Use of reverse‐phase liquid chromatography, linked to tandem mass spectrometry, to profile the Calvin cycle and other metabolic intermediates in Arabidopsis rosettes at different carbon dioxide concentrations

Abstract

SUMMARYA platform using reverse-phase liquid chromatography coupled to tandem mass spectrometry was developed to measure 28 metabolites from photosynthetic metabolism. It was validated by comparison with authentic standards, with a requirement for distinct and clearly separated peaks, high sensitivity and repeatability in Arabidopsis rosette extracts. The recovery of authentic standards added to the plant material before extraction was 80-120%, demonstrating the reliability of the extraction and analytic procedures. Some metabolites could not be reliably measured, and were extracted and determined by other methods. Measurements of 37 metabolites in Arabidopsis rosettes after 15 min of illumination at different CO 2 concentrations showed that most Calvin cycle intermediates remain unaltered, or decrease only slightly (<30%), at compensation point CO 2 , whereas dedicated metabolites in end-product synthesis pathways decrease strongly. The inhibition of end-product synthesis allows high levels of metabolites to be retained in the Calvin cycle to support a rapid cycle with photorespiration.

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

“…Indeed, hot water was shown to extract efficiently phosphorylated metabolites such as nucleotides (Lundin and Thore, 1975) and intermediaries of glycolysis (Hiller et al, 2007). The recoveries that we obtained after hot water extraction were similar to those obtained by extraction with aqueous formic acid saturated with n-butanol on tobacco (Nicotiana tabacum) suspension cell cultures and cultured pollen tubes (Schlü pmann et al, 1994) and ice-cold methanol/chloroform (3:7, v/v) performed on Arabidopsis rosettes (Arrivault et al, 2009), and twice as high as the methanol/chloroform/formic acid/water (12:5:1:2, v/v/v/v) carried out on tobacco leaves (Cruz et al, 2008).…”

“…To quantify the levels and the mass isotopomer distribution of hexose-Ps and NDP-sugars, we used a triple-quad Quattro Premier (Waters) operating in the negative ion mode. The combination of a specific retention time with a specific parent molecular ion, and a unique fragment (or daughter) ion, can be a sensitive method to unambiguously monitor and quantify metabolites of interest (van Dam et al, 2002;Huck et al, 2003;Grange et al, 2005;Bajad et al, 2006;Luo et al, 2007;Cruz et al, 2008;Arrivault et al, 2009). Since all cell wall precursors are phosphorylated, we optimized the collision parameters (Table I) for the formation of phosphate (m/z 79, 97) and diphosphate (m/z 159) ions.…”

“…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.…”

“…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). …”

Leaf Starch Turnover Occurs in Long Days and in Falling Light at the End of the Day

“…For total starch content, Glc was assayed enzymatically. For 13 C starch content, Glc was converted to Glc6P by incubation with hexokinase and excess ATP, and 13 C in Glc6P was measured by LC-MS/MS (Arrivault et al, 2009; Supplemental Text S1).…”