Capillary electrophoresis mass spectrometry identifies new isomers of inositol pyrophosphates in mammalian tissues

Abstract

Technical challenges have to date prevented a complete profiling of the levels of myo-inositol phosphates (InsPs) and pyrophosphates (PP-InsPs) in mammalian tissues. Here, we have deployed capillary electrophoresis mass spectrometry to identify and record the levels of InsPs and PP-InsPs in several tissues obtained from wild type mice and a newly created PPIP5K2 knockout strain. We observe that the mouse colon harbours unusually high levels of InsPs and PP-InsPs. Additionally, the PP-InsP profile is considerably more complex than previously reported for animal cells: using chemically synthesized internal stable isotope references and high-resolution mass spectra, we characterize two new PP-InsP isomers as 4/6-PP-InsP₅ and 2-PP-InsP₅. The latter has not previously been described in nature. The analysis of feces and the commercial mouse diet suggests that the latter is one potential source of noncanonical isomers in the colon. However, we also identify both molecules in the heart, indicating unknown synthesis pathways in mammals. We also demonstrate that the CE-MS method is sensitive enough to measure PP-InsPs from patient samples such as colon biopsies and peripheral blood mononuclear cells (PBMCs). Strikingly, PBMCs also contain 4/6-PP-InsP₅ and 2-PP-InsP₅. In summary, our study substantially expands PP-InsP biology in mammals.

Fig. 1. Main metabolic reactions that determine the turnover of inositol pyrophosphates in mammalian cells. Three isoforms of IP6Ks phosphorylate the 5-position of InsP₆ and two isoforms of PPIP5Ks phosphorylate the 1-position with a preference for 5-PP-InsP₅ over InsP₆. The question mark indicates that an unknown pathway is responsible for the synthesis of the 4/6-PP-InsP₅ and 2-PP-InsP₅ identified in the current study (inside dotted box, note that 4/6-PP-InsP₅ are enantiomers). PPIP5Ks also harbour a phosphatase domain catalyzing dephosphorylation 1,5-(PP)₂-InsP₄. DIPPs are specialized phosphatases that degrade the phosphoric anhydrides in PP-InsPs.

Fig. 2. Profiling of PP-InsPs in mouse tissues (wild type vs. PPIP5K2 knockout) and observation of new isomers. (A) Profiling of InsP₆, 2-OH InsP₅, and the total for all other InsP₅ isomers (4/6-OH InsP₅, 1/3-OH InsP₅, and 5-OH InsP₅). (B) PP-InsP₅-a and PP-InsP₅-b refer to two base-line resolved peaks. The tentative identification of the components of each peak (in bold font) is described in the text. Data for (A) and (B) are indicated as means ± SEM, n = 7 and for the colon, n = 6. *P < 0.05 and **P < 0.01, Student's t-test. (C) Extracted ion electropherograms (EIEs) of [¹³C] labeled internal reference compounds (black lines) plus a 2-PP-InsP₅ standard (red trace) or PP-InsP₅ extracts from the mouse colon, heart and liver, resolved with BGE A containing 35 mM ammonium acetate titrated with ammonium hydroxide to pH 9.7. D EIEs of [¹³C]-labeled internal reference compounds (black lines) plus (left panel) a 6-PP-InsP₅ standard (red trace) or PP-InsP₅ extracts from the mouse colon, heart and liver (red trace), resolved with BGE B containing 40 mM ammonium acetate titrated with ammonium hydroxide to pH 9.0.

Fig. 3. (A) Synthesis and application of ¹⁸O labeled P-amidites with diverse protecting group patterns and their application to a late-stage labeling 4-PP-InsP₅ synthesis. AB: acetoxybenzyl and PMB: para-methoxybenzyl. (B) Separation of 5-PP-InsP₅ and 4/6-PP-InsP₅ (filled red plots) from mouse colon and heart samples using BGE-B and assignment of the isomer with the new internal reference compound [¹⁸O₂] 4-PP-InsP₅ (blue plot) as either 4-PP-InsP₅ or 6-PP-InsP₅. EIEs (PP-InsP₅ and [¹⁸O₂] PP-InsP₅) are scaled to the largest peak indicated as 100%.

Fig. 4. (A) CE-MS analysis of PP-InsP₅ in mouse feces (filled red plots) with internal references of [¹³C₆]5-PP-InsP₅ and [¹³C₆]1-PP-InsP₅ (black plot) and [¹⁸O₂]4-PP-InsP₅ (blue plot) using BGE B. 4/6-PP-InsP₅ isomer is identified in mouse feces as well. (B) Analysis of PP-InsP₅ in mouse food the same as in (A), which contains high levels of this 4/6-PP-InsP₅ isomer. (C) Profiling of PP-InsPs and InsPs in mouse feces (wild type vs. PPIP5K2 knockout). Data are indicated as means ± SEM, n = 5. *P < 0.05, and Student's t-test.

Fig. 5. (A) CE-MS analysis of a human colon tissue biopsy (18 mg) enables the identification of several important inositol phosphate (InsP₆ and InsP₅) and pyrophosphate (5-PP-InsP₅) isomers. (B) 4/6-PP-InsP₅ is identified in PBMCs by CE-MS analysis. The electropherograms are representative of independent biological triplicates giving comparable results. (C) 4/6-PP-InsP₅ is enriched in a CD8⁺ T-cell preparation and is also present in the CD8⁺ depleted PBMC pool (D). It is assigned by its exact same migration time as [¹⁸O₂] 4-PP-InsP₅. PP-InsP₅ (filled red plot) isomer identification is achieved with the aid of [¹³C₆]5-PP-InsP₅, [¹³C₆]1-PP-InsP₅ (black plot) and [¹⁸O₂] 4-PP-InsP₅ (blue plot).