Materials and Reagents

HPLC Grade Methanol (Fisher Chemical, catalog number: A452-4 )
Water, CHROMASOLV^TM, for HPLC (Honeywell Riedel-de Haen^TM, Fisher Chemical, catalog number: 60-048-247 )
FlowLogic U Scintillation Cocktail (LabLogic, catalog number: SG-BXX-05 )

Equipment

HPLC Equipment: Agilent 1260 Infinity II High Performance Liquid Chromatography System
Flow Radio Detector: LabLogic β-Ram 6
Thermo Scientific Accucore C18 Column; 3 x 150 mm, 2.5 μm spherical solid core particles. (Thermo Fisher, catalog number: 17126-153030 )
Accucore C18 Guard Column; 3 x 10 mm, 2.6 μm spherical solid core particles (Thermo Fisher, catalog number: 17126-013005 )

Software

LabLogic Laura version 6.0.1.40
GraphPad Prism version 8.1.2 (332)

Procedure

Sample preparation
1. Isolate MGDG from the leaf lipid extract via normal phase HPLC method using a time based fraction collection method, as disclosed in Zhou et al. (2020), or by thin layer chromatography (Karki et al., 2019).
2. Dry collected MGDG fractions under a gentle N₂ stream.
3. Dissolve the isolated MGDG into a small volume (e.g., 25 µl) HPLC grade methanol for analysis via Radio-HPLC. The volume of injection solvent for the sample analysis illustrated in Figure 1 is 10 μl and the total radioactivity per injection is approximately 17,500 Disintegrations Per Minute (DPM). For the analysis of MGDG molecular species, it is ideal to have at least 5,000 DPM of total MGDG radioactivity per injection of a minimal volume of solvent into the HPLC.
4. Transfer the MGDG in methanol to an autosampler vial with a small volume insert for HPLC analysis. Spring bottom inserts are ideal for small volumes (10-100 µl) where nearly the entire sample may be injected.

Reverse phase Radio HPLC-¹⁴C MGDG molecular species analysis
1. Power on the HPLC modules; pump, autosampler, multi column compartment, Ultra Violet Diode-Array Detector (UV-DAD), and flow radio detector.
2. The HPLC parameters and the online liquid scintillation counter are both controlled by the LabLogic Laura software.
3. Set up the sequence in Laura with a blank methanol as the first sample in the sequence. Allow the column to equilibrate for 20 min prior to the start of analysis. Since the method uses isocratic conditions, column equilibration is not necessary between each sample.
4. Once the sequence is saved, the method for the analysis will be loaded. The method conditions for the HPLC analysis are as described below:
  1. Quaternary Pump: Solvent A: Methanol and Solvent B: Water. The method uses Isocratic elution with mobile phase–95%A/5%B. The flow is set at 0.35 ml/min and the run time is 35 min.
  2. Autosampler: Autosampler temperature is set at 20 °C. Sample Injection solvent: Methanol. Set the injector settings in the method to as follows: Needle Draw Speed–200 μl/min, Needle eject Speed–400 μl/min, Wait Time after draw–1.2 s.
  3. Multi Column Compartment: The temperature of the column compartment is set at 35 °C. The mobile phase is allowed to pass through a Quick-Connect Heat Exchanger prior to entering the column. A short 10 mm guard column containing same material as that of the stationary phase is installed immediately prior to the analytical column.
  4. UV-DAD: Direct the flow of eluate from the column to UV-DAD. UV absorption at 210 nm will provide retention times of the molecular species containing unsaturated acyl chains with significant mass abundance in the sample as illustrated in Figure 1A and the original research paper (Zhou et al., 2020), Supplementary Figure S4A.
  5. β-Ram 6 Radio Detector: Direct the eluate from UV-DAD to the radio detector. Set the type of analysis to Non-Stop Radio HPLC. The radio detector is equipped with a 500 μl adjustable volume flow cell that facilitates the radio counting process. Set the flow cell volume to 300 μl. Prior to entering the flow cell, the eluate from the UV-DAD is mixed with the scintillation cocktail whose flow is controlled by a scintillant pump. Set the scintillant flow to 1.05 ml/min (a 1:3 for eluate:scintillation cocktail ratio). The resulting residence time in the flow cell for these flow settings is 12.9 s. The recommended residence time for flow counting is above 5 s. The eluate mixed with scintillant from the radio detector is directed to the properly labeled radioactive HPLC waste container secured in a secondary waste container. In the method, adjust the detector settings of Radio 1 channel as follows: Measurement units–CPM, Dwell Time–1 s, Shift Time–0 s, Lower Limit–200, Upper Limit–9000.
5. After the HPLC data acquisition is completed, proceed to data analysis.
  
  Figure 1. HPLC Analysis-MGDG molecular species from wild type tobacco leaves continuously labeled with [¹⁴C]acetate for 2 h. A. UV chromatogram at 210 nm. B. radioactivity in counts per minute (CPM). Identities of individual molecular species from B are as follows: 1-18:3/16:3, 2-18:3/16:2>18:2/16:3, 3-18:3/18:3, 4-18:2/16:2>18:3/16:1, 5-18:3/18:2, 6-18:2/16:1, 7-18:3/16:0>18:2:18:2, 8-18:3/18:1, 9-18:2/18:1, 10-18:2/16:0, 11-18:1/16:0. Figure reproduced from Zhou, X. R., Bhandari, S., Johnson, B. S., Kotapati, H. K., Allen, D. K., Vanhercke, T. and Bates, P. D. (2020). Reorganization of acyl flux through the lipid metabolic network in oil-accumulating tobacco leaves.Plant Physiol 182(2): 739-755. www.plantphysiol.org. “Copyright American Society of Plant Biologists”.

Data analysis

Analyze radio HPLC data via LabLogic Laura, and a sample chromatogram after processing is illustrated in Figure 1B and original research paper (Zhou et al., 2020) Supplementary Figure S4B.
Load the first MGDG sample in the sequence and select all the significant peaks in the order of elution.
Each peak will be designated as a region and numbers are assigned in the same elution order.
Multiple samples in the sequence via batch analysis where all the regions in a chromatogram will be selected based on the first chromatogram. It is ideal to go through each chromatogram and make sure all significant peaks are selected. This also helps to identify any peak drifts due to errors in the analysis.
The data table for the processed chromatogram provides the following information: retention time for each peak, area under the peak in CPM, % total and % region of interest (%ROI).
Export the data table to Microsoft Excel by simple copy paste function.
%Total for each identified peak in a chromatogram is used to determine the relative labeling of each molecular species in the MGDG sample. See notes for additional information on the MGDG molecular species analysis.
Identify the individual molecular species based on the retention times of the peaks as shown in Table 1. MGDG species are identified based on the carbon chain length and unsaturation number in the acyl chains. The data of relative %¹⁴C label between the molecular species plotted in Prism and is illustrated in original research paper (Zhou et al., 2020) Supplementary Figures S5 and S6.

Table 1. Retention times of identified ¹⁴C MGDG molecular species in WT tobacco

Notes

For the molecular species identification, it is always ideal to let the analysis go for longer than our reported method run time. This is to make sure there are no other species besides the reported ones.
If the retention times are off, then the identity of each peak needs to be determined. One procedure is to collect each ¹⁴C peak, convert to fatty acid methyl esters, and identify the ¹⁴C fatty acids via argentation TLC and phosphorimaging analysis as reported in Bates et al. (2009), this procedure was used for original identification of each labeled MGDG molecular species in Zhou et al. (2020).
Further method optimization by adjusting method parameters such as using different stationary phase (e.g., a RP column with longer carbon chain) or same column with different dimensions (e.g., increased length) might help with the separation of co-eluting molecular species of MGDG.

Acknowledgments

The research is supported by U.S. Department of Agriculture National Institute of Food and Agriculture (grant nos. 2017-67013- 26156, 2017-67013-29481, and Hatch umbrella project no. 1015621), and the National Science Foundation (grant no. 1930559).

Competing interests

The authors report no competing interests.

References

Bates, P. D., Durrett, T. P., Ohlrogge, J. B. and Pollard, M. (2009). Analysis of acyl fluxes through multiple pathways of triacylglycerol synthesis in developing soybean embryos. Plant Physiology 150(1): 55-72.
Karki, N., Johnson, B. S. and Bates, P. D. (2019). Metabolically distinct pools of phosphatidylcholine are involved in trafficking of fatty acids out of and into the chloroplast for membrane production. Plant Cell 31(11): 2768-2788.
Yamauchi, R., Kojima, M., Isogai, M., Kato, K. and Ueno, Y. (1982). Separation and purification of molecular species of galactolipids by high performance liquid chromatography. Agricultural and Biological Chemistry 46(11): 2847-2849.
Zhou, X. R., Bhandari, S., Johnson, B. S., Kotapati, H. K., Allen, D. K., Vanhercke, T. and Bates, P. D. (2020). Reorganization of acyl flux through the lipid metabolic network in oil-accumulating tobacco leaves. Plant Physiol 182(2): 739-755.