Resolving Hexose-Phosphates by LC-MS Leads to New Insights in PGM1-CDG Pathophysiology

Area of Science:

• Metabolomics
• Biochemistry
• Analytical Chemistry

Background:

• Hexose-phosphates are crucial in metabolic pathways like glycolysis and glycosylation.
• Understanding hexose-phosphate levels is vital for studying diseases such as congenital disorders of glycosylation (CDG).
• Differentiating structural isomer hexose-phosphates using mass spectrometry (MS) is challenging due to similar physicochemical properties.

Purpose of the Study:

• To develop and compare optimized liquid chromatography-MS methods for identifying human hexose-phosphates.
• To enable accurate quantification of hexose-phosphates for tracer metabolomics applications.
• To analyze hexose-phosphate profiles in the context of CDG and treatment responses.

Main Methods:

• Development and comparison of two optimized liquid chromatography-MS methods.
• Validation of methods for linearity (3-4 orders of magnitude) and limits of quantification (0.5-50 nM).
• Application of the methods to analyze hexose-phosphate profiles in phosphoglucomutase 1 (PGM1)-CDG fibroblasts treated with galactose.

Main Results:

• Successful identification and quantification of eight hexose-monophosphates and two hexose-bisphosphates.
• Methods demonstrated high linearity and sensitivity suitable for cellular concentrations.
• First report of hexose-phosphate profiles in CDG fibroblasts and their changes upon galactose treatment.

Conclusions:

• The developed LC-MS methods are effective for differentiating and quantifying human hexose-phosphates in tracer metabolomics.
• These methods provide valuable insights into the molecular basis of CDG and other metabolic disorders.
• The study demonstrates the utility of these methods in analyzing disease-specific metabolic alterations and treatment effects.