Extensive Thiol Profiling for Assessment of Intracellular Redox Status in Cultured Cells by HPLC-MS/MS
Oxidative stress may contribute to the pathology of many diseases, and endogenous thiols, especially glutathione (GSH) and its metabolites, play essential roles in the maintenance of normal redox status. Understanding how these metabolites change in response to oxidative insult can provide key insig...
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| Format: | Article |
| Language: | English |
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MDPI AG
2021-12-01
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| Series: | Antioxidants |
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| Online Access: | https://www.mdpi.com/2076-3921/11/1/24 |
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| author | Jiandong Wu Anna Chernatynskaya Annalise Pfaff Huari Kou Nan Cen Nuran Ercal Honglan Shi |
| author_facet | Jiandong Wu Anna Chernatynskaya Annalise Pfaff Huari Kou Nan Cen Nuran Ercal Honglan Shi |
| author_sort | Jiandong Wu |
| collection | DOAJ |
| description | Oxidative stress may contribute to the pathology of many diseases, and endogenous thiols, especially glutathione (GSH) and its metabolites, play essential roles in the maintenance of normal redox status. Understanding how these metabolites change in response to oxidative insult can provide key insights into potential methods of prevention and treatment. Most existing methodologies focus only on the GSH/GSH disulfide (GSSG) redox couple, but GSH regulation is highly complex and depends on several pathways with multiple redox-active sulfur-containing species. In order to more fully characterize thiol redox status in response to oxidative insult, a high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) method was developed to simultaneously determine seven sulfur-containing metabolites, generating a panel that systematically examines several pathways involved in thiol metabolism and oxidative stress responses. The sensitivity (LOQ as low as 0.01 ng/mL), accuracy (88–126% spike recovery), and precision (≤12% RSD) were comparable or superior to those of existing methods. Additionally, the method was used to compare the baseline thiol profiles and oxidative stress responses of cell lines derived from different tissues. The results revealed a previously unreported response to oxidative stress in lens epithelial (B3) cells, which may be exploited as a new therapeutic target for oxidative-stress-related ocular diseases. Further application of this method may uncover new pathways involved in oxidative-stress-related diseases and endogenous defense mechanisms. |
| first_indexed | 2024-03-10T03:01:56Z |
| format | Article |
| id | doaj.art-989d0bf3a36d420189a91692889c609b |
| institution | Directory Open Access Journal |
| issn | 2076-3921 |
| language | English |
| last_indexed | 2024-03-10T03:01:56Z |
| publishDate | 2021-12-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Antioxidants |
| spelling | doaj.art-989d0bf3a36d420189a91692889c609b2023-11-23T12:46:07ZengMDPI AGAntioxidants2076-39212021-12-011112410.3390/antiox11010024Extensive Thiol Profiling for Assessment of Intracellular Redox Status in Cultured Cells by HPLC-MS/MSJiandong Wu0Anna Chernatynskaya1Annalise Pfaff2Huari Kou3Nan Cen4Nuran Ercal5Honglan Shi6Department of Chemical and Biochemical Engineering, Missouri University of Science and Technology, Rolla, MO 65409, USADepartment of Chemical and Biochemical Engineering, Missouri University of Science and Technology, Rolla, MO 65409, USADepartment of Chemistry, Missouri University of Science and Technology, Rolla, MO 65409, USADepartment of Chemical and Biochemical Engineering, Missouri University of Science and Technology, Rolla, MO 65409, USADepartment of Computer Science, Missouri University of Science and Technology, Rolla, MO 65409, USADepartment of Chemistry, Missouri University of Science and Technology, Rolla, MO 65409, USADepartment of Chemistry, Missouri University of Science and Technology, Rolla, MO 65409, USAOxidative stress may contribute to the pathology of many diseases, and endogenous thiols, especially glutathione (GSH) and its metabolites, play essential roles in the maintenance of normal redox status. Understanding how these metabolites change in response to oxidative insult can provide key insights into potential methods of prevention and treatment. Most existing methodologies focus only on the GSH/GSH disulfide (GSSG) redox couple, but GSH regulation is highly complex and depends on several pathways with multiple redox-active sulfur-containing species. In order to more fully characterize thiol redox status in response to oxidative insult, a high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) method was developed to simultaneously determine seven sulfur-containing metabolites, generating a panel that systematically examines several pathways involved in thiol metabolism and oxidative stress responses. The sensitivity (LOQ as low as 0.01 ng/mL), accuracy (88–126% spike recovery), and precision (≤12% RSD) were comparable or superior to those of existing methods. Additionally, the method was used to compare the baseline thiol profiles and oxidative stress responses of cell lines derived from different tissues. The results revealed a previously unreported response to oxidative stress in lens epithelial (B3) cells, which may be exploited as a new therapeutic target for oxidative-stress-related ocular diseases. Further application of this method may uncover new pathways involved in oxidative-stress-related diseases and endogenous defense mechanisms.https://www.mdpi.com/2076-3921/11/1/24thiolglutathionebiomarkerHPLC-MS/MSlens epithelial cellscancer cells |
| spellingShingle | Jiandong Wu Anna Chernatynskaya Annalise Pfaff Huari Kou Nan Cen Nuran Ercal Honglan Shi Extensive Thiol Profiling for Assessment of Intracellular Redox Status in Cultured Cells by HPLC-MS/MS Antioxidants thiol glutathione biomarker HPLC-MS/MS lens epithelial cells cancer cells |
| title | Extensive Thiol Profiling for Assessment of Intracellular Redox Status in Cultured Cells by HPLC-MS/MS |
| title_full | Extensive Thiol Profiling for Assessment of Intracellular Redox Status in Cultured Cells by HPLC-MS/MS |
| title_fullStr | Extensive Thiol Profiling for Assessment of Intracellular Redox Status in Cultured Cells by HPLC-MS/MS |
| title_full_unstemmed | Extensive Thiol Profiling for Assessment of Intracellular Redox Status in Cultured Cells by HPLC-MS/MS |
| title_short | Extensive Thiol Profiling for Assessment of Intracellular Redox Status in Cultured Cells by HPLC-MS/MS |
| title_sort | extensive thiol profiling for assessment of intracellular redox status in cultured cells by hplc ms ms |
| topic | thiol glutathione biomarker HPLC-MS/MS lens epithelial cells cancer cells |
| url | https://www.mdpi.com/2076-3921/11/1/24 |
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