Absolute Quantitation of Inositol Pyrophosphates by Capillary Electrophoresis Electrospray Ionization Mass Spectrometry

1. Setting up the CE-ESI-MS system

1. Set up a CE-ESI-MS system consisting of a commercial CE system – and a triple quadrupole tandem mass spectrometer, equipped with an Agilent Jet Stream (AJS) electrospray ionization (ESI) source. A CE-ESI-MS sprayer kit and an isocratic LC (Liquid Chromatography) pump are requisite.
2. Connect the sheath flow 1:100 splitter (included in the CE-MS sprayer kit) and the isocratic LC pump outlet.
3. Ensure that the CE system inlet vial is at the same height as the sprayer tip of the mass analyzer.
4. Utilize the MassHunter Workstation (Version 10.1) or comparable MS software to control the entire system, and for data acquisition and analysis.

2. Preparing buffer, capillary, and CE-MS system

1. Prepare CE running buffer: Adjust the pH of 40 mM ammonium acetate to 9.0 with ammonium hydroxide. A 250 mL volumetric flask is recommended. Filter the 250 mL of buffer with 0.2 µm pore-sized membrane filters. Ensure to use only ultra-pure deionized water and MS-grade reagents.
   NOTE: This buffer can be kept at room temperature for 2-3 weeks or for several months in a fridge.
2. Prepare sheath liquid: Mix 100 mL of ultra-pure water and 100 mL of LC-MS grade isopropanol in a 250 mL bottle. Change the sheath liquid at least once a week. Add mass reference into the sheath liquid when employing a high-resolution mass spectrometer.
3. Install sheath liquid: Purge at 5 mL/min for 5 min and set the flow rate at 1 mL/min (10 µL/min into the CE-MS sprayer). The pump pressure will be at ca. 180 bar. Ensure that the recycle tubing connects back into the sheath liquid bottle to reuse the solvent.
4. Prepare capillary: Purchase a CE-MS capillary (50 µm i.d. and 365 µM o.d. with a length of 125 cm) with a UV detection window. The user can also obtain much cheaper bar-fused silica capillaries from specialized distributors. Cut a capillary with a length of 100 cm. Properly cut both capillary ends with a capillary column cutter with a rotating diamond blade and remove 2-3 cm of polyimide coating on both ends with a lighter. Clean the capillary surface with isopropanol.
5. Install capillary: Match the capillary into the CE-MS cassette. Click on the Change Cassette button and install the cassette into the CE device. The inlet end of the capillary is around 2 mm lower than the electrode. Ensure that the inlet end is lower than the sample's surface during the injection process.
6. Activate capillary: Prior to first use, flush the capillary with 1 M NaOH, followed by water for 10 min, and CE running buffer for 15 min.
7. Insert the capillary end into the CE-MS sprayer: Gently put the capillary into the CE-MS sprayer and ensure that the capillary end protrudes approximately 0.1 mm out of the sprayer tip. Ensure to make precise adjustment of the capillary outlet end with a magnifying glass and the adjustment screw in the sprayer. Insert the sprayer back into the ion source and avoid touching the adjustment screw. The MS is on Standby mode when performing this operation.
8. Check ESI spray: Check the stability of the ESI sprayer under full scan mode. The fluctuation of total ion electropherograms must be within 5%.
9. Perform a test run with InsP standards: Employ a mixture of 2 µM InsP₃-InsP₈ standards (adjusted by quantitative ³¹P NMR^15,27) for test runs with an injection at 50 mbar for 10 s (10 nL). Set the detailed ESI and MS parameters as shown in Table 1. CE current is ca. 26 µA. The peak width is around 0.4-0.5 min. Ensure that the signal-to-noise ratio reaches at least 400.

3. Extraction of soluble inositol phosphates from mammalian cells

NOTE: HCT116^NIH cells were a kind gift from Stephen Shears²⁸. HCT116^UCL cells were from Saiardi's Lab²⁶.

1. Seeding cells
   1. Culture HCT116^NIH or HCT116^UCL cells in T75 flasks at 37 °C in a 5% high humidity CO₂ atmosphere (further referred to as standard conditions) in Dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS).
   2. Wash the HCT116^NIH and HCT116^UCL stock cultures with phosphate-buffered saline (PBS) (5 mL) and incubate the cells with trypsin-ethylenediamine tetraacetic acid (EDTA) (3 mL, 0.25%) under standard conditions until they are completely detached. Quench trypsin activity by adding medium (7 mL), collect the cells into a centrifuge tube, and centrifuge (200 x g, 3 min).
   3. Remove the supernatant and resuspend the cells in medium (10 mL). Count the cells and determine the viability via trypan blue exclusion.
   4. Seed the cells (6 million HCT116 cells per assay) into a 150 mm dish and adjust 20 mL of cell culture medium in total. Premix the medium and the cells in a centrifuge tube prior to seeding to achieve an equal distribution of the cells in the dish. Prepare a parallel dish when normalization by cell number is required.
   5. Culture the cells under standard conditions for 72 h. The cells will reach about 80%-90% confluence.
2. Modulation of inositol phosphate levels with NaF and cell harvesting
   1. NaF treatment: Add NaF (10 mM) 1 h before harvesting into the medium. Mix the medium by swirling the plate/pipetting and incubate the cells for 60 min under standard conditions.
   2. After the NaF treatment, remove the medium from the cells and place the cells on ice.
   3. Wash the cells twice with PBS (5 mL, 4 °C) and remove the PBS completely from the dish.
   4. Add perchloric acid (PA) (1 mL, 1 M, 4 °C). Ensure to cover the whole surface with PA (the cells will turn white as proteins precipitate). Incubate the cells for 10 min on a tilt table at 4 °C.
   5. Collect PA into a centrifuge tube and remove the contaminating debris by centrifugation (17,000 x g, 5 min, 4 °C). Add the supernatant to the prepared TiO₂ beads for the pulldown of InsPs.
   6. Wash the post-extraction dish twice with PBS (5 mL, r.t.) for deacidification; remove PBS completely from the dish.
   7. Solubilize the proteins on the plate via the addition of cell lysis buffer (1.5 mL, r.t.; 0.1% sodium dodecyl sulfate [SDS] in 0.1 M NaOH). Incubate the dish for 15 min on a tilt table at r.t. Transfer the cell lysate into a centrifuge tube and centrifuge (17,000 x g, 5 min, 4 °C). Store the supernatant at -80 °C until the protein concentration is determined via the DC protein assay using bovine serum albumin as calibration standard (usually, one 150 mm dish contains around 10 mg of proteins).
   8. Determination of cell numbers from parallel dishes: Harvest the cells of the parallel dish via trypsin as described in step 3.1.2 (use 5 mL trypsin-EDTA for the 150 mm dish) and remove the medium. Resuspend the cell pellet in PBS (5 mL), mix properly, and count the cells. Perform this step right before harvest via direct quenching to obtain representative cell counts. Additionally, measure the volume of the cells with an appropriate method (e.g., with a multisizer machine).
3. TiO₂ enrichment of inositol phosphates
   NOTE: To avoid acidic decomposition of phosphorylated compounds, perform all the enrichment steps until elution on ice, and cool all reagents to 4 °C. Keep the time for the extraction to a minimum (1.5-2 h). Perform all the extraction steps with 1 M PA.
   1. Preparation of beads: Wash TiO₂ beads (5 mg per sample) with ddH₂O (1 mL) and centrifuge (3,500 x g, 1 min, 4 °C). Remove ddH₂O and wash the beads with PA (1 mL). Remove PA by centrifugation (3,500 x g, 1 min, 4 °C). Resuspend the beads in PA (50 µL per sample).
   2. Add the supernatant containing phosphorylated compounds (compare section 3.2, step 3.2.5) to the bead suspension, vortex, and then rotate the sample for 20 min at 4 °C.
   3. Centrifuge the sample (3,500 x g, 1 min, 4 °C) and discard the supernatant. Wash the beads with PA (500 µL) and centrifuge (3,500 x g, 1 min, 4 °C). Discard the supernatant and repeat the washing step.
   4. Add NH₄OH (200 µL, 3%) to the beads and resuspend. Rotate the sample for 5 min at r.t.
   5. Centrifuge the sample (3,500 x g, 1 min) and transfer the supernatant into a new centrifuge tube.
   6. Repeat elution steps 3.3.4 and 3.3.5 and combine the eluents. Discard the beads.
   7. Centrifuge the combined eluents (17,000 x g, 1 min, 4 °C) to remove any insoluble residues.
   8. Completely dry the supernatant under vacuum evaporation (70 min, 60 °C, V-AQ). Add ddH₂O (50 µL) to the dried extracts containing InsPs. Vortex to mix the sample until completely dissolved. Store the sample at -20 °C until CE-ESI-MS analysis.

4. Performing the CE-ESI-MS runs

1. Prepare a mixture of internal standards containing 40 µM [¹³C₆]1,5-(PP)₂-InsP₄, 80 µM [¹³C₆]5-PP-InsP₅, 80 µM [¹³C₆]1-PP-InsP₅, 400 µM [¹³C₆]InsP₆ and 400 µM [¹³C₆]Ins(1,3,4,5,6)P₅. Determine the concentrations of SIL IS solutions by quantitative ³¹P and ¹H NMR, with the aid of a certified reference standard, i.e., phosphoacetic acid.
   NOTE: All above SIL internal standards (IS) with purities higher than 96% were synthesized and provided by Fiedler group^15,27. Same as with nucleotides, these IS could carry many crystal water molecules and diverse counter ions. Instead of weighing the substance and calculating the concentration, concentration determination of ¹³C₆ standard solutions by quantitative ³¹P NMR is recommended.
2. Mix 10 µL of the sample with 0.5 µL of internal standards mixture in a CE sample vial. 2 µM [¹³C₆]1,5-(PP)₂-InsP₄, 4 µM [¹³C₆]5-PP-InsP₅, 4 µM [¹³C₆]1-PP-InsP₅, 20 µM [¹³C₆]InsP₆, and 20 µM [¹³C₆]Ins(1,3,4,5,6)P₅ are the final concentrations inside the samples.
3. When using the replenishment system, click on the Change Bottle button, put the prepared 250 mL of CE running buffer into the electrolyte bottle, and click on Clean Tubes. Keep the replenishment needle in a water vial.
4. Set ESI and MS parameters as shown in Table 1. Optimize the source parameters using a Source Optimizer with a mixture of inositol polyphosphate standards. Obtain the multiple reaction monitoring (MRM) settings using Masshunter Optimizer with all standards. Adjust the ESI and MS/MS settings for different instrumentation.
5. Perform a run for the InsP extracts and check the result (Figure 2). Set a sequence when there are more samples.
6. Let the MS be on Standby mode after measurements. Do not turn off the LC pump. The flow of sheath liquid protects the sprayer needle. Replace the CE-ESI-MS Sprayer with an LC-ESI-MS Sprayer when there is no sheath liquid supply.

5. Data analysis

1. Open Quantitative Analysis (for QQQ) software, create a batch for all samples.
2. Create a new method from the acquired MRM data. Set the [¹³C₆]InsPs as internal standards – (ISTD). Check MRM compound setup, retention time setup, ISTD setup, concentration setup, and qualifier setup. Pass the validation and exit to apply the method to the current batch. Save the method.
3. Check whether each peak in the batch is properly integrated; otherwise, manually integrate the peak.
4. Export the results into a spreadsheet. Perform the quantitation of inositol (pyro)phosphates by comparing the analyte peak response with the respective peak response of SIL IS with known concentrations. Theoretical and experimental calibration curves are shown in Table 2 for 5-PP-InsP₅, InsP_6, and Ins(1,3,4,5,6)P₅ with the linear range.
   NOTE: The precondition applying theoretical calibration curve is the usage of a high-quality isotopic pattern of targeted analytes and with credible concentration. Evaluate the linear range. A calibration curve is essential for absolute quantitation when the full acquisition of the above-mentioned isotopic standards is impractical.
5. With the measured concentration in the InsP extract solution and its volume, calculate the absolute amounts. Further, normalize the amount by cell counts or protein content. Calculate the cellular concentration based on cell counts and average cell volume of HCT116 (1.68 fL).