Two projects: Improved prediction of metabolite retetion times and analysis of ammonium transporters from Marchantia polymorpha

Abstract

Gradient retention times are difficult to project from the underlying retention factor (k) vs. solvent composition (φ) relationships. A major reason for this difficulty is that gradients produced by HPLC pumps are imperfect – gradient delay, gradient dispersion, and solvent mis-proportioning are all difficult to account for in calculations. However, we recently showed that a gradient “back-calculation” methodology, Retention Projection with Back-calculation (RPwB), can measure these imperfections and take them into account. In RPLC (Reverse Phase Liquid Chromatography), when the back-calculation methodology was used, error in projected gradient retention times is as low as could be expected based on repeatability in the k vs. φ relationships. To extend the application of RPwB, we test the prediction accuracy in HILIC (Hydrophilic Interaction Liquid Chromatography) too. Compared with RPLC, HILIC presents a new challenge: the selectivity of HILIC columns drift strongly over time. Retention is repeatable in short time, but selectivity frequently drifts over the course of weeks. In this study, we set out to understand if the issue of selectivity drift can be avoided by doing our experiments quickly, and if there are any other factors that make it difficult to predict gradient retention times from isocratic k vs. φ relationships when gradient imperfections are taken into account with the back-calculation methodology. While in past reports, the accuracy of retention projections was >5%, the back-calculation methodology brought our error down to ~1%. This result was 6-43 times more accurate than projections made using ideal gradients and 3-5 times more accurate than the same retention projections made using offset gradients (i.e., gradients that only took gradient delay into account). Still, the error remained higher in our HILIC projections than in RPLC. Based on the shape of the back-calculated gradients, we suspect the higher error is a result of prominent gradient distortion caused by strong, preferential water uptake from the mobile phase into the stationary phase during the gradient – a factor our model did not properly take into account. It appears that, at least with the stationary phase we used, column distortion is an important factor to take into account in retention projection in HILIC that is not usually important in RPLC. However, this methodology has only been approved its robustness in preferred sample (solvents 73% acetonitrile and 27% water) in RPLC. Biological samples are often complicated and sample solvents vary. In this study, we set out to explore the sensitivity of RPwB under a limited set of extreme conditions. First, we selected nine sample solvents representing a wide range of polarities (methanol, ethanol, isopropanol, acetonitrile, acetone, dichloromethane, ethyl acetate, tetrahydrofuran, and toluene), and studied their effects on peak shapes, retention times, and prediction accuracy of RPwB. We found isopropanol, acetonitrile, and ethyl acetate were the top three that distorted peak shapes the most, and all these nine sample solvents shifted retention times and poorly retained compounds suffered more than well retained ones. Only ethyl acetate broke the prediction accuracy of RPwB and had a prediction error of 4.3 sec, which was more than the 3 sec that was the maximum deviation allowed for the successful application of RPwB in RPLC. Second, five plant samples (Solanum lycopersicum fruit, Solanum lycopersicum stems and leafs, Nicotiana flowers, Nicotiana leafs, and Nicotiana forsteri leafs) were randomly chosen and extracted using the four sample solvents (water, 70% ethanol, dichloromethane, and isopropanol) that are the most common ones and that did not change the prediction accuracy of RPwB, and obtained the most concentrated plant extracts possible. The effect of plant matrices on retention times of nearly all analytes was negligible, despite their high concentrations. However, we observed a buildup of some plant matrix solutes in the column that lowered retention projection accuracy for two charged analytes, tetrabutylammonium and tetrapentylammonium. However, this buildup could be removed (and the accuracy of retention projections restored) by either flushing the column with a stronger mobile phase or decreasing the concentration of the injected sample. The second part of the dissertation is in a different field- protein structure and function. Nitrogen is a limiting nutrient for plants. To understand the mechanisms that plants use to acquire nitrogen from the environment, it is useful to study diverse plants. Basal plants contain a small gene family of ammonium transporters within the AMT/MEP/Rh superfamily and transporters in the same family are used to take up ammonium in angiosperms. Here we characterized the transport activity of two ammonium transporters from Marchantia polymorpha, a liverwort and a representative of the most basal land plants. Ammonium transporter MpAMT1;2 was shown to localize to the plasma membrane in Marchantia gametophyte thallus by stable transformation using a C-terminal citrine fusion. MpAMT1;2 expression was studied using qRT-PCR and shown to be higher when plants were N deficient and lower when plants were grown on media containing ammonium, nitrate, or the amino acid glutamine. Expression in Xenopus oocytes and analysis by electrophysiology revealed that MpAMT1;2 is an electrogenic ammonium transporter with a very high affinity for ammonium (7 µM at pH 5.6 and a membrane potential of -137 mV). A conserved inhibitory phosphorylation site identified in angiosperm AMT1s is also present in all AMT1s in Marchantia. Here we show that a phosphomimetic mutation T475D in MpAMT1;2 completely inhibits ammonium transport activity. The results indicate that MpAMT1;2 may be important for ammonium uptake into cells in the Marchantia thallus. A second ammonium transporter, MpAMT1;5 was functionally expressed in both yeast and Xenopus oocytes. It showed a low-affinity for ammonium (K0.5 of 0.38 mM at pH 5.6 and a membrane potential of -121 mV) when expressed in Xenopus oocytes and assayed using two-electrode voltage clamping. MpAMT1;5 was localized to the plasma membrane in gametophyte thalli of transgenic Marchantia polymorpha expressing a MpAMT1;5-citrine fusion. MpAMT1;5 expression was studied using M. polymorpha transformed with a MpAMT1;5 promoter-GUS fusion.