# Establishing 18O‑Labeled Inositol Phosphates for Quantitative Capillary Electrophoresis-Mass Spectrometry: Fragmentation Pathways and Comparison with 13C‑Labeled Analogs

## TL;DR

This paper introduces a new method using 18O-labeled inositol phosphates for accurate and affordable quantitative analysis using CE-MS.

## Contribution

The study introduces 18O labeling as a cost-effective alternative to 13C labeling for quantifying inositol phosphates and pyrophosphates.

## Key findings

- 18O-labeled inositol phosphates and pyrophosphates were synthesized and evaluated for CE-MS quantitation.
- Two major MS2 fragmentation pathways were identified, with the loss of HPO3 minimizing isotope redistribution.
- The method achieved accurate quantitation in yeast, human cells, and plant extracts comparable to 13C-based methods.

## Abstract

Capillary electrophoresis mass spectrometry (CE-MS) allows for
the rapid and accurate quantitative analysis of inositol phosphates
(InsPs) and inositol pyrophosphates (PP-InsPs). The recent discovery
of new InsPs and PP-InsPs isomers in plants and mammals necessitates
new heavy isotope references for quantitative analysis of complex
cellular extracts. Here, we evaluate 18O-labeled InsPs
and PP-InsPs as alternatives to 13C labeled internal standards
for quantitation by CE-MS. In contrast to 13C labels, the 18O labels are introduced at the end of a synthetic campaign
and not at the beginning, rendering 18O much more accessible
and affordable as a label. A series of 18O-labeled InsPs
and PP-InsPs with different numbers and positions of 18O atoms were synthesized, enabling systematic investigation of MS2
fragmentation pathways. We propose two major dissociation pathways
to elucidate the 18O redistribution of the dominant product
ion (the loss of H3PO4). Based on these insights,
we identified the loss of HPO3 as a suitable transition
for minimizing isotope redistribution in MS2 analysis. The ratios
of this alternative product ion and dominant product ion were reproducible
across replicates, concentration, and measurement days, supporting
the use of this alternative product ion as a reliable product ion
for quantitative analysis. Application to Saccharomyces
cerevisiae, HCT116 cells, and Arabidopsis
thaliana extracts confirmed accurate quantitation
and precision comparable to 13C-based methods.