Reducing time and increasing sensitivity in sample preparation for adherent mammalian cell metabolomics

Area of Science:

• Metabolomics
• Cellular Biology
• Analytical Chemistry

Background:

• Accurate metabolite quantification is vital for understanding cellular processes.
• Existing sample preparation methods can be time-consuming and affect metabolite stability.
• High-performance liquid chromatography-mass spectrometry (HPLC-MS) is a powerful tool for metabolite analysis.

Purpose of the Study:

• To develop a simple, fast, and reproducible sample preparation method for relative metabolite quantification in adherent mammalian cells.
• To optimize sample preparation for enhanced metabolite signal and stability using INS-1 cells.
• To evaluate the impact of different preparation steps on metabolite recovery and detection.

Main Methods:

• Developed a rapid sample preparation protocol involving a water rinse and liquid nitrogen quench.
• Evaluated 27 metabolites in glycolysis and the tricarboxylic acid cycle using directed and undirected HPLC-MS.
• Compared different extraction solvents (methanol:chloroform, acetonitrile, ethanol, methanol) and extraction times.

Main Results:

• A water rinse increased signal for 26/27 targeted metabolites and doubled detected features (237 to 452).
• Liquid nitrogen quenching allowed sample storage for at least 7 days at -80°C without significant metabolite loss.
• A 9:1 methanol:chloroform extraction solvent provided superior recovery and stability in a rapid, single-step extraction.

Conclusions:

• The developed ~5-minute method significantly improves metabolite detection and quantification in mammalian cells.
• This rapid and robust protocol enhances experimental convenience and sample stability for metabolomic studies.
• The optimized method enables precise metabolite measurements, facilitating the study of dynamic cellular processes like insulin secretion.