High-throughput MS-based cholesterol quantitation assay for Niemann-Pick Disease type C (NPC)

Authors

• WH Hibler
• YL Yu
• G Merlino

Abstract

Quantification of cellular lipids is a key step for the development of therapeutics for lipid storage diseases. For Niemann-Pick Disease Type C (NPC), a genetic disorder characterized by the accumulation of unesterified cholesterol in late endosome/lysosome, filipin staining was used traditionally for cholesterol quantification. However, due to the non-specific binding of filipin to other sterol derivatives and its sensitivity to photobleaching, using filipin staining in high-throughput assay for compound screening is not ideal. We have developed an automated protocol for extracting cellular cholesterol and quantifying it using the high-throughput LC/MS system RapidFire 360. Neuronal stem cells derived from human NPC fibroblasts were optimized to grow in 384-well format, and cholesterol was extracted using methyl-tert-butyl ether (MTBE). 13C-labeled cholesterol was added to MTBE so that signal of native cholesterol could be normalized to it to account for variations introduced during multiple liquid transfer steps. This assay allows lipid extraction and analyses by MS for 384 samples to be completed in 1.5-2 hours. Using this assay, we screened a 248-compound library of investigational agents for NPC in dose-response, benchmarking this efficient, label-free cellular cholesterol quantification assay for high-throughput applications.

Scientific Focus Area: Chemical Biology

This page was last updated on Monday, September 25, 2023