Dataset Information

Assessment of biarsenical dye-reactive cysteine residues of human metallothionein MT1E

ABSTRACT: Metallothioneis are low-molecular weight proteins engaged in zinc and copper ion homeostasis and redox potential regulation of the cell. Their clinical importance lies in their role in cancer resistance to cisplatin related drugs, which is related to their capability of binding sole toxic metal ions, as well as their complexes. Due to metallothioneins' intrinsic properties a satisfactory analytical method to detect, measure and quantify this family of proteins is lacking. In the study project at hand a fluorescent biarsenical probe, CrAsH-EDT2, is presented as specific means for the detection of human metallothioneins. Biarsencial dyes comprise of two arsenic atoms incorporated covalently into a fluorophore structure. Such chemical species show exemplary affinity for thiol groups of cysteine side chains separated in a specific spatial manner; a -CCXXCC- motif being the model one where each arsenic atom is coordinated by two adjacent cysteine thiols. Mass spectrometry analysis was employed in the project in order to pin-point the MT1E metallothionein's cysteine residues responsible for interaction with the CrAsH dye upon pure protein in vitro labelling. Using the extremely tight binding of CrAsH to MT1E to the advantage, a simple approach was undertaken, where the protein was first incubated with the dye at 1:1, 1:2 and 1:3 protein:dye molar ratio, and an alkylation (iodoacetamide (IAA)) and reduction (β-mercaptoethanol (βME)) procedure was carried out on the sample. As controls, dye-untreated non-alkylated and alkylated samples were also processed. Carbamidomethylation of cysteines within MT1E's tryptic peptides was then monitored and used as negative evidence of given cysteine’s participation in dye coordination, assuming the residue as free for the IAA reaction, and thus not occupied by the dye. Previous experiments in the project suggested three possible binding sites of CrAsH within MT1E, hence the analysis comprised of the mentioned three molar ratio steps.

INSTRUMENT(S): Synapt MS

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Michał Tracz

LAB HEAD: Michal Tracz

PROVIDER: PXD048982 | Pride | 2024-12-16

REPOSITORIES: pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	240117_ZCHB_APO_E0N_r0_nr10.mgf	Mgf
	240117_ZCHB_APO_E0N_r0_nr10.raw.zip	Raw
	240117_ZCHB_APO_E0N_r0_nr10_MSe_MT1E_wrkflow_IAA_enriched_5MsClvg_2.mzid.gz	Mzid
	240117_ZCHB_APO_E0_r0_nr10.mgf	Mgf
	240117_ZCHB_APO_E0_r0_nr10.raw.zip	Raw

Items per page:

1 - 5 of 19

Similar Datasets

Project description:The use of integrated approaches in genetic toxicology, including the incorporation of gene expression data to determine the molecular pathways involved in the response, is becoming more common. In a companion paper, a genomic biomarker was developed in human TK6 cells to classify chemicals as genotoxic or non-genotoxic. Because TK6 cells are not metabolically competent, we set out to broaden the utility of the biomarker for use with chemicals requiring metabolic activation. Specifically, chemical exposures were conducted in the presence of rat liver S9. The ability of the biomarker to classify genotoxic (benzo[a]pyrene, BaP; aflatoxin B1, AFB1) and non-genotoxic (dexamethasone, DEX; phenobarbital, PB) agents correctly was evaluated. Cells were exposed to increasing chemical concentrations for 4h and collected 0h, 4h and 20h post-exposure. Relative survival, apoptosis, and micronucleus frequency were measured at 24h. Transcriptome profiles were measured with Agilent microarrays. Statistical modeling and bioinformatics tools were applied to classify each chemical using the genomic biomarker. BaP and AFB1 were correctly classified as genotoxic at the mid- and high concentrations at all three time points, whereas DEX was correctly classified as non-genotoxic at all concentrations and time points. The high concentration of PB was misclassified at 24h, suggesting that cytotoxicity at later time points may cause misclassification. The data suggest that the use of S9 does not impair the ability of the biomarker to classify genotoxicity in TK6 cells. Finally, we demonstrate that the biomarker is also able to accurately classify genotoxicity using a publicly available dataset derived from human HepaRG cells. This experiment examined the whole genome transcriptional response of TK6 cells exposed to Aflatoxin B1 for 4 hours followed by a 0h, 4h and 20h recovery in fresh media (4h, 8h, and 24h time points, respectively) at 3 different concentrations, including a low (0.025 Î¼M), a medium (0.075 Î¼M), and a high (0.1 Î¼M) concentration, in addition to negative controls (Â±S9), vehicle controls (Â±S9) and a direct-acting positive control, cisplatin (-S9) at 1 Î¼g/ml (PC-1) and 24 Î¼g/ml (PC-24). Each concentration and time point had 3 biological replicates. There were a total 30 samples (60 arrays) included in the final analysis using a two-colour dye swap design. Please note that each sample data table contains normalized data combined from two replicates and the sample-dye assignment information for each of the raw data files is included in the 'readme.txt' (available as Series supplementary file).

Dataset Information

Assessment of biarsenical dye-reactive cysteine residues of human metallothionein MT1E

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets