Rapid and sensitive quantitation of glucose and glucose phosphates derived from storage carbohydrates using gas chromatography mass spectrometry

Abstract

Glycogen is the primary storage carbohydrate in mammals and it is synthesized in most tissues. Glycogen contains covalently attached phosphate groups at C2-, C3-, and C6-hydroxyls of the glucose units. The addition of phosphate modulates branching pattern, granular size, and crystallinity of a glycogen molecule, which all impact its accessibility to glycogen interacting enzymes. As glycogen architecture modulates its role in metabolism, it is essential to accurately evaluate and quantify each parameter. Simultaneous quantitation of glucose and its phosphate esters is challenging and requires an assay with high sensitivity and a robust dynamic range. Currently, this method is lacking in the field. Herein, we describe a highly sensitive method for the detection of both glycogen-derived glucose and glucose-phosphate esters utilizing gas-chromatography coupled mass spectrometry. Using this method, we observed higher glycogen levels in the liver compared to skeletal muscle, but skeletal muscle contained much more phosphate esters. These results confirm previous findings and establish the validity of the method. Importantly, this method can detect femtomole levels of glucose and glucose phosphate esters within an extremely robust dynamic range with excellent accuracy and reproducibility. The method can also be easily adapted for the quantification of glucose from plant starch, amylopectin or other biopolymers as well as covalently attached phosphate within them.

Improved workflow for mass spectrometry–based metabolomics analysis of the heart

“…We have employed this cryomilling method previously and shown robust reproducibility during metabolite extraction from whole tissue (Fig. 2A) (37,38).…”

Loss of Rb1 Enhances Glycolytic Metabolism in Kras-Driven Lung Tumors In Vivo

“…Relative abundance was corrected for recovery using L-norvaline and adjusted to protein input. The rapid quantitation of derivatized protein/DNA/RNA/glycogen pellet was performed as described in [45]. An unsupervised hierarchical clustering analysis of steady-state metabolite levels was performed using the online Morpheus software tool from the Broad Institute.…”