Isomer-selective analysis of inositol phosphates with differential isotope labelling by phosphate methylation using...

Inositol phosphates belong to a family of structurally diverse signaling molecules playing crucial role in Ca²⁺ release from intracellular storage vesicles. There are many possibilities of phosphorylation, including their degree and position. Inositol (1,4,5) trisphosphate has been well recognized as the most important second messenger among this family. It remains a challenge to analyse the entire inositol phosphate metabolite family due to its structural complexity, high polarity, and high phosphate density. In this study, we have established an improved UHPLC-ESI-MS/MS method based on a differential isotope labelling methylation strategy. An SPE extraction kit composed of TiO2 and PTFE filter was employed for sample preparation which provided good extraction performance. Samples were methylated (light label) to neutralize the phosphate groups and give better performance in liquid chromatography. Regioisomers and inositol phosphates differing in their number of phosphate residues were successfully separated after optimization on a core-shell cholesterylether-bonded RP-type column (Cosmocore 2.6Cholester) using methanol as organic modifier. Triple quadrupole MS detection was based on selected reaction monitoring (SRM) acquisition with characteristic fragments. Stable isotope labeling methylation was performed to generate internal standards (heavy label). Limits of quantification from 0.32 to 0.89 pmol on column was achieved. This method was validated to be suitable for inositol phosphate profiling in biological samples. After application in cultured HeLa cells, NIST SRM1950 plasma, and human platelets, distinct inositol profiles were obtained. This newly established method exhibited improved analytical performance, holding the potential to advance the understanding of inositol phosphate signaling.