • Rose K.L.
        • Galligan J.J.
        • Mitchener M.M.
        • Rouzer C.A.
        • Tallman K.A.
        • Lamberson C.R.
        • Wang X.
        • Hill S.
        • Ivanova P.T.
        • Brown H.A.
        • Zhang B.
        • Porter N.A.
        • Marnett L.J.
        Protein modification by endogenously generated lipid electrophiles: mitochondria as the source and target.
        ACS Chem. Biol. 2017; 12: 2062-2069
        • Vizcaino J.A.
        • Csordas A.
        • Del-Toro N.
        • Dianes J.A.
        • Griss J.
        • Lavidas I.
        • Mayer G.
        • Perez-Riverol Y.
        • Reisinger F.
        • Ternent T.
        • Xu Q.W.
        • Wang R.
        • Hermjakob H.
        2016 update of the PRIDE database and its related tools.
        Nucleic Acids Res. 2016; 44: 11033