An Integrated Platform for High-Throughput Extraction and Mass Spectrometry-Based Quantification of Cholesterol and...

Area of Science:

• Biochemistry
• Cell Biology
• Analytical Chemistry

Background:

• Lipid storage diseases, such as Niemann-Pick Disease Type C (NPC), involve cellular lipid accumulation, primarily unesterified cholesterol.
• Current methods like the filipin fluorescence assay have limitations including nonspecific binding, multiple steps, and poor quantitation accuracy, hindering high-throughput screening.
• Accurate and efficient cellular cholesterol quantification is crucial for developing novel therapeutics for these genetic disorders.

Purpose of the Study:

• To develop an integrated, semiautomated, high-throughput protocol for cellular cholesterol extraction and quantification.
• To establish a label-free assay for accurate cholesterol measurement in a 384-well plate format.
• To validate the method's efficiency and applicability for screening investigational compounds and multiplexing lipid analysis.

Main Methods:

• Development of an integrated workflow using a liquid handling platform and high-throughput mass spectrometry (MS).
• Lipid extraction from neural stem cells using methyl tert-butyl ether (MTBE) with 13C-cholesterol as an internal standard.
• Semiautomated protocol enabling lipid extraction and MS analysis within 2 hours in a 384-well plate format.

Main Results:

• The integrated platform demonstrated excellent quantification linearity and reproducibility (intraday and interday, R2 > 0.99).
• High recovery rates (83-107%) were achieved for cholesterol extraction.
• The method was successfully applied to screen 241 compounds and showed capability for multiplexing sphingosine and cholesterol quantification.

Conclusions:

• The developed integrated and semiautomated platform provides an efficient, label-free, and high-throughput method for cellular cholesterol quantification.
• This assay overcomes limitations of traditional methods, enabling accurate screening for lipid storage disease therapeutics.
• The workflow's adaptability extends its utility to other lipid storage diseases through multiplexed lipid analysis.