Targeted analysis of sugar phosphates from glycolysis pathway by phosphate methylation with liquid chromatography coupled to tandem mass spectrometry

Monitoring the glycolysis pathway remains an analytical challenge as most metabolites involved are sugar phosphates. Structural similarity, instability, high polarity, and rich negative charges of sugar phosphates make LC-MS based analysis challenging. Here, we developed an improved workflow integrating uniformly ¹³C-labeled yeast metabolite extract, TiO₂-based enrichment, differential stable isotope labeling phosphate methylation, porous graphic carbon column, and selected reaction monitoring acquisition. Uniformly ¹³C labeled yeast metabolite extract was used as internal standards while differential stable isotope labeled sugar phosphates worked as calibrants. The established method was validated in human plasma, platelet and cultured HeLa cells. The limits of quantification ranged between 0.25 and 0.54 pmol on column. The method was adapted and its applicability tested for human platelets in which activation with collagen-related peptide (CRP) clearly showed the upregulation of some SPx metabolites. The results document that this newly established method can be successfully used to monitor glycolysis in different biological samples. As an extension, more phosphorylated and carboxylated metabolites from the central carbon metabolism (pentose phosphate cycle, TCA cycle) were tested as well. This method showed superior performance, especially for multiple phosphorylated and carboxylated metabolites. For quantitative purpose, the concept of SPx in three sets (¹²C-analytes, U-¹³C-IS, deuterated calibrants) has the potential to be adapted for more anionic metabolites.