Quantifying the Labeling and the Levels of Plant Cell Wall Precursors Using Ion Chromatography Tandem Mass Spectrometry

The biosynthesis of cell wall polymers involves enormous fluxes through central metabolism that are not fully delineated and whose regulation is poorly understood. We have established and validated a liquid chromatography tandem mass spectrometry method using multiple reaction monitoring mode to separate and quantify the levels of plant cell wall precursors. Target analytes were identified by their parent/daughter ions and retention times. The method allows the quantification of precursors at low picomole quantities with linear responses up to the nanomole quantity range. When applying the technique to Arabidopsis (Arabidopsis thaliana) T87 cell cultures, 16 hexose-phosphates (hexose-Ps) and nucleotide-sugars (NDP-sugars) involved in cell wall biosynthesis were separately quantified. Using hexose-P and NDP-sugar standards, we have shown that hot water extraction allows good recovery of the target metabolites (over 86%). This method is applicable to quantifying the levels of hexose-Ps and NDP-sugars in different plant tissues, such as Arabidopsis T87 cells in culture and fenugreek (Trigonella foenum-graecum) endosperm tissue, showing higher levels of galacto-mannan precursors in fenugreek endosperm. In Arabidopsis cells incubated with [U-¹³C_Fru]sucrose, the method was used to track the labeling pattern in cell wall precursors. As the fragmentation of hexose-Ps and NDP-sugars results in high yields of [PO₃]⁻/or [H₂PO₄]⁻ ions, mass isotopomers can be quantified directly from the intensity of selected tandem mass spectrometry transitions. The ability to directly measure ¹³C labeling in cell wall precursors makes possible metabolic flux analysis of cell wall biosynthesis based on dynamic labeling experiments.Plant cell walls are the most abundant renewable resources (Pauly and Keegstra, 2008a). Much of the current biotechnological research on plant cell wall synthesis involves manipulating these biosynthetic processes to obtain higher concentrations of starches or oil, which show much promise in biofuel production, or to alter cell wall composition for easier breakdown. A detailed knowledge of these processes is essential to understanding and utilizing plant cell wall materials as well as for progress in understanding plant growth and structural development (Pauly and Keegstra, 2008b). However, research into cell wall biosynthesis has been hindered by our limited understanding of the metabolic processes that produce cell walls and particularly their regulation. Progress in this area is limited by the difficulty of differentiating among the compounds involved and of analyzing the fluxes through the biochemical network of wall biosynthesis. Many of the metabolic steps involve isomeric sugars, including hexose-Ps and nucleotide-sugars (NDP-sugars) that serve as direct precursors to plant cell wall biosynthesis. Separate quantification of these sugars has been difficult to achieve.Much of the current research on identifying and differentiating among different metabolic pathways involves the use of chromatography and mass spectrometry (MS; Wolfender et al., 2009). It has been found that liquid chromatography (LC), when linked to a triple quadrupole mass spectrometer (tandem MS [MS/MS]), can be a powerful tool to detect and specifically quantify several classes of metabolic compounds (Allwood and Goodacre, 2010). After initial compound separation by LC, analytes are directed to a triple quadrupole mass spectrometer (MS/MS), where the initial two quadrupoles separate the compounds for detection in the third quadrupole, first by selection of particular mass-to-charge (m/z) ratios of the ionized parent compounds in the first quadrupole, then by fragmentation of the compounds in the second quadrupole (Arrivault et al., 2009). This coupling method of LC-MS/MS to identify compounds has been used with several metabolites involved in plant primary metabolism recently (Cruz et al., 2008; Arrivault et al., 2009).Several studies have reported the separation of phosphorylated metabolites using LC-MS/MS. We are particularly interested in analyzing such compounds because plant cell wall biosynthesis involves a range of phosphorylated precursors, mainly NDP-sugars and hexose-Ps (Feingold and Barber, 1990; Fry 2000, 2004; Seifert, 2004; Somerville et al., 2004; Sharples and Fry, 2007). For example, Huck et al. (2003) and Luo et al. (2007) were able to separate, respectively, six and 28 intracellular metabolites involved in glycolysis, the oxidative pentose-P pathway, and the tricarboxylic acid cycle, some of which are phosphorylated. Bajad et al. (2006) were able to separate a large number of water-soluble cellular metabolites by hydrophilic interaction chromatography, but this method does not appear to separate isomers. Anion exchange chromatography was shown to be effective at separating phosphorylated intermediates involved in glycolysis (van Dam et al., 2002) and in the Calvin cycle (Cruz et al., 2008; Arrivault et al., 2009), especially when coupled with the high specificity and sensitivity of triple quadrupole MS. Moreover, anion-exchange chromatography coupled with MS/MS can be used to determine the mass isotopomer distribution of labeled compounds and Kiefer et al. (2007) were able to quantify isotope abundances in six phosphorylated metabolites in Escherichia coli.However, some of the methods that achieved good separation of four NDP-sugars did not allow quantification by MS/MS because of the eluents used (Räbinä et al., 2001), and none of the methods using coupled LC-MS/MS developed to date separates all or nearly all of the hexose-Ps and NDP-sugars known to be involved in plant cell wall biosynthesis (Turnock and Ferguson, 2007). This presents a special challenge given the fact that many of these sugar compounds are diastereoisomers and ionize similarly in traditional LC-MS/MS methods. Current methods of separating hexose-Ps and NDP-sugars also involve multiple steps of chromatographic and enzymatic separation. In a notable recent study, Sharples and Fry (2007) separated many of the compounds involved in plant cell wall biosynthesis, including hexose-Ps and NDP-sugars, and used radioactive [U-¹⁴C]Fru and [1-³H]Gal as substrates to determine their relative contributions to different cell wall components. The method used in that study involved high-voltage paper electrophoresis separation followed by mild acid hydrolysis and/or phosphatase digestion of different fractions to release neutral hexoses that were then separated by a second paper chromatography procedure. At the cost of considerable effort, this approach allowed eight compounds to be separated. However, neither this nor many of the other approaches used to date appear to have yielded absolute metabolite levels or specific activities in labeling. Metabolic flux analysis requires quantifying these compounds, their fractional and preferably also positional labeling, and the ability to analyze many time point samples. These requirements necessitated the development of a method that can be performed in medium to high throughput and achieves compound separation and quantitation, such as LC-MS/MS, and that also yields detailed labeling information.In this study we have developed and validated a robust and sensitive LC-MS/MS method that successfully allows us to separate and quantify the levels and isotopic labeling of plant cell wall precursors. Using plant tissues from fenugreek (Trigonella foenum-graecum) endosperms and Arabidopsis (Arabidopsis thaliana) cell cultures, 12 hexose-Ps and NDP-sugars known to be involved in plant cell wall biosynthesis were separated and quantified. The direct analysis of intracellular cell wall precursors and their isotopic labeling significantly expands the set of tools for assessing the dynamics and regulation of cell wall biosynthesis, including the potential for dynamic metabolic flux analysis.