High-Coverage Strategy for Multi-Subcellular Metabolome Analysis Using Dansyl-Labeling-Based LC-MS/MS.

Area of Science:

• Cell Biology
• Metabolomics
• Biochemistry

Background:

• Subcellular compartmentalization is crucial for cellular metabolic efficiency.
• Understanding organelle-specific metabolism offers deep insights into cellular functions.
• Sensitive measurement of multi-subcellular metabolic profiles remains a challenge.

Purpose of the Study:

• To develop a comprehensive strategy for subcellular fractionation and metabolome analysis.
• To establish and validate a sensitive method for measuring metabolites in subcellular fractions.
• To investigate organelle-specific metabolic changes in acute liver injury.

Main Methods:

• Six subcellular fractions (nuclei, mitochondria, lysosomes, peroxisomes, microsomes, cytoplasm) were isolated from liver homogenate.
• A dansyl-labeling-assisted liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to quantify 151 metabolites.
• The strategy was applied to a rat model of carbon tetrachloride (CCl4)-induced acute liver injury (ALI).

Main Results:

• The developed method successfully profiled metabolites across six subcellular fractions.
• Organelle-specific metabolic profiles revealed unique changes not detected in whole liver tissue.
• Tissue-level analysis masked significant organelle-specific metabolic alterations due to a leveling effect.

Conclusions:

• This study presents a feasible approach for broad-spectrum targeted metabolomic profiling of subcellular fractions.
• Subcellular metabolomic analysis is essential for accurate understanding of cellular metabolism in health and disease.
• The findings highlight the limitations of tissue-level metabolomics and the importance of organelle-specific investigations.