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A proteomics analysis to evaluate cytotoxicity in NRK-52E cells caused by unmodified Nano-Fe₃O₄.

Lin YR, Kuo CJ, Lin HY, Wu CJ, Liang SS - ScientificWorldJournal (2014)

Bottom Line: Through global proteomics analysis using dimethyl labeling techniques and liquid phase chromatography coupled with a tandem mass spectrometer (LC-MS/MS), we characterized 435 proteins including the programmed cell death related proteins, ras-related proteins, glutathione related proteins, and the chaperone proteins such as heat shock proteins, serpin H1, protein disulfide-isomerase A4, endoplasmin, and endoplasmic reticulum resident proteins.From the statistical data of identified proteins, we believed that NPs treatment causes cell death and promotes expression of ras-related proteins.In order to avoid apoptosis, NRK-52E cell lines induce a series of protective effects such as glutathione related proteins to reduce reactive oxygen species (ROS), and chaperone proteins to recycle damaged proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Fooyin University, 151 Jinxue Road, Kaohsiung 83102, Taiwan.

ABSTRACT
We synthesized unmodified Fe₃O₄ nanoparticles (NPs) with particles size from 10 nm to 100 nm. We cultured NRK-52E cell lines (rat, kidney) and treated with Fe₃O₄ NPs to investigate and evaluate the cytotoxicity of NPs for NRK-52E cells. Through global proteomics analysis using dimethyl labeling techniques and liquid phase chromatography coupled with a tandem mass spectrometer (LC-MS/MS), we characterized 435 proteins including the programmed cell death related proteins, ras-related proteins, glutathione related proteins, and the chaperone proteins such as heat shock proteins, serpin H1, protein disulfide-isomerase A4, endoplasmin, and endoplasmic reticulum resident proteins. From the statistical data of identified proteins, we believed that NPs treatment causes cell death and promotes expression of ras-related proteins. In order to avoid apoptosis, NRK-52E cell lines induce a series of protective effects such as glutathione related proteins to reduce reactive oxygen species (ROS), and chaperone proteins to recycle damaged proteins. We suggested that, in the indigenous cellular environment, Fe₃O₄ NPs treatment induced an antagonistic effect for cell lines to go to which avoids apoptosis.

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Related in: MedlinePlus

Simplified flow chart of sample pretreatment, fractionation, and analysis using LC-MS/MS.
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Related In: Results  -  Collection


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fig2: Simplified flow chart of sample pretreatment, fractionation, and analysis using LC-MS/MS.

Mentions: For protein quantitation, the procedures and using reagents including formaldehyde-H2, formaldehyde-D2, and sodium cyanoborohydride (NaBCNH3) were obeyed as following the previous literature [19]. By utilization of dimethyl labeling, the differences of protein expressions in treatment with and without NPs was enabled to be determined. In the mass spectrum, the proteins of NRK-52E treated NPs were labeled as formaldehyde-D2 and proteins of untreated NPs were labeled as formaldehyde-H2. In the same sequential peptides, however, different treatments had a mass difference 4 Da or 8 Da depending on the use of the labeling agent. Off-line HILIC fractionation was utilized to decrease the complication of the mixed samples [16, 18]. Through LC-MS/MS, the proteins identification and quantitative ratios of D/H peptides were identified and calculated using the bioinformatic Mascot Distiller software to finally generate a protein list. The schematic flow chart of the process is shown in Figure 2 from sample pretreatment and protein identification to protein quantitation.


A proteomics analysis to evaluate cytotoxicity in NRK-52E cells caused by unmodified Nano-Fe₃O₄.

Lin YR, Kuo CJ, Lin HY, Wu CJ, Liang SS - ScientificWorldJournal (2014)

Simplified flow chart of sample pretreatment, fractionation, and analysis using LC-MS/MS.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4150542&req=5

fig2: Simplified flow chart of sample pretreatment, fractionation, and analysis using LC-MS/MS.
Mentions: For protein quantitation, the procedures and using reagents including formaldehyde-H2, formaldehyde-D2, and sodium cyanoborohydride (NaBCNH3) were obeyed as following the previous literature [19]. By utilization of dimethyl labeling, the differences of protein expressions in treatment with and without NPs was enabled to be determined. In the mass spectrum, the proteins of NRK-52E treated NPs were labeled as formaldehyde-D2 and proteins of untreated NPs were labeled as formaldehyde-H2. In the same sequential peptides, however, different treatments had a mass difference 4 Da or 8 Da depending on the use of the labeling agent. Off-line HILIC fractionation was utilized to decrease the complication of the mixed samples [16, 18]. Through LC-MS/MS, the proteins identification and quantitative ratios of D/H peptides were identified and calculated using the bioinformatic Mascot Distiller software to finally generate a protein list. The schematic flow chart of the process is shown in Figure 2 from sample pretreatment and protein identification to protein quantitation.

Bottom Line: Through global proteomics analysis using dimethyl labeling techniques and liquid phase chromatography coupled with a tandem mass spectrometer (LC-MS/MS), we characterized 435 proteins including the programmed cell death related proteins, ras-related proteins, glutathione related proteins, and the chaperone proteins such as heat shock proteins, serpin H1, protein disulfide-isomerase A4, endoplasmin, and endoplasmic reticulum resident proteins.From the statistical data of identified proteins, we believed that NPs treatment causes cell death and promotes expression of ras-related proteins.In order to avoid apoptosis, NRK-52E cell lines induce a series of protective effects such as glutathione related proteins to reduce reactive oxygen species (ROS), and chaperone proteins to recycle damaged proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Fooyin University, 151 Jinxue Road, Kaohsiung 83102, Taiwan.

ABSTRACT
We synthesized unmodified Fe₃O₄ nanoparticles (NPs) with particles size from 10 nm to 100 nm. We cultured NRK-52E cell lines (rat, kidney) and treated with Fe₃O₄ NPs to investigate and evaluate the cytotoxicity of NPs for NRK-52E cells. Through global proteomics analysis using dimethyl labeling techniques and liquid phase chromatography coupled with a tandem mass spectrometer (LC-MS/MS), we characterized 435 proteins including the programmed cell death related proteins, ras-related proteins, glutathione related proteins, and the chaperone proteins such as heat shock proteins, serpin H1, protein disulfide-isomerase A4, endoplasmin, and endoplasmic reticulum resident proteins. From the statistical data of identified proteins, we believed that NPs treatment causes cell death and promotes expression of ras-related proteins. In order to avoid apoptosis, NRK-52E cell lines induce a series of protective effects such as glutathione related proteins to reduce reactive oxygen species (ROS), and chaperone proteins to recycle damaged proteins. We suggested that, in the indigenous cellular environment, Fe₃O₄ NPs treatment induced an antagonistic effect for cell lines to go to which avoids apoptosis.

Show MeSH
Related in: MedlinePlus