Quantification of Pentose Phosphate Pathway (PPP) Metabolites by Liquid Chromatography-Mass Spectrometry (LC-MS)

Abstract

The pentose phosphate pathway plays an important role in several cellular processes including biosynthesis and catabolism of five-carbon sugars and generation of reducing power through NADPH synthesis. Although the pentose phosphate metabolic reaction network has been mapped in substantial detail, the comprehensive quantitative analysis of the rates and regulation of individual reactions remains a major interest for various biofields. Here we describe a simple method for comprehensive quantitative analysis of pentose phosphate pathway intermediates. The method is based on Group Specific Internal Standard Technology (GSIST) labeling in which an experimental sample and corresponding internal standards are derivatized in vitro with isotope-coded reagents in separate reactions, then mixed and analyzed in a single LC-MS run. The use of co-eluting isotope-coded internal standards and experimental molecules eliminates potential issues with ion suppression and allows for precise quantification of individual metabolites. Derivatization also increases hydrophobicity of the metabolites enabling their effective separation using reversed-phase chromatography.