Quantitative HPLC-MS analysis of nucleotide sugars in plant cells following off-line SPE sample preparation

Abstract

An analytical workflow was developed for the absolute quantification of uridine diphosphate (UDP)-sugars in plant material in order to compare their metabolism both in wild-type Arabidopsis thaliana and mutated plants (ugd2,3) possessing genetic alterations within the UDP-glucose dehydrogenase genes involved in UDP-sugar metabolism. UDP-sugars were extracted from fresh plant material by chloroform-methanol-water extraction and further purified by solid-phase extraction with a porous graphitic carbon adsorbent with extraction efficiencies between 80 ± 5 % and 90 ± 5 %. Quantitative determination of the UDP-sugars was accomplished through HPLC separation with a porous graphitic carbon column (HypercarbTM) which was interfaced to electrospray ionization Orbitrap mass spectrometry. The problem of instable retention times due to redox processes on the stationary phase were circumvented by grounding of the column effluent and incorporation of a column regeneration procedure using acetonitrile-water containing 0.10 % trifluoroacetic acid. The method was calibrated using external calibration and UDP as internal standard. Calibration functions were approximated by first- or second-order regression analysis for concentrations spanning three orders of magnitude. Upon injecting sample volumes of 2.65 μL, the limits of detection for the UDP-sugars were in the 70 nmol L−1 range. Six different UDP-sugars, including UDP-glucose, UDP-galactose, UDP-arabinose, UDP-xylose, UDP-glucuronic acid, and UDP-galacturonic acid were found in concentrations of 0.4 to 38 μg/g plant material. Data evaluation by analysis of variance (ANOVA) revealed statistically significant differences in UDP-sugar concentrations between wild-type and mutant plants, which were found to conclusively mirror the impaired metabolic pathways in the mutant plants.FigureᅟElectronic supplementary materialThe online version of this article (doi:10.1007/s00216-014-7746-3) contains supplementary material, which is available to authorized users.

A Bifunctional UDP-Sugar 4-Epimerase Supports Biosynthesis of Multiple Cell Surface Polysaccharides in Sinorhizobium meliloti

“…Separation of UDP-glucose and UDP-galactose was achieved according to a method described by Behmüller et al (62) using an Agilent 1100 quaternary HPLC system equipped with a HYPERCARB 150-mm by 1-mm column (Thermo Scientific) at a temperature of 60°C, a flow rate of 0.1 ml/min with a gradient of A (water), B (water-0.3% formic acid [pH adjusted to 9 using NH 3 ; aqueous]), C (acetonitrile), and D (79.95% acetonitrile-19.95% water-0.1% TFA). The gradient is shown in Table S3.…”

KONJAC1 and 2 Are Key Factors for GDP-Mannose Generation and Affect l-Ascorbic Acid and Glucomannan Biosynthesis in Arabidopsis

“…The extracts were combined, dried with a centrifugal vacuum concentrator, and resuspended in 1 mL of water. Solid-phase extraction with Supelclean ENVI-Carb SPE tubes (Sigma-Aldrich) was performed as described by Behmüller et al (2014). Samples were dried using a centrifugal vacuum concentrator, resuspended in 500 mL of water, and analyzed immediately.…”

Profiling Cell Wall Monosaccharides and Nucleotide‐Sugars from Plants

“…By contrast, we have found that the application of porous graphitic carbon a reliable and robust separation technique that is highly compatible with MS. The approach generates baseline separation of all reported plant nucleotide sugars and we have not observed the reported retention time instabilities (Behmuller, Forstenlehner, Tenhaken, & Huber, ). The stability of porous graphitic carbon we have observed over many hundreds of samples may be because we do not regenerate the column with trifluoroacetic acid, as has been reported for this technique (Pabst et al., ).…”