This application introduces a separation of inositol polyphosphates using a polymer-based amino column, HILICpak VG-50 2D, with a triple quadrupole (QQQ) mass spectrometry (LC-MS/MS).
Diphosphoinositol pentakisphosphate (IP₇) is biosynthesized from inositol hexakisphosphate (phytic acid; IP₆) through intracellular kinases. Accumulation of intracellular IP₇ is observed when human colorectal adenocarcinoma cell line HCT116 is treated with sodium fluoride (NaF), an inhibitor of pyrophosphatases.
Use of VG-50 2D with LC-MS/MS made it possible for an advanced sensitive analysis of highly phosphorylated molecules. Presence of phosphate groups in molecules may cause metal ions to adsorb on the LC connecting tubing. Addition of methylenediphosphonic acid in the mobile phase prevents this adsorption, and allows the detection of both IP₆ and IP₇ at around 10 pmol level.

Sample Preparation

1. (1)Wash cells twice with PBS, then lyse with cell lysis buffer (0.01 %Triton X-100, 1 mM EDTA, 20 mM Tris-HCl).
2. (2)Treat the cell lysate with 2-M perchloric acid solution to remove proteins, then add titanium oxide beads to the obtained supernatant to let the beads adsorb inositol polyphosphates.
3. (3)Incubate the titanium oxide beads at 4 ℃ for 30 minutes, then wash the beads twice with 2-M perchloric acid solution.
4. (4)Add 10% ammonia solution to obtain inositol polyphosphates bound to the titanium oxide beads.
5. (5)Repeat step (4), dry the total eluate from steps (4) and (5). Reconstitute the remaining solid in 100-mM ammonium carbonate aqueous solution/CH₃CN = 60/40 and use it as a sample solution.

Sample: 50 μL

1.