Dataset Information

Identification of Cytosolic Protein Targets of Catechol Estrogens in Breast Cancer Cells Using a Click Chemistry-based Workflow

ABSTRACT: Catechol estrogens (CEs) are known to be toxic metabolites and the initiators in the oncogenesis of breast cancers via forming covalent adducts with DNAs. CEs shall also react with proteins but their cellular protein targets remain un-explored. Here we reported the identification of protein targets of CEs in the soluble cytosol of estrogen-sensitive breast cancer cells by multiple comparative proteomics using liquid chromatography tandem mass spectrometry (LC-MS/MS) coupled with an improved click chemistry-based workflow. Multiple comparative proteomics composed of experimental pair (probe versus solvent) and two control pairs (solvent versus solvent and probe versus solvent without enrichment), were studied using stable isotope dimethyl labeling. The use of 4-hydroxyethynylestradiol (4OHEE2) probe with an amide-free linker coupled with on-bead digestion and re-digestion of the proteins cleaved from the beads was shown to greatly improve the recovery and identification of CEs-adducted peptides. A total of 310 protein targets and 35 adduction sites were repeatedly (n≥2) identified with D/H (probe/solvent) ratio >4 versus only one identified with D/H >4 from the two control pairs, suggesting that our workflow imposes only a very low background. Meanwhile, multiple comparative D/H ratios revealed that CEs may down-regulate many target proteins involved in the metabolism or detoxification, suggesting a negative correlation between CEs-induced adduction and expression of proteins acting on the alleviation of stress-induced cellular damages. The reported method and data shall provide opportunities to study the progression of estrogen metabolism-derived diseases and biomarkers.

INSTRUMENT(S): LTQ Orbitrap Elite, Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Erythrocyte, Cell Culture

DISEASE(S): Breast Cancer

SUBMITTER: Quynh-Trang Do

LAB HEAD: Shu-Hui Chen

PROVIDER: PXD017731 | Pride | 2020-09-28

REPOSITORIES: Pride

ACCESS DATA

Publications

Identification of Cytosolic Protein Targets of Catechol Estrogens in Breast Cancer Cells Using a Click Chemistry-Based Workflow.

Do Quynh-Trang QT Huang Ting-En TE Liu Yi-Chen YC Tai Jung-Hsiang JH Chen Shu-Hui SH

Journal of proteome research 20201001 1

Catechol estrogens (CEs) are known to be toxic metabolites and the initiators of the oncogenesis of breast cancers via forming covalent adducts with DNAs. CEs shall also react with proteins, but their cellular protein targets remain unexplored. Here, we reported the identification of protein targets of CEs in the soluble cytosol of estrogen-sensitive breast cancer cells by multiple comparative proteomics using liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with an improved cli ...[more]

PMID: 32951420

Similar Datasets

Project description:Cysteine is unique among all protein-coding amino acids owing to its intrinsically high nucleophilicity. The cysteinyl thiol group can be covalently modified by both a broad range of redox mechanisms and various electrophiles derived from exogenous or endogenous sources. Measuring proteomic cysteines response to redox perturbation or electrophiles is critical for understanding the underlying mechanisms involved. Activity-based protein profiling (ABPP) based on thiol-reactive probes has been the method of choice for such analyses. We therefore adapted this approach and developed a new chemoproteomic platform, termed as QTRP (Quantitative Thiol Reactivity Profiling), that relies on the ability of a commercially available thiol-reactive probe IPM to covalently label, enrich, and quantify the reactive cysteinome in cells and tissues. Here, we provide a detailed and updated workflow of QTRP that includes procedures for (i) labeling of the reactive cysteinome from cell or tissue samples (e.g. control vs treatment) with IPM, (ii) processing the protein samples into tryptic peptides and tagging the probe-modified peptides with isotopically labeled azido-biotin reagents containing a photo-cleavable linker via click chemistry reaction, (iii) capturing biotin-conjugated peptides with streptavidin beads, (iv) identifying and quantifying the photo-released peptides by mass spectrometry (MS)-based shotgun proteomics, (v) interpreting MS data by a streamlined informatic pipeline using a proteomics software, pFind 3, and an automatic post-processing algorithm. We also outline here how to use QTRP for measuring the changes on proteomic cysteines upon redox perturbation and for identifying targeted cysteine sites of thiol-reactive electrophiles. We anticipate that this protocol should find broad applications in both redox/chemical biology and drug discovery. The protocol for sample preparation takes 3 days, whereas MS measurements and data analyses require 75 min and <30 min, respectively, per sample.

Dataset Information

Identification of Cytosolic Protein Targets of Catechol Estrogens in Breast Cancer Cells Using a Click Chemistry-based Workflow

Publications

Identification of Cytosolic Protein Targets of Catechol Estrogens in Breast Cancer Cells Using a Click Chemistry-Based Workflow.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure