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Obtusaquinone is a cysteine modifying compound that targets Keap1 for degradation

ABSTRACT: Bovine catalase: This is an experiment to use differential cysteine alkylation to map the nature of binding of a cysteine-targeting small molecule OBT (for details of the method, please see the Method Section of the manuscript). The alkylating reagents uses besides the studied drug were iodoacetamide (IAA) and N-ethylmaleimide (NEM). As described in the manuscript the experiment includes three routes of serial alkylation experiments termed OBT, IAA, and NEM. Samples were prepared in triplicate and TMT10-plex reagents were used for labeling as follows: OBT replicate 1 (OBT1), 129c; OBT2, 130n; OBT3, 130c; IAA1, 126; IAA2, 127n; IAA3, 127c; NEM1, 128n; NEM2, 128c; NEM3, 129n. An SPS-MS3 method was used for data acquisition on an Orbitrap Fusion Lumos mass spectrometer. The RAW file for these data is catalase_diffAlk.RAW. A differential alkylation experiment was also performed as described above for bovine catalase but using recombinant human KEAP1 as substrate. One set of experiments (OBT, IAA, NEM) was performed. TMT10-plex labeling was performed as follows: OBT, 129n; IAA, 128c; NEM, 128n. An SPS-MS3 method was used for data acquisition on an Orbitrap Fusion mass spectrometer. The RAW file for these data is RAW_file: keap1_diffAlk.RAW.

INSTRUMENT(S): Orbitrap Fusion Lumos, Orbitrap Fusion

ORGANISM(S): Bos Taurus (ncbitaxon:9913) Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Bakhos Tannous

PROVIDER: MSV000084638 | MassIVE | Sat Nov 30 14:53:00 GMT 2019

REPOSITORIES: MassIVE

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Project description:The small molecule YC-1 was identified as identified as a selectively active agent against subsets of the two major live cancer subtypes, intrahepatic cholangiocarcinoma and hepatocellular carcinoma. Response to YC-1 is dependent on the expression of the sulfotransferase SULT1A1, which activates YC-1 through sulfonation. The contributions of mass spectrometry to the study were: (i) identifying SULT1A1 as the key player in YC-1 response through observing the protein in a cell line with acquired resistance to the small molecule treatment, (ii) corroborating the role of SULT1A1 in the YC-1 activity by correlating protein expression profiles and drug responses across 37 biliary cancer cell lines, (iii) showing that SULT1A1 expression was associated with IDH mutations, FGFR2 fusion, and BAP1 loss in vivo studying patient-derived xenograft (PDX) models, and (iv) identifying protein targets in a protein enrichment experiment with an immobilized drug. (iii) PDX samples were analyzed using TMT multiplexing on an Orbitrap Fusion mass spectrometer using the SPS-MS3 method. Prior to analysis, samples were fractionated offline using high pH reversed chromatography (12 fractions per TMT set). Three TMT sets were analyzed (ShiBardeesy_PDX_TMT1, ShiBardeesy_PDX_TMT2, and ShiBardeesy_PDX_TMT3), and samples were labeled as follows with channel (bridge channel, 130c): ShiBardeesy_PDX_TMT1: SS101 (126), MG12 (127c), SS106 (127n), MG62 (128c), MG26 (128n), LCCH13 (129c), LCCH3 (129n), Q12050 (130n). ShiBardeesy_PDX_TMT2: SS102 (126), SS110 (127n), MG34 (128c), MG17 (128n), LCCH4 (129c), MG68 (129n), HBT110 (130n). ShiBardeesy_PDX_TMT3: SS103 (126), MG10 (127n), MG59 (128c), MG25 (128n), LCCH10 (129c), MG73 (129n), HBT211 (130n). (iv) Samples from experiments using immobilized small molecules to determine direct interactors of YC-1 were performed using TMT10-barcoding and the SPS-MS3 method on an Orbitrap Lumos mass spectrometer. No off-line fraction was performed. Samples were grouped into two TMT sets (ShiBardeesy_ImmobilizedDrug_TMT1 and ShiBardeesy_ImmobilizedDrug_TMT2). Channel 131N was used as the bridge channel. Samples were labeled as follows: ShiBardeesy_ImmobilizedDrug_TMT1: immobilized_inactiveYC-1_rep_1 (126), immobilized_inactiveYC-1_rep_2 (128n), immobilized_inactiveYC-1_rep_3 (128c), immobilized_YC-1_rep_1 (129n), immobilized_YC-1_plus_soluble_YC-1_competitor_rep_1 (130c). ShiBardeesy_ImmobilizedDrug_TMT2: immobilized_YC-1_rep_2 (127n), immobilized_YC-1_plus_soluble_YC-1_competitor_rep_2 (128n), immobilized_YC-1_plus_soluble_YC-1_competitor_rep_3 (129c), immobilized_YC-1_rep_3 (130c).

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Obtusaquinone is a cysteine modifying compound that targets Keap1 for degradation

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