Quantitative and Site-Specific Chemoproteomic Profiling of Targets of

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Quantitative and site-specific chemoproteomic profiling of targets of acrolein ying chen, Yuan Liu, Xiaomeng Hou, zi Ye, and Chu Wang Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.8b00343 • Publication Date (Web): 03 Jan 2019 Downloaded from http://pubs.acs.org on January 7, 2019

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Chemical Research in Toxicology

Quantitative and site-specific chemoproteomic profiling of targets of acrolein

Ying Chena,b, Yuan Liua,b, Xiaomeng Houa,b, Zi Yea,c, Chu Wanga,b,c* Addresses: aSynthetic

and Functional Biomolecules Center, Beijing National Laboratory for

Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education bCollege

of Chemistry and Molecular Engineering

cPeking-Tsinghua

Center for Life Sciences, Peking University, Beijing 100871, China

Correspondence: [email protected]

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TOC

Abstract Acrolein exists in common pollutants, such as cigarette smoke and car exhaust, which has been implicated with many pathological processes. It is also one type of endogenous lipid-derived electrophiles (LDEs) generated from lipid peroxidation when cells are under oxidative stress. Chemically, acrolein is able to covalently modify nucleophilic residues in proteins so as to influence its structure and function, and identification of targets of acrolein modification in proteomes is very critical for understanding its biological roles. Here we reported a quantitative chemoproteomic method to globally profile acrolein modifications using an aldehyde-directed aniline-based probe. Collectively, we identified >2300 proteins and >500 cysteine sites that are targeted by acrolein. Our data provide valuable information for understanding acrolein-mediated toxicity and expanding our knowledge of oxidative stress-mediated damage and signaling.


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Introduction Acrolein is a ubiquitous toxic pollutant, which mainly exist in cigarette smoke1, cooking smoke2 and car exhaust from combustion of petroleum fuels3. It can also be endogenously generated from lipid peroxidation of polyunsaturated fatty acid when cells are under oxidative stress4,

5

or alcohol stress from long-term liquor over-

consumption6. As the structurally smallest but chemically most reactive α, βunsaturated electrophile, acrolein can rapidly conjugate with intracellular glutathione upon exposure, influencing dramatically on the redox homeostasis4. It can also form irreversible Michael adducts with nucleophilic residues in proteins (cysteines, histidines and lysines)7-9, as well as Schiff base products with side-chain amines of lysines to cause intra- or inter-protein crosslinks10. These modifications have huge influence on protein structures and functions, which could contribute to its toxicity11. For example, protein tyrosine phosphatase 1B12, glyceraldehyde-3-phosphate dehydrogenase (GAPDH)13, Cu,Zn-superoxide dismutase14 and epidermal growth factor receptor (EGFR)15 have been reported to be inactivated by acrolein. As a result, acrolein has been implicated with many diseases including alcoholic liver damage9, Alzheimer’s disease16 , diabetes17 and lung cancer18. In order to mechanistically elucidate the pathological roles of acrolein in oxidativestress associated diseases, it’s highly desired to identify protein targets and specific sites of acrolein modification. Combining intact protein analysis and LC-MS/MS fragmentation, Spickett and colleagues identified acrolein modified sites directly in model proteins of lysozyme and human serum albumin (HSA)19. When it comes to complex cellular proteome samples, enrichment of the modified proteins is usually necessary before they are analyzed by shotgun proteomics, which is a standard pipeline for activity-based protein profiling (ABPP)20. Such “chemoproteomic” strategies have been developed for mapping targets of LDEs7, 8. For example, a competitive ABPP strategy was developed by Cravatt and colleagues to enable quantitative profiling of >1000 LDE-modified cysteines in complex proteomes, however, the method does not identify direct adducts of LDE modification in proteomes21. While biorthogonal



analogue probes were designed to directly profile modifications by other LDEs22-24, the strategy is not amenable for acrolein due to its small molecular size and special structure. For proteomic profiling of acrolein-modified targets, most of the approaches have been based on the aldehyde-directed selective capture strategy25-27. Van der Vliet and colleagues used a biotin hydrazide probe to globally profile protein adducts formed in acrolein-exposed bronchial epithelial cells. As a result, 769 proteins were identified and 161 of them were mapped with a specific adduct containing the biotin tag25. In another study, an aldehyde/ketone selective probe with a hydroxylamine warhead enabled identification of 39 endogenous lipoxidation sites modified by acrolein and other 2alkenals in cardiac mitochondria26. In order to avoid the harsh conditions during biotin elution of the modified peptides, a cleavable alkoxyamine-PEG4-SS-PEG4-biotin was used to identify acrolein modification sites in a model protein of HSA27. Lastly, commercially available hydrazine-functionalized beads were used to enrich and release of oxylipid modified sites in cardiac mitochondrial28. We recently reported an aniline-based probe m-APA with improved sensitivity than the hydrazide and hydroxylamine warheads for capturing protein carbonylations in proteomes29. In combination with a site-specific quantitative chemical proteomic strategy, we identified >1000 sites of modification when cell lysates were treated exogenously with another common LDE molecule, HNE. The improved probe sensitivity also enabled identification of endogenous LDE modifications from cells under ferroptosis, including some sites modified by acrolein29. These results inspired us to test the performance of m-APA in capturing acrolein modifications for quantitative and site-specific chemoproteomic profiling. Herein, we applied the m-APA probe for the detection of acrolein modifications in a model of non-small cell lung cancer (NSCLC) H1299 cell line. We provided to date the most comprehensive databases of acrolein targets which contains >2300 acrolein modified proteins and >500 modified residue sites. Our large-scale chemproteomic profiling study should greatly promote future investigation on functional roles of acrolein in mediating various pathological processes.


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Materials and Methods Cell culture Non-small cell lung cancer H1299 cells and HT1080 cells were cultured at 37℃ under 5% CO2 atmosphere in DMEM culture medium supplemented with 10% FBS and 1% PS. Gel-based profiling of acrolein modifications H1299 cells were lysed by sonication in ice-cold PBS containing 0.1% TritonX-100 and centrifuged at 100,000g for 30 mins to remove cell debris. The protein concentrations were determined by the BCA protein assay and normalized to 2 mg/mL. The proteomes were treated with 0, 10, 50, 100 or 250 μM acrolein at room temperature for 1 h. The proteomes were then labeled by 0.5 mM m-APA in presence of 60 mM NaBH3CN at pH 5.0 for 1h and precipitated by methanol/chloroform. The precipitations were washed using cold methanol, resuspended in 0.4% SDS/PBS and reacted with 1 mM CuSO4, 100 μM TBTA ligand, 100 μM TAMRA-N3 and 1 mM TCEP for 1 h at room temperature. Finally, the proteomes were boiled with a gel loading buffer at 90 ℃ for 5 mins, resolved on 10% SDS-PAGE gels and imaged by ChemiDoc (Bio-Rad). For the free aniline competition experiment, 0, 0.1, 0.5, 1, 5 or 10 mM of aniline was added at the same time of the mAPA probe labeling of cell lysates treated with 100 μM acrolein. Fluorescent imaging of acrolein modifications. HT1080 cells were treated with 50 μM acrolein for 1 h with serum-free medium. The cells were washed with PBS, fixed with 4% formaldehyde/PBS at 37 ℃ for 15 mins, rinsed three times with PBS and permeabilized in 0.2% TritonX-100/PBS at 37 ℃ for 10 mins. The cells were again rinsed three times with PBS and labeled with 0.5 mM m-APA and 60mM NaBH3CN for 1 h. After the cells were washed three times using PBS, they were conjugated with cy5-N3 via click chemistry using a BTTAA-CuSO4 complex for 15 mins at room temperature. Finally, the cells were stained with 25 μg/ml Hoechst 33342 for 10 minutes and images were acquired with a Zeiss LSM 700 laser scanning confocal microscope equipped with 63x/1.4 NA oil immersion objective lens.



Profiling of acrolein modified proteins by RD-ABPP. In the enrichment experiment, acrolein or DMSO treated proteomes were separately labeled by the m-APA probe and then clicked with 1 mM CuSO4, 100 μM TBTA ligand, 100 μM biotin-(PEG)2-N3 and 1 mM TCEP. After 1h, the proteomes were precipitated with methanol/chloroform and resuspended in 1.2% SDS/PBS. The proteomes were boiled at 90℃ for 5 mins and centrifuged at 20000 g for 1 min. The supernatant is diluted to 0.2% SDS/PBS and subjected to streptavidin enrichment. The enriched proteins are digested by trypsin in 100 mM TEAB buffer and subjected to reductive dimethylation labeling30. After 1 h, the reaction is sequentially quenched by 1% ammonia and 5% formic acid. The light labeled (acrolein treated) and heavy labeled (DMSO treated) proteomes were mixed, concentrated, separated by Fast-seq31, and analyzed on a Q-Exactive mass spectrometer. In the competition experiment, the m-APA labeling procedure was done by adding 10 mM free aniline at the same time. In the dimethylation labeling procedure, the control proteomes were labeled by light aldehyde and the aniline-competed proteomes were labeled by heavy aldehyde. Profiling of acrolein modified sites by isoTOP-ABPP. For the in vitro experiments, two proteomes were independently treated with 100 μM acrolein at room temperature for 1h. For the in situ experiments, H1299 cells were treated with 100 μM acrolein for 1 h in serum-free media. The cells were collected, lysed in ice-cold 0.5% TritonX100/PBS, centrifuged at 100,000g for 30 mins to remove cell debris and normalized to 2 mg/mL. The acrolein-treated proteomes were then labeled by 0.5 mM m-APA for 1h. Afterwards, the proteomes were reacted with 1 mM CuSO4, 100 μM TBTA ligand, 100 μM light or heavy biotin-TEV-N3 and 1 mM TCEP for another 1h. The proteomes were precipitated with methanol/chloroform, combined, resuspended in 1.2% SDS/PBS and diluted to 0.2% SDS/PBS. The samples were subjected to streptavidin enrichment, and on-bead trypsin digestion. Then the trypsin digest is collected and the beads are further digested by TEV protease. Finally, the supernatant is collected, desalted, and analyzed on a Q Exactive mass spectrometer using MudPIT32.


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LC-MS/MS and data analysis Samples were analyzed by a Q Exactive series Orbitrap mass spectrometer. Under the positive-ion mode, full-scan mass spectra were acquired over the m/z range from 350 to 1800 with mass resolution of 70000. MS/MS fragmentation is performed in a data-dependent mode, of which TOP 20 most intense ions are selected for MS2 analysis a resolution of 17500 using collision mode of HCD. Other important parameters: isolation window, 2.0 m/z units; default charge, 2+; normalized collision energy, 28%; maximum IT, 50 ms; dynamic exclusion, 20.0 s. LCMS/MS data was analyzed by ProLuCID33 with static modification of cysteine (+57.0215 Da) and variable oxidation of methionine (+15.9949 Da). For RD-ABPP data, the isotopic modifications (28.0313 and 34.0631 Da for light and heavy labeling respectively) are set as variable modifications on the N-terminal of a peptide and lysines. For isoTOP-ABPP data, 484.2910 and 490.3048 Da are set as variable modifications on cysteines. The ratios of reductive dimethylation and isoTOP-ABPP were quantified by the in-house CIMAGE software with default parameters as described previously29, 34, 35. The MS1 chromatogram resolution is set as 50 ppm and the retention time window width is 10 minutes. Additionally, the isotope envelope score cutoff is 0.8 and the coelution profile R2 cutoff is 0.7. Validation of individual acrolein targeted protein HEK-293T cells overexpressing EEF2WT and EEF2C41A were lysed in 0.1% TritonX-100/PBS, centrifuged at 100,000g for 30 minutes to remove cell debris. Protein concentrations were determined by BCA protein assay. Proteomes were normalized to 2 mg/mL, incubated with 100 μM acrolein for 1h at room temperature and labeled by 0.5 mM m-APA at pH 5.0 for 1h. Proteomes were precipitated by methanol/chloroform and resuspended in 0.4% SDS/PBS. The proteomes were reacted with 1 mM CuSO4, 100 μM TBTA ligand, 100 μM biotin(PEG)2-N3 and 1 mM TCEP for 1 h. After a second precipitation, the proteomes were enriched with streptavidin beads for 3 hours. The beads were washed with PBS for 3 times, and eluted with loading buffer at 95℃for 10 min. Samples were separated on a SDS-PAGE gel and immunoblotted with an anti-6xHis antibody.



Results and Discussion Capture of acrolein modifications by m-APA probe A general scheme for detecting acrolein modifications using the m-APA probe is presented in Figure 1A. After acrolein reacts with nucleophilic residues including cysteines, histidines and lysines via Michael addition, it leaves a free aldehyde group on the target proteins, which can be captured by m-APA to form Schiff base and subsequently reduced by sodium cyanoborohydride. The bioorthogonal alkyne handle of m-APA enables either fluorescence-based visualization or biotin-based enrichment of acrolein modifications depending on the type of the azide-functionalized reporter group conjugated via copper catalyzed click chemistry36. Firstly, we evaluated the labeling of the m-APA probe for acrolein modifications in vitro. Lysates from H1299 cells were incubated with a series of concentrations of acrolein for 1 h and labeled with 0.5 mM of m-APA probe. After reduction by NaBH3CN, the m-APA-labeled proteins were conjugated with an azide-TAMRA. Ingel fluorescence scanning revealed the labeling intensity of m-APA was correlated with the acrolein concentration, indicating the acrolein modifications were successfully captured by the probe (Figure 1B). Co-administration of a non-clickable aniline competitor suppressed the fluorescence signal, confirming that the labeling is specifically mediated by the aniline warhead but not by other potential side reactions (Figure 1C). Moreover, we incubated cells with 50 μM of acrolein for 1 h and fixed them by formaldehyde. The fixed cells were labeled by the m-APA probe and clicked with cy5 fluorophores. Fluorescent imaging detected much stronger signals in the acrolein-treated cells than that in the control cells (Figure 1D). These results collectively demonstrated that the m-APA probe is capable of capturing acrolein modifications on proteins efficiently.


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Figure 1. Labeling of acrolein modifications by the m-APA probe. A. Scheme of reaction between acrolein modified proteins and m-APA probe. B. Detection of concentration-dependent acrolein modifications in proteomes by m-APA. C. The labeling of m-APA can be effectively competed by free aniline. D. Imaging protein carbonylations in acrolein-treated cells using m-APA by fluorescence confocal microscopy.

Identification of acrolein-modified proteins by a reductive dimethyl-Activitybased protein profiling (RD-ABPP) strategy We next set out to identify protein targets of acrolein by a RD-ABPP strategy. Two types of profiling experiments were performed in H1299 cell lysates (Figure 2A). In the first “enrichment” experiment, two aliquots of H1299 cell lysates were treated with 100 μM of acrolein or DMSO, respectively, and labeled by the m-APA probe. After conjugation with azide-biotin tags and enrichment on streptavidin beads, the probe-labeled proteins were digested into



peptides by trypsin, which were further subjected to reductive dimethyl labeling by light (for the acrolein-treated samples) or heavy (for the DMSO-treated sample) formaldehyde. The “light” and “heavy” samples were combined, desalted and analyzed by LC-MS/MS. A total of 3397 proteins with an enrichment (light/heavy) ratio over 4.0 were identified from three biological replicates (Supplementary Figure 1A and Table S1). In the second “competition” experiment, both aliquots of H1299 cell lysates were treated with 100 μM of acrolein and labeled by the m-APA probe, however, one aliquot was co-treated with free aniline as the competitor. The samples were subjected to the rest of RD-ABPP protocol as described above, with those with or without the aniline competition labeled by heavy or light formaldehyde, respectively. In three biological replicates, a total of 2850 proteins were identified with a competition ratio (light/heavy) over 4.0 (Supplementary Figure 1B and Table S1). By crosschecking the “enrichment” and “competition” dataset with each other, we finally assigned 2301 proteins as highly reliable acrolein modified targets (Figure 2B and Table S1). Comparison of our dataset with the proteins identified by Van der Vliet and colleagues using the biotin hydrazide probe[ref] led to 198 common targets (Supplementary Figure 2A and Table S1), including important proteins involved in redox homeostasis such as peroxiredoxin 1 (PRDX1), peroxiredoxin 6 (PRDX6) and a selenoprotein thioredoxin reductase 2 (TXNRD2)25. In addition, we identified three more members of the peroxiredoxin family (PRDX3, PRDX4 and PRDX5) with pronounced ratios in both the “enrichment” and “competition” experiments (Figure 2C). We also identified three other selenoproteins as targets of acrolein besides TXNRD2, including phospholipid hydroperoxide glutathione peroxidase (GPX4), selenoprotein S (SELS) and thioredoxin reductase 1 (TXNRD1). These suggested that acrolein could damage the anti-oxidant defense in cells by both depleting GSH and targeting important redox-regulating enzymes. Cellular component analysis shows that acrolein modified proteins are more enriched in cytosol and nucleoplasm than membrane (Supplementary Figure 2B), which is consistent with its good water


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solubility. Molecular function analysis of these proteins reveals that 650 (28.7%) of them are involved in various types of “binding” activity including protein binding, RNA binding and GTP binding etc. 703 (31%) of them possess catalytic activity and 103 (4.5%) of them function as structural proteins. The rest are annotated with receptor activity, transporter activity and antioxidant activity (Figure 2D).

Figure 2. Chemoproteomic profiling of acrolein-modified proteins. A. Scheme of the “enrichment” and “competition” experiments to identify acrolein modified proteins. B. Venn diagram of the number of acrolein modified proteins identified from the “enrichment” and “competition” experiments. C. Chromatographic peaks of selected proteins modified by acrolein. Red and blue are traces of light and heavy dimethylated



peptides. The protein names and quantified ratios are shown above and below, respectively. D. Molecular function analysis of the acrolein modified proteins. Identification of acrolein-modified sites by an isotopic TOP-ABPP (isoTOP-ABPP) strategy We next applied m-APA labeling in an isoTOP-ABPP strategy to identify specific residue sites of modification by acrolein (Figure 3A). In such experiments, the isotopic signature was used to generate unique “doublet” signatures in full MS spectra in order to increase the confidence of adduct identification, which has been implemented in previous studies24,

37, 38.

Two identical aliquots of acrolein-treated

lysates were labeled by equal amounts of m-APA and conjugated with a pair of isotopeencoded azido-biotin tags with a TEV protease cleavable linker (biotin-TEV-N3), respectively. The “light” and “heavy” samples were mixed and subjected to standard TOP-ABPP analysis. After quantification, we defined the adducted peptides with a light/heavy ratio in the range of 0.67-1.5 as targets modified by acrolein. Using this criterion, a total of 865 cysteine sites were identified in common from two biological replicates (Figure 3B, Supplementary Figure 3A and Table S2). Interestingly, we did not identify any adducted histidine and lysine sites with creditable scores (data not shown), even though these two nucleophilic residues could react with acrolein via Michael addition as well. The “cysteine-only” data is consistent with a previous theoretical calculation showing that cysteines are more preferred targets of LDEs than histidines and lysines39. However, considering the van der Vliet’work where the identified adducts were 36% lysines and 16% histidines, this may also reflect a residue bias inherent to the m-APA probe for enriching more acrolein-modified cysteines25. It should also be noted that acrolein might react with sidechain primary amines in lysines by Schiff-base formation which could not detected by the m-APA probe. We also performed the same analysis to profile acrolein modified sites in living cells. H1299 cells were treated with 100 μM acrolein and the m-APA-based isoTOP-ABPP analysis identified a total of 824 cysteines in common from at least two biological replicates (Figure 3B, Supplementary Figure 3B and Table S2). Crosschecking the in vitro and in situ profiling data together, we identified in common a total of 541


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cysteines, which could be assigned as acrolein modified sites with higher confidence (Figure 3B and Table S2). To our best knowledge, this is the most comprehensive sitespecific profiling study of acrolein modifications in proteomes to date. Our dataset contains several previously reported sites of modification by acrolein. For example, C65 of voltage-dependent anion-selective channel protein 3 (VDAC3) was identified by Maier and colleagues in the cardiac mitochondria26 and the active site C152 of GAPDH was previously identified in mouse mammary carcinoma FM3A cells13. We also compared this data with our previously reported HNE modified peptides29, and found 161 common ones, which account for 30% of all the acrolein modified sites (Supplementary Figure 4A and Table S2). These sites include several HNE-hyper-sensitive targets including C210 of voltage-dependent anion-selective channel protein 2 (VDAC2), C369 of D-3-phosphoglycerate dehydrogenase (PHGDH) and C41 of elongation factor 2 (EEF2) (Figure 3C and Supplementary Figure 4B, 4C)29. Interestingly, EEF2 is among the list of acrolein target proteins with more than 3 modifications (C41, C136, C290 and C812) (Figure 3D). To test if C41 is the major site of acrolein modifications in EEF2, we overexpressed the wild-type protein along with its C41A mutant in HEK-293T cells and labeled the lysates with m-APA after they were treated with acrolein. After the following biotin pull-down experiment, samples were separated by SDS-PAGE and immunoblotted with an anti-6xHis antibody. The result showed that, upon acrolein treatment, the labeling signal of m-APA probe on the C41A mutant dramatically decreased compared to that on the wild-type protein, which confirmed that C41 is a major site for acrolein modification in EEF2 (Figure 3E). As EEF2 is a key regulator of translation elongation40, 41, the acrolein modification of C41 may interfere with translational fidelity and protein synthesis. Moreover, we also identified some other proteins with more than 4 acrolein modified sites including TP53-binding protein 1 (TP53BP1), neuroblast differentiationassociated protein (AHNAK) and anillin (ANLN). TP53BP1 is a protein functionally involved with DNA repair42,

43,

AHNAK is reported to support neuronal cell

differentiation44 and ANLN plays an important role in cytokinesis45. Functional



characterization of acrolein modifications on these proteins might help unveil new insights on acrolein-mediated signaling and toxicity.

Figure 3. Site-specific chemoproteomic profiling of acrolein modifications. A. Scheme of isoTOP-ABPP to identify acrolein modified sites. B. Venn diagram of the number of acrolein-modified sites identified from the in vitro and in situ experiments. C. Tandem MS/MS spectra supporting the acrolein modification on C41 of EEF2. D. Bar graph showing the number of proteins with single or multiple acrolein modified sites. E. The pull-down experiment showed m-APA could selectively label acrolein modifications on C41 of EEF2 in proteomes. Alignment of peptide sequences flanking the site of modifications revealed that acrolein prefers to modify cysteines when a second cysteine is not available at position X+2 or X-2, which is the representative motif of the thioredoxin family (Supplementary Figure 5). The observation is consistent with the data that few thioredoxin family


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members were identified as targets of acrolein. We also analyzed the secondary structure features of acrolein modified peptides and found acrolein biases against cysteines at α-helix (Supplementary Figure 6), which is similar to HNE but different from iodoacetamide as we have observed previously29, 34. Since, acrolein is structurally very small, these analyses collectively suggested that cysteine hyper-reactivity alone is not sufficient to define acrolein’s modification profile and electrophiles with distinct chemical properties would target different sub-populations of cysteines in proteomes. Conclusions In summary, we have systematically profiled acrolein modifications in proteomes with site specificity using an aniline-based m-APA probe. Applying two quantitative ABPP methods, RD-ABPP and isoTOP-ABPP, we successfully identified >2300 acrolein modified proteins and >500 modified cysteines in H1299 cells. It represents the most comprehensive database of acrolein targets up to date. Our data provide valuable resource for further exploring the biological roles of acrolein in oxidative stress-mediated damage and signaling. We envision that the m-APA probe should be will be readily applicable tool for depicting the global portrait of protein carbonylations in proteomes beyond those induced by HNE and acrolein.

ASSOCIATED CONTENT Supporting Information Supplementary Figures S1-S6, Tables S1-S2. AUTHOR INFORMATION Corresponding Author *Correspondence: [email protected] ACKNOWLEDGMENT We thank Prof. Xing Chen and Ms. Pinou Lv for help with fluorescence imaging. We thank the Computing Platform of the Center for Life Science for supporting the proteomic data analysis. This work was supported by National Science Foundation of China (21472008, 81490741 and 21521003), Ministry of Science and Technology of China (2016YFA0501500), and a "1000 Talents Plan" Young Investigator Award (C.W.).



Notes The authors declare no competing financial interest.

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Quantitative and Site-Specific Chemoproteomic Profiling of Targets of

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