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.

• PMID: 41191388
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Journal Article

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Liu G, Dürr-Mayer T, Lu M, Jessen HJ

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