Liquid Chromatography Tandem Mass Spectrometry Quantification of 13C-Labeling in Sugars

Abstract

Subcellular compartmentation has been challenging in plant ¹³C-metabolic flux analysis. Indeed, plant cells are highly compartmented: they contain vacuoles and plastids in addition to the regular organelles found in other eukaryotes. The distinction of reactions between compartments is possible when metabolites are synthesized in a particular compartment or by a unique pathway. Sucrose is an example of such a metabolite: it is specifically produced in the cytosol from glucose 6-phosphate (G6P) and fructose 6-phosphate (F6P). Therefore, determining the ¹³C-labeling in the fructosyl and glucosyl moieties of sucrose directly informs about the labeling of cytosolic F6P and G6P, respectively. To date, the most commonly used method to monitor sucrose labeling is by nuclear magnetic resonance, which requires substantial amounts of biological sample. This study describes a new methodology that accurately measures the labeling in free sugars using liquid chromatography tandem mass spectrometry (LC-MS/MS). For this purpose, maize embryos were pulsed with [U-¹³C]-fructose, intracellular sugars were extracted, and their time-course labeling was analyzed by LC-MS/MS. Additionally, extracts were enzymatically treated with hexokinase to remove the soluble hexoses, and then invertase to cleave sucrose into fructose and glucose. Finally, the labeling in the glucosyl and fructosyl moieties of sucrose was determined by LC-MS/MS.

Keywords: 13C-labeling; 13C-metabolic flux analysis; LC-MS/MS; fructose 6-phosphate; glucose 6-phosphate; hexokinase; invertase; subcellular compartmentation; sucrose.

Figure 1

Representative example of maize embryos to illustrate the subcellular compartmentation of hexose-phosphates in plant cells. Developing maize embryos mainly import glucose and fructose as carbon sources. These sugars get phosphorylated into hexose-phosphate in the cytosol. The resulting hexose-phosphates are involved in major pathways: glycolysis (purple), and the oxidative pentose-phosphate pathway (OPPP; green). Full and dashed arrows represent one reaction or a multiple-step reaction, respectively: 1 is hexokinase; 2, phosphoglucose isomerase; 3 and 7, cytosolic and plastidial aldolases, respectively; 4 and 8, exchange of hexose-phosphates and triose-phosphates between cytosol and plastid, respectively; 5, starch synthesis; 6, oxidative part of the OPPP; 9, glucose 6-phosphatase; 10, sucrose phosphate synthase; and 11, invertase. Abbreviations in alphabetical order: E4Pp, plastidic erythrose 4-phosphate, FRU(ext), (extracellular) fructose; F6P, fructose 6-phosphate; GLC(ext), (extracellular) glucose; G6P, glucose 6-phosphate; H6Pp, plastidic hexose-phosphates; P5Pp, plastidic pentose-phosphates; S7Pp, plastidic sedoheptulose 7-phosphate; TP(p), (plastidic) triose-phosphates.

Figure 2

LC-MS/MS chromatograms of free sugars before and after enzymatic treatments. (A) shows the chromatographic separation and mass spectrometric detection of fructose, glucose (transition 179/89), and sucrose (transition 341/59) after boiling water extraction of unlabeled maize embryos. (B) and (C) represent the LC-MS/MS chromatograms obtained after hexokinase and invertase treatments, respectively. LC-MS/MS chromatograms for hexoses and sucrose were obtained using MRM scan survey approach as indicated in the Materials and Methods.

Figure 3

Relative abundance of isotopomers for intracellular fructose and glucose during pulse labeling with [U-¹³C]fructose. (A,C) show the relative abundance of mass isotopomers at all time points for fructose and glucose, respectively. (B,D) represent the relative abundance of mass isotopomers from 0 to 80 min for fructose and glucose, respectively. Intracellular glucose and fructose were extracted, and analyzed as described in the “Materials and Methods” section. Mass relative abundances (in %) were reported as the average ± SD (n = 4 biological replicates). Mass isotopomers m₀, m₃, and m₆ are depicted as blue diamond, red square, and green triangle, respectively.

Figure 4

Relative abundance of isotopomers for ¹³C-fructosyl and ¹³C-glucosyl units from sucrose during the pulse labeling experiment. (A,C,E) represent the relative abundance of mass isotopomers at all time points for sucrose, SUC_Fructosyl, and SUC_Glucosyl, respectively. (B,D,F) depict the relative abundance of mass isotopomers from 0 to 80 min for sucrose, SUC_Fructosyl, and SUC_Glucosyl, respectively. Sucrose was extracted, cleaved into its fructosyl and glucosyl units, and analyzed as described in the Materials and Methods section. Mass relative abundances (in %) were reported as the average ± SD (n = 4 biological replicates). Mass isotopomers m₀, m₃, m₆, m₉, m₁₂ are shown as blue diamond, red square, green triangle, orange cross, and black circle, respectively. SUC_Fructosyl: Sucrose fructosyl; SUC_Glucosyl: Sucrose glucosyl.