The protocol demonstrated here can help to elucidate the biosynthesis of inositol polyphosphates of plants and to answer questions regarding their role in certain aspects of plant metabolism and development. This method is highly sensitive because the use of the radioactively labeled precursor of inositol phosphates allows for reliable detection of all inositol phosphate species in plants. Visual demonstration of this method is important because most plant biologists are unfamiliar with HPLC analysis and the needed setup.
In addition, critical steps of this protocol, like the extraction of inositol phosphates, are difficult to follow without proper demonstration. Begin by setting up a system consisting of two independent HPLC pumps, one for buffer A and one for buffer B.Both pumps need to be controlled together via a computer or a master pump. Implement a piston seal wash for both pumps, either via gravitational force or through a third low pressure pump.
Connect both pumps to a dynamic mixer, then connect the mixer to an injection valve with a sample loop that has at least one milliliter capacity. Use capillaries to connect the injection valve to the column and the column to the fraction collector. Sow out lotus seeds in one row on square Petri dishes filled with solid growth media consisting of 0.8%bacteriological agar in deionized water.
Allow the seeds to stratify for at least three days at four degrees Celsius in the dark. Place the seeds in a growth incubator. Transfer 10 to 20 seedlings into one well of a 12-well clear flat bottom cell culture plate filled with two milliliters of half-strength MS salt solution that is supplemented with 1%sucrose and adjusted to pH 5.7.
Add 45 microcurie of 3H myo-inositol to the plate and swirl it gently. Cover the plate with a lid and seal it with microporous surgical tape. Then place it back into the growth incubator.
After five days of labeling, remove seedlings from the media and wash them briefly with deionized water. Dry them with paper towels and transfer them into a 1.5 milliliter microcentrifuge tube, making sure not to overfill the tube. Snap freeze the tube in liquid nitrogen and store it at minus 80 degrees Celsius until extraction.
Take the samples from the freezer and keep them in liquid nitrogen until ready to use. Grind them with a microcentrifuge tube pestle until they start thawing, then add 500 microliters of ice cold extraction buffer. Continue grinding until the sample is homogenized and the solution is colored.
Centrifuge the samples for 10 minutes at 18, 000 times G and four degrees Celsius. Then transfer the supernatant into a fresh 1.5 milliliter tube. The tubes used for extraction are considered solid radioactive waste and need to be disposed of accordingly.
Carefully add 300 microliters of neutralization buffer to the extract, which will immediately cause precipitation of proteins and bubbling. After a minute, mix the sample with a pipette tip and pipette five microliters onto pH paper to make sure that the pH is between seven and eight. If necessary, add small amounts of either neutralization buffer or extraction buffer until the desired pH is reached and let the samples rest on ice for at least one hour with an open lid.
Then centrifuge them for 10 minutes and transfer the supernatant into a fresh 1.5 milliliter tube. Equip the fraction collector with 96 small scintillation vials and fill each vial with two milliliters of a suitable scintillation cocktail that is compatible with low pH buffers and high ammonium phosphate concentrations. Start the HPLC system, then activate the piston seal wash and keep it activated during the whole run.
Manually inject the sample with a suitable syringe. Start the pumps and the gradient. While the HPLC run is ongoing, check the pressure regularly.
The starting pressure should be around 18 to 24 bar and should slowly rise to 50 to 60 bar once 100%buffer B is reached. After the run, close the vials tightly and vigorously shake the fractions with the scintillation cocktail to mix. If not proceeding directly with the measurement, keep the vials in an upright position in the dark.
To measure the fractions, insert the vials into the scintillation counter racks using racks that fit the small vials and measure each vial for five minutes in a liquid scintillation counter. Prepare a 2D line chart where the measured counts per minute or CPM are plotted against the retention time. To compare samples with each other, normalize the data by summing up the CPM from each eluted fraction from minute 25 to 96 for each individual sample and by dividing the total CPM from that sample by the total CPM of the other samples.
To perform relative quantifications of certain inositol polyphosphate peaks and to subsequently create bar graphs that contain data of replications for statistical analyses, continue the analysis with a specialized software that can calculate peak areas of chromatograms. A complete inositol polyphosphate spectrum obtained from A.thaliana extracts after scintillation counting is shown here. The peaks are nicely separated and can be assigned to different isomers.
When an aged column is used, a clear reduction of inositol hexakisphosphate and the absence of inositol pyrophosphate is visible. SAX-HPLC analysis was also performed on L.japonica seedlings that were grown and labeled under the same conditions as the Arabidopsis seedlings. While presumably all inositol polyphosphate species and peaks that are known from Arabidopsis can be seen, there are differences in the relative amounts of specific inositol polyphosphate isomers between the two species.
To demonstrate that samples do not have to be analyzed immediately after extraction, one sample was split in two and half of it was analyzed on the next day after storage at minus 80 degrees Celsius. The differences between SAX-HPLC profiles of the fresh and frozen samples were not significant, demonstrating that one freeze-thaw cycle does not harm the sample and that the method itself generates reproducible results. When attempting this protocol, always grind the samples thoroughly, frozen and with extraction buffer, and aim for close to neutral pH after neutralization to ensure a maximum recovery of radio-labeled inositol phosphate for SAX-HPLC analysis.
It is possible to purify radio-labeled from a SAX-HPLC run for later use, for example in mutual reactions by collecting fractions without scintillation cocktails and measuring only small aliquots.
