Ultrasensitive Determination of Sugar Phosphates in Trace Samples by Stable Isotope Chemical Labeling Combined with RPLC-MS

Sugar phosphates are important metabolic intermediates in organisms and play a vital role in energy and central carbon metabolism. Profiling of sugar phosphates is of great significance but full of challenges due to their high structural similarity and low sensitivities in liquid chromatography (LC)-mass spectrometry (MS). In this study, we developed a novel stable isotope chemical labeling combined with the reversed-phase (RP)LC-MS method for ultrasensitive determination of sugar phosphates at the single-cell level. By chemical derivatization with 2-(diazo-methyl)-N-methyl-N-phenyl-benzamide (2-DMBA) and d(5)-2-DMBA, sugar phosphate isomers can obtain better separation and identification, and the detection sensitivities of sugar phosphates increased by 3.5-147 folds. The obtained limits of detection of sugar phosphates ranged from 5 to 16 pg/mL. Using this method, we achieved ultrasensitive and accurate quantification of 12 sugar phosphates in different trace biological samples. Benefiting from the improved separation and detection sensitivity, we successfully quantified five sugar phosphates (D-glucose 1-phosphate, D-mannose 6-phosphate, D-fructose 6-phosphate, D-glucose 6-phosphate, and seduheptulose 7-phosphate) in a single protoplast of Arabidopsis thaliana.