Dataset Information

Quantitative chemical proteomics profile target proteins of ITCs

ABSTRACT: Quantitative chemical proteomics was performed to profile the target proteins of ITCs.For the forward SILAC experiment, heavy-labeled cells were treated with the ITC probe (20 μM) for 30 min and light-labeled cells were pre-treated with the natural ITC (60 μM) for 30 min, followed by the ITC probe (20 μM) for another 30 min. For the reverse SILAC experiment, light-labeled cells were treated with the ITC probe (20 μM) for 30 min and heavy-labeled cells were treated with the natural ITC (60 μM) for 30 min, followed by the ITC probe (20 μM) for another 30 min.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

DISEASE(S): Breast Cancer

SUBMITTER: Tao Peng

LAB HEAD: Tao Peng

PROVIDER: PXD051941 | Pride | 2025-05-06

REPOSITORIES: Pride

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Publications

Quantitative chemical proteomics reveals that phenethyl isothiocyanate covalently targets BID to promote apoptosis.

Dong Xiaoshu X Yu Xinqian X Lu Minghao M Xu Yaxin Y Zhou Liyan L Peng Tao T

Cell death discovery 20241029 1

Naturally occurring isothiocyanates (ITCs) found in cruciferous vegetables, such as benzyl isothiocyanate (BITC), phenethyl isothiocyanate (PEITC), and sulforaphane (SFN), have attracted significant research interest for their promising anti-cancer activity in vitro and in vivo. While the induction of apoptosis is recognized to play a key role in the anti-cancer effects of ITCs, the specific protein targets and associated upstream events underlying ITC-induced apoptosis remain unknown. In this s ...[more]

PMID: 39472556

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Project description:In vitro H2O2-sensitivity of CLOCK cysteines was measured with a quantitative chemoproteomic approach termed as QTRP (Quantitative Thiol Reactivity Profiling) as previously reported. In brief, 5 μg of recombinant CLOCK proteins were reduced with 1 mM DTT for 30 min at room temperature. After reaction, excess DTT was removed by Zeba™ Spin Desalting Column (Thermo Scientific) pre-equilibrated with PBS, pH 7.4. Reduced CLOCK proteins were then reacted with or without H2O2 (150 μM) for 10 min at room temperature. The reaction was quenched with catalase. Proteins were labeled with 100 μM thiol-reactive IPM probe for 30 min at room temperature. The reversibly oxidized cysteines were reduced with 1 mM DTT at 75°C for 15 min and alkylated with 5 mM IAM for 30 min in the dark at the room temperature. Proteins were then digested with sequencing grade trypsin (Promega) at a 1:50 (enzyme/ substrate) ratio overnight at 37°C. The resulting tryptic peptides were desalted with C18 tips, evaporated to dryness under vacuum, and reconstituted in a solution containing 30% ACN at pH 6. An isotopically labeled and UV-cleavable azido-biotin tag (Az-UV-Biotin) was then conjugated to the IPM-modified peptides using copper catalyzed alkyne azide cycloaddition reaction (CuAAC, click chemistry). Click chemistry was performed by the addition of 0.8 mM either light Az-UV-biotin for H2O2 treated sample or heavy Az-UV-biotin for untreated sample, 8 mM sodium ascorbate, 1 mM TBTA, and 8 mM CuSO4. Samples were allowed to react at room temperature for 2 h with rotation in the dark. The light and heavy biotinylated peptide samples were then mixed together, cleaned with SCX spin columns to remove excess reagents, and captured with pre-washed streptavidin sepharose beads for 2 h at room temperature. Streptavidin beads then was washed with 50 mM NaAc, 50 mM NaAc containing 2 M NaCl, and water twice, respectively, and resuspended in 25 mM ammonium bicarbonate buffer. The probe-modified peptides were then photoreleased under 365 nm UV light, desalted with C18 tips, evaporated to dryness, and stored at -20°C until LC-MS/MS analysis. One microgram of recombinant CLOCK protein (Creative Biomart) was incubated with 1 mM hydrogen peroxide for 10 min at 37°C, at which point dimedone was added to a final concentration of 5 mM and the samples were incubated for an additional 1 h. After the dimedone treatment, 10 mM DTT was added, and the samples were incubated for an additional 30 min at 37°C. Subsequently, samples were incubated with 20 mM indole-3-acetic acid (IAA) for an additional 30 min, after which trypsin digestion was performed overnight. LC-MS/MS analyses were performed using a Q-Exactive Plus instrument equipped with an Easy-nLC1000 system.

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Dataset Information

Quantitative chemical proteomics profile target proteins of ITCs

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Publications

Quantitative chemical proteomics reveals that phenethyl isothiocyanate covalently targets BID to promote apoptosis.

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