Hydrophilic interaction liquid chromatography–tandem mass spectrometry for the quantitative analysis of mammalian-derived inositol poly/pyrophosphates

Abstract

Although myo-inositol pyrophosphates such as diphosphoinositol pentakisphosphate (InsP) are important in biology, little quantitative information is available regarding their presence in mammalian organisms owing to the technical difficulties associated with accurately detecting these materials in biological samples. We have developed an analytical method whereby InsP and its precursor inositol hexakisphosphate (InsP) are determined directly and sensitively using tandem mass spectrometry coupled with hydrophilic interaction liquid chromatography (HILIC). InsP and InsP peak symmetry is influenced greatly by the buffer salt composition and pH of the mobile phase used in HILIC analysis. The use of 300 mM ammonium carbonate (pH 10.5) as an aqueous mobile phase resolves InsP and InsP on a polymer-based amino HILIC column with minimal peak tailing. Method validation shows that InsP and InsP can be quantitated from 20-500 pmol with minimal intra-day/inter-day variance in peak area and retention time. The concentration of InsP in C57BL/6J mouse brain (40.68 ± 3.84 pmol/mg wet weight) is successfully determined. HILIC‒MS/MS analysis using HEK293 culture cells confirms previous observations that InsP is induced by NaF treatment and ectopic expression of InsPK2, a primary kinase for InsP synthesis. Furthermore, this analysis reveals the abundance of InsP (50.46 ± 18.57 pmol/10 cells) and scarcity of InsP in human blood cells. The results demonstrate that HILIC‒MS/MS analysis can quantitate endogenous InsP and InsP in mouse and human samples, and we expect that the method will contribute to further understanding of InsP functions in mammalian pathobiology.

Inositol Pyrophosphates: Signaling Molecules with Pleiotropic Actions in Mammals

“…Establishing a tight link between PP-IPs and clinical findings will be important to translate scientific knowledge into clinical diagnostic and therapeutic applications. Mass spectrometric measurement of PP-IPs was recently attempted [95,96], applying a highly sensitive platform to the detection of PP-IPs. We strongly believe that advanced detection techniques and a comprehensive understanding of the roles of PP-IPs in cellular signaling networks will lead to the development of valuable tools to manage leading human diseases, like metabolic syndromes and cancer.…”

Analysis of inositol phosphate metabolism by capillary electrophoresis electrospray ionization mass spectrometry

“…However, standard SAX-HPLC using water-salt-based gradients is incompatible with MS detection and MS-compatible volatile buffers do not currently enable isomer assignment 15 . HPLC-MS/MS and hydrophilic interaction liquid chromatography coupled to MS (HILIC-MS/ MS) unfortunately result in a suboptimal separation of the analytes, obliterating InsP isomer identity 16 .…”

Analyses of Lipid A Diversity in Gram-Negative Intestinal Bacteria Using Liquid Chromatography–Quadrupole Time-of-Flight Mass Spectrometry

“…The difficulty in the analysis of intact lipid A by LC-MS is due to the diverse physical properties arising from different numbers of acyl chains and phosphate groups [21]. In particular, phosphate groups tend to form chelates with metals in LC columns and stainless-steel piping, hindering the acquisition of sharp chromatographic peaks [22,23]. To overcome this problem, ion-pair reagents have been often used.…”