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Data-independent proteome analysis of ARPE-19 cells.

ABSTRACT: We have performed a proteomics analysis of a human retinal pigment epithelial cell line (ARPE-19), which represents a widely used model for in vitro studies of cellular and molecular mechanisms related to human RPE cells (Dunn et al., 1996; Weigel et al., 2002) [1], [2]. Whole cell protein extracts were separated in four gel fractions via short (10?min) SDS-PAGE runs. Following fractionation and trypsin digestion, the resulting peptides were separated on a nano UPLC LC system and analyzed on-line with a QTof-IMS instrument: a tandem mass spectrometer with ion mobility separation (Synapt G2-Si). Data were acquired in data-independent mode (UDMSE), which allows for absolute and/or relative post-acquisition protein quantification (Silva et al., 2006) [3]. The proteome profile data obtained from this study can be used as a protein reference database with qualitative and quantitative protein information related to ARPE-19 cells under normal growth conditions.

SUBMITTER: Koirala D

PROVIDER: S-EPMC6111057 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Data-independent proteome analysis of ARPE-19 cells.

Koirala Diwa D Beranova-Giorgianni Sarka S Giorgianni Francesco F

Data in brief 20180704

We have performed a proteomics analysis of a human retinal pigment epithelial cell line (ARPE-19), which represents a widely used model for <i>in vitro</i> studies of cellular and molecular mechanisms related to human RPE cells (Dunn et al., 1996; Weigel et al., 2002) [1], [2]. Whole cell protein extracts were separated in four gel fractions via short (10 min) SDS-PAGE runs. Following fractionation and trypsin digestion, the resulting peptides were separated on a nano UPLC LC system and analyzed ...[more]

PMID: 30167441

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Project description:Age related macular degeneration (AMD) is one of the leading causes of blindness. Genetics, environmental insult, and age-related factors all play a key role in altering proteostasis, the homeostatic process regulating protein synthesis, degradation and processing. These factors also play a role in the pathogenesis of AMD and it has been well established that cigarette smoking (CS) initiates AMD pathogenic mechanisms. The primary goal of this study is to elucidate whether CS can induce proteostasis/autophagy-impairment in retinal pigment epithelial (RPE) cells. In our preliminary analysis, it was found that cigarette smoke extract (CSE) induces accumulation of ubiquitinated proteins in the insoluble protein fraction (p < 0.01), which was subsequently mitigated through cysteamine (p < 0.01) or fisetin (p < 0.05) treatment. Further, it was verified that these CSE induced ubiquitinated proteins accumulated in the peri-nuclear spaces (p<0.05) that were cleared- off with cysteamine (p < 0.05) or fisetin (p < 0.05). Moreover, CSE-induced aggresome-formation (LC3B-GFP and Ub-RFP co-localization) and autophagy-flux impairment was significantly (p<0.01) mitigated by cysteamine (p<0.05) or fisetin (p<0.05) treatment, indicating the restoration of CSE-mediated autophagy-impairment. CSE treatment was also found to induce intracellular reactive oxygen species (ROS, p < 0.001) while impacting cell viability (p < 0.001), which was quantified using CMH2DCFDA-dye (ROS) and MTS (proliferation) or propodium iodide staining (cell viability) assays, respectively. Moreover, cysteamine and fisetin treatment ameliorated CS-mediated ROS production (p < 0.05) and diminished cell viability (p < 0.05). Lastly, CSE was found to induce cellular senescence (p < 0.001), which was significantly ameliorated by cysteamine (p < 0.001) or fisetin (p < 0.001). In conclusion, our study indicates that CS induced proteostasis/autophagy-impairment regulates mechanisms associated with AMD pathogenesis. Moreover, autophagy-inducing drugs such as cysteamine or fisetin can ameliorate AMD pathogenesis mechanisms that warrant further investigation in pre-clinical murine models.

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Data-independent proteome analysis of ARPE-19 cells.

Publications

Data-independent proteome analysis of ARPE-19 cells.

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