This provide high-quality results for the inositol phosphate metabolism in all the organisms we tested so far. It will also provide insight into new cellular biosynthetic pathway that we are presently are aware of. Analysis of inositol phosphate metabolism by CE-ESI-MS is faster, highly sensitive, and capable of providing structure information for inositol phosphate assemblies.
To begin, set up a Capillary Electrophoresis Electrospray Ionization Mass Spectrometry or CE-ESI-MS system consisting of a commercial CE system, and a triple quadrupole tandem mass spectrometer, equipped with an Agilent Jet Stream ESI source. Next, connect the sheath flow 1:100 splitter and the isocratic liquid chromatography pump outlet, and ensure that the CE system inlet vial is at the same height as the sprayer tip of the mass analyzer. Then, utilize the MassHunter Workstation version or similar MS software, to control the entire system and data acquisition and analysis.
After preparing the CE running buffer and sheath liquid, install the sheath liquid by purging at five milliliters per minute for five minutes. Then set the flow rate to one milliliter per minute and the pump pressure to 180 bar. Ensure that the recycled tubing connects back to the sheath liquid bottle to reuse the solvent.
Next, using a capillary column cutter with a rotating diamond blade, properly cut both ends of a CE-MS capillary with a UV-detection window. Then remove two to three centimeters of polyimide coating on both ends with a lighter, and clean the capillary surface with Isopropanol. To install the capillary, match the capillary into the CE-MS cassette.
Then click on the Change Cassette button and install the cassette into the CE device. To activate the capillary, first flush the capillary with one molar sodium hydroxide, followed by water for 10 minutes, and then CE running buffer for 15 minutes. Next, insert the capillary into the CE-MS sprayer.
Using a magnifying glass and the adjustment screw in the sprayer, precisely adjust the capillary outlet, ensuring that the capillary end protrudes approximately 0.1 millimeters out of the sprayer tip. Check the stability of the ESI sprayer under Full Scan mode. The fluctuation of total ion electropherograms should be within 5%Then, perform a test run with inositol phosphate standards.
After preparing the internal standards mixture, mix 10 microliters of the sample with 0.5 microliters of the internal standards mixture and a CE sample vial. When using the replenishment system, click on the Change Bottle button. Put the prepared 250 milliliters of CE running buffer into the electrolyte bottle.
Then click on Clean Tubes keeping the replenishment needle in a water vial. Set the ESI and MS parameters. Then optimize the source parameters using a source optimizer with a mixture of inositol polyphosphate standards, and multiple reaction monitoring settings using MassHunter Optimizer with all standards.
Next, perform a run for the inositol phosphate extracts and check the results. Set a sequence when there are more samples. After measurement, let the MS be on Standby mode and do not turn off the liquid chromatography pump.
Replace the CE-ESI-MS sprayer with an LC-ESI-MS sprayer when there is no sheath liquid supply. For data analysis, open Quantitative Analysis software and create a batch for all samples. Next, create a new method from the acquired multiple reaction monitoring data, and set the Carbon-13 inositol phosphates as internal standards.
Then, check the Multiple Reaction Monitoring Compound, Retention Time, internal standards, Concentration and Qualifier setup. Pass the Validation and Exit to apply the method to the current batch. Save the method.
Then check whether each peak in the batch is integrated correctly. Otherwise, manually integrate the peak. Export the results into a spreadsheet, and quantify the inositol pyrophosphates, by comparing the anolyte peak response with the respective peak response of the stable isotope labeled internal standards with known concentrations.
Finally, with the measured concentration in the inositol phosphate, extract solution and its volume, calculate the absolute amounts and further normalize the amount by cell counts or protein content. Calculate the cellular concentration based on cell counts and average cell volume of HCT116 cells. The extracted ion electropherograms of inositol pyrophosphate standards at a concentration of two micromoles are shown here.
Metabolism of inositol pyrophosphates in mammals with their simplified structures is inserted. A CE-ESI-MS run of HCT116 cells from University College London or UCL is shown here. CE-MS was used to quantify variation in IP8 levels between HCT116 cells from UCL and those from NIH.
The HCT116 cells from UCL contains sevenfold higher levels of IP8 than those from NIH. Significant accumulation of 1, 5-IP8 in the HCT116 UCL cells is paralleled by a considerable increase in 1-IP7. CE-ESI-MS analysis of sodium fluoride treated HCT116 cells from NIH demonstrated an elevation of 5-IP7, along with a reduction in IP6 and an appearance of 5-PPIP4.
Although levels of 1-IP7 decreased to some degree, it is not completely absent in sodium fluoride treated cells, but mostly under the limit of detection. It is important to form a stable CE-ESI-MS grade. We recommend checking the repeatability and sensitivity of the system before measurements.
This technique allows for providing inositol phosphate metabolism, implants and tissues, and also provide an opportunity for closely studying the biologically relevant inositol pyrophosphate.
