Accurate and sensitive quantitation of glucose and glucose phosphates derived from storage carbohydrates by mass spectrometry

Abstract

The addition of phosphate groups into glycogen modulates its branching pattern and solubility which all impact its accessibility to glycogen interacting enzymes. As glycogen architecture modulates its metabolism, it is essential to accurately evaluate and quantify its phosphate content. Simultaneous direct quantitation of glucose and its phosphate esters requires an assay with high sensitivity and a robust dynamic range. Herein, we describe a highly-sensitive method for the accurate 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 many more phosphate esters. 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 plant starch, amylopectin or other biopolymers.

Keywords: GCMS; Glucose; Glucose phosphate esters; Glycogen; Lafora disease; Laforin; Starch.

Fig. 1.. Derivatization and fragmentation pattern of glucose, G2P, G3P, G6P.

Trimethylsilylation of analytical grade standards of glucose (A), G2P (B), G3P (C), and G6P (D) (−6TMS;1MEOX). Two μmoles of each standard were derivatized with 20mg/ml MEOX in pyridine for 1-hour (reaction 1), followed by silylation by MSTFA for 1-hour (reaction 2). Both reaction steps took place in a 60° C dry heat block. Fragmentation pattern for each silylated standard was obtained on a single quadrupole mass spectrometer with an electron ionization (EI) energy of 70 eV, mass range of 30-650 AMU, and 1.47 scan/s.

Fig. 2.. Standard and rapid gas chromatography separation of glucose, G2P, G3P, G6P.

(A) Temperature gradient for standard separation of glucose, G3P, and G6P: Initial temperature was 60° C, held for 1 minute, rising at 10° C/minute to 325° C, held for 10 minutes. Total run time: 37.5 minutes. Grey bar indicates window of separation. (B) Stacked chromatography spectra for silylated glucose, G2P, G3P, and G6P using the temperature setting in (A). Twenty nmoles of each silylated standard were injected into the GC column. (C) Temperature gradient setting for rapid separation of glucose, G3P, and G6P: initial temperature was 60° C, held for 1 minute, rising at 60° C/minute to 220° C, continued rising at 30° C/minute to 270° C, and finished rising at 30° C/minute to 325° C, held for 5 minutes. Total run time: 12.2 minutes. Grey bar indicates window of separation. (D) Stacked chromatography spectra for silylated glucose, G2P, G3P, and G6P combined into one sample using the temperature setting in (C). Two nmoles of each silylated standard were injected into the GC column.

Fig. 3.. Dynamic range of GCMS analysis of glucose, G3P, and G6P.

Silylated glucose (A), G3P (B), G6P (C), and G2P (D) standards at multiple concentrations were injected into the GC using split mode with a ratio of 10:1.

Figure 4:. Extraction and GCMS analysis of liver and muscle glycogen

(A) Schematics of glycogen extraction: mouse liver or skeletal muscle were milled to 10μm particles by liquid N2 Freezer/Mill Cryogenics Grinder magnetic assisted tissue-grinding mill, followed by polar and organic solvent removal of free polar metabolites and lipids. Glycogen was then extracted by 10% TCA. Isolated glycogen was hydrolyzed to monomers by mild hydrolysis, derivatized by MEOX and MSTFA, and analyzed by GCMS (B). Quantitation of liver or muscle derived glucose (C), G6P (D), G3P (E), G2P (F) the third wash of tissue pellet is served as blank, and free polar metabolite fraction serves as negative control for G3P and G2P and a positive control for G6P. (F) Muscle contains higher G2P, G3P and G6P than liver when standardized to tissue weight. Data shown in (C-G) are from three experiments and are shown as mean ±SEM. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001; two-tailed t-test.

Fig. 5.. Comparison between plant starch and mammalian liver glycogen.

Stacked spectra overlay between WT potato starch, gwd-/- Arabidopsis starch, and mammalian liver glycogen to demonstrate versatility of the approach. All three samples were standardized to total glucose. The region between 7-7.8 minutes demonstrates that WT plant starch contains 10-fold higher phosphate than liver glycogen, while no detectable phosphate was observed in the gwd-/- Arabidopsis starch.