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Development of a Label-Free LC-MS/MS-Based Glucosylceramide Synthase Assay and Its Application to Inhibitors Screening for Ceramide-Related Diseases
Ceramide metabolism is known to be an essential etiology for various diseases, such as atopic dermatitis and Gaucher disease. Glucosylceramide synthase (GCS) is a key enzyme for the synthesis of glucosylceramide (GlcCer), which is a main ceramide metabolism pathway in mammalian cells. In this articl...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
The Korean Society of Applied Pharmacology
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6430231/ https://www.ncbi.nlm.nih.gov/pubmed/30231605 http://dx.doi.org/10.4062/biomolther.2018.122 |
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author | Fu, Zhicheng Yun, So Yoon Won, Jong Hoon Back, Moon Jung Jang, Ji Min Ha, Hae Chan Lee, Hae Kyung Shin, In Chul Kim, Ju Yeun Kim, Hee Soo Kim, Dae Kyong |
author_facet | Fu, Zhicheng Yun, So Yoon Won, Jong Hoon Back, Moon Jung Jang, Ji Min Ha, Hae Chan Lee, Hae Kyung Shin, In Chul Kim, Ju Yeun Kim, Hee Soo Kim, Dae Kyong |
author_sort | Fu, Zhicheng |
collection | PubMed |
description | Ceramide metabolism is known to be an essential etiology for various diseases, such as atopic dermatitis and Gaucher disease. Glucosylceramide synthase (GCS) is a key enzyme for the synthesis of glucosylceramide (GlcCer), which is a main ceramide metabolism pathway in mammalian cells. In this article, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to determine GCS activity using synthetic non-natural sphingolipid C8-ceramide as a substrate. The reaction products, C8-GlcCer for GCS, could be separated on a C18 column by reverse-phase high-performance liquid chromatography (HPLC). Quantification was conducted using the multiple reaction monitoring (MRM) mode to monitor the precursor-to-product ion transitions of m/z 588.6 → 264.4 for C8-GlcCer at positive ionization mode. The calibration curve was established over the range of 0.625–160 ng/mL, and the correlation coefficient was larger than 0.999. This method was successfully applied to detect GCS in the human hepatocellular carcinoma cell line (HepG2 cells) and mouse peripheral blood mononuclear cells. We also evaluated the inhibition degree of a known GCS inhibitor 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) on GCS enzymatic activity and proved that this method could be successfully applied to GCS inhibitor screening of preventive and therapeutic drugs for ceramide metabolism diseases, such as atopic dermatitis and Gaucher disease. |
format | Online Article Text |
id | pubmed-6430231 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | The Korean Society of Applied Pharmacology |
record_format | MEDLINE/PubMed |
spelling | pubmed-64302312019-03-25 Development of a Label-Free LC-MS/MS-Based Glucosylceramide Synthase Assay and Its Application to Inhibitors Screening for Ceramide-Related Diseases Fu, Zhicheng Yun, So Yoon Won, Jong Hoon Back, Moon Jung Jang, Ji Min Ha, Hae Chan Lee, Hae Kyung Shin, In Chul Kim, Ju Yeun Kim, Hee Soo Kim, Dae Kyong Biomol Ther (Seoul) Original Article Ceramide metabolism is known to be an essential etiology for various diseases, such as atopic dermatitis and Gaucher disease. Glucosylceramide synthase (GCS) is a key enzyme for the synthesis of glucosylceramide (GlcCer), which is a main ceramide metabolism pathway in mammalian cells. In this article, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to determine GCS activity using synthetic non-natural sphingolipid C8-ceramide as a substrate. The reaction products, C8-GlcCer for GCS, could be separated on a C18 column by reverse-phase high-performance liquid chromatography (HPLC). Quantification was conducted using the multiple reaction monitoring (MRM) mode to monitor the precursor-to-product ion transitions of m/z 588.6 → 264.4 for C8-GlcCer at positive ionization mode. The calibration curve was established over the range of 0.625–160 ng/mL, and the correlation coefficient was larger than 0.999. This method was successfully applied to detect GCS in the human hepatocellular carcinoma cell line (HepG2 cells) and mouse peripheral blood mononuclear cells. We also evaluated the inhibition degree of a known GCS inhibitor 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) on GCS enzymatic activity and proved that this method could be successfully applied to GCS inhibitor screening of preventive and therapeutic drugs for ceramide metabolism diseases, such as atopic dermatitis and Gaucher disease. The Korean Society of Applied Pharmacology 2019-02 2018-09-20 /pmc/articles/PMC6430231/ /pubmed/30231605 http://dx.doi.org/10.4062/biomolther.2018.122 Text en Copyright ©2019, The Korean Society of Applied Pharmacology http://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Original Article Fu, Zhicheng Yun, So Yoon Won, Jong Hoon Back, Moon Jung Jang, Ji Min Ha, Hae Chan Lee, Hae Kyung Shin, In Chul Kim, Ju Yeun Kim, Hee Soo Kim, Dae Kyong Development of a Label-Free LC-MS/MS-Based Glucosylceramide Synthase Assay and Its Application to Inhibitors Screening for Ceramide-Related Diseases |
title | Development of a Label-Free LC-MS/MS-Based Glucosylceramide Synthase Assay and Its Application to Inhibitors Screening for Ceramide-Related Diseases |
title_full | Development of a Label-Free LC-MS/MS-Based Glucosylceramide Synthase Assay and Its Application to Inhibitors Screening for Ceramide-Related Diseases |
title_fullStr | Development of a Label-Free LC-MS/MS-Based Glucosylceramide Synthase Assay and Its Application to Inhibitors Screening for Ceramide-Related Diseases |
title_full_unstemmed | Development of a Label-Free LC-MS/MS-Based Glucosylceramide Synthase Assay and Its Application to Inhibitors Screening for Ceramide-Related Diseases |
title_short | Development of a Label-Free LC-MS/MS-Based Glucosylceramide Synthase Assay and Its Application to Inhibitors Screening for Ceramide-Related Diseases |
title_sort | development of a label-free lc-ms/ms-based glucosylceramide synthase assay and its application to inhibitors screening for ceramide-related diseases |
topic | Original Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6430231/ https://www.ncbi.nlm.nih.gov/pubmed/30231605 http://dx.doi.org/10.4062/biomolther.2018.122 |
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