Biomol Ther.  2019 Feb;27(2):193-200. 10.4062/biomolther.2018.122.

Development of a Label-Free LC-MS/MS-Based Glucosylceramide Synthase Assay and Its Application to Inhibitors Screening for Ceramide-Related Diseases

• Fu Z
• Yun SY
• Won JH
• Back MJ
• Jang JM
• Ha HC
• Lee HK
• Shin IC
• Kim JY
• Kim HS
• Kim DK

• KMID: 2442011
• DOI: http://doi.org/10.4062/biomolther.2018.122

Abstract

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.

Keyword

Ceramide; Chemotherapeutic multi-drug resistance; Gaucher disease; Glucosylceramide; Glucosylceramide synthase; LC-MS/MS

MeSH Terms

Animals
Calibration
Carcinoma, Hepatocellular
Cell Line
Chromatography, Liquid
Dermatitis, Atopic
Gaucher Disease
Humans
Mass Screening*
Mass Spectrometry
Metabolism
Methods
Mice