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SILAC-Based Quantitative Proteomic Analysis Unveils Arsenite-Induced Perturbation of Multiple Pathways in Human Skin Fibroblast Cells.

ABSTRACT: Humans are exposed to arsenic species through inhalation, ingestion, and dermal contact, which may lead to skin, liver, and bladder cancers as well as cardiovascular and neurological diseases. The mechanisms underlying the cytotoxic and carcinogenic effects of arsenic species, however, remain incompletely understood. To exploit the mechanisms of toxicity of As(III), we employed stable isotope labeling by amino acids in cell culture (SILAC) together with LC/MS/MS analysis to quantitatively assess the As(III)-induced perturbation of the entire proteome of cultured human skin fibroblast cells. Shotgun proteomic analysis on an LTQ-Orbitrap Velos mass spectrometer facilitated the quantification of 3880 proteins, 130 of which were quantified in both forward and reverse SILAC-labeling experiments and displayed significant alterations (>1.5 fold) upon arsenite treatment. Targeted analysis on a triple-quadrupole mass spectrometer in multiple-reaction monitoring (MRM) mode confirmed the quantification results of some select proteins. Ingenuity pathway analysis revealed the arsenite-induced alteration of more than 10 biological pathways, including the Nrf2-mediated oxidative stress response pathway, which is represented by the upregulation of nine proteins in this pathway. In addition, arsenite induced changes in expression levels of a number of selenoproteins and metallothioneins. Together, the results from the present study painted a more complete picture regarding the biological pathways that are altered in human skin fibroblast cells upon arsenite exposure.

SUBMITTER: Zhang F

PROVIDER: S-EPMC5616127 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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SILAC-Based Quantitative Proteomic Analysis Unveils Arsenite-Induced Perturbation of Multiple Pathways in Human Skin Fibroblast Cells.

Zhang Fan F Xiao Yongsheng Y Wang Yinsheng Y

Chemical research in toxicology 20170214 4

Humans are exposed to arsenic species through inhalation, ingestion, and dermal contact, which may lead to skin, liver, and bladder cancers as well as cardiovascular and neurological diseases. The mechanisms underlying the cytotoxic and carcinogenic effects of arsenic species, however, remain incompletely understood. To exploit the mechanisms of toxicity of As(III), we employed stable isotope labeling by amino acids in cell culture (SILAC) together with LC/MS/MS analysis to quantitatively assess ...[more]

PMID: 28140569

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Project description:BackgroundTriglyceride deposit cardiomyovasculopathy (TGCV) is a rare disease, characterized by the massive accumulation of triglyceride (TG) in multiple tissues, especially skeletal muscle, heart muscle and the coronary artery. TGCV is caused by mutation of adipose triglyceride lipase, which is an essential molecule for the hydrolysis of TG. TGCV is at high risk for skeletal myopathy and heart dysfunction, and therefore premature death. Development of therapeutic methods for TGCV is highly desirable. This study aims to discover specific molecules responsible for TGCV pathogenesis.MethodsTo identify differentially expressed proteins in TGCV patient cells, the stable isotope labeling with amino acids in cell culture (SILAC) method coupled with LC-MS/MS was performed using skin fibroblast cells derived from two TGCV patients and three healthy volunteers. Altered protein expression in TGCV cells was confirmed using the selected reaction monitoring (SRM) method. Microarray-based transcriptome analysis was simultaneously performed to identify changes in gene expression in TGCV cells.ResultsUsing SILAC proteomics, 4033 proteins were quantified, 53 of which showed significantly altered expression in both TGCV patient cells. Twenty altered proteins were chosen and confirmed using SRM. SRM analysis successfully quantified 14 proteins, 13 of which showed the same trend as SILAC proteomics. The altered protein expression data set was used in Ingenuity Pathway Analysis (IPA), and significant networks were identified. Several of these proteins have been previously implicated in lipid metabolism, while others represent new therapeutic targets or markers for TGCV. Microarray analysis quantified 20743 transcripts, and 252 genes showed significantly altered expression in both TGCV patient cells. Ten altered genes were chosen, 9 of which were successfully confirmed using quantitative RT-PCR. Biological networks of altered genes were analyzed using an IPA search.ConclusionsWe performed the SILAC- and SRM-based identification-through-confirmation study using skin fibroblast cells derived from TGCV patients, and first identified altered proteins specific for TGCV. Microarray analysis also identified changes in gene expression. The functional networks of the altered proteins and genes are discussed. Our findings will be exploited to elucidate the pathogenesis of TGCV and discover clinically relevant molecules for TGCV in the near future.

Dataset Information

SILAC-Based Quantitative Proteomic Analysis Unveils Arsenite-Induced Perturbation of Multiple Pathways in Human Skin Fibroblast Cells.

Publications

SILAC-Based Quantitative Proteomic Analysis Unveils Arsenite-Induced Perturbation of Multiple Pathways in Human Skin Fibroblast Cells.

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