Abstract
Stable isotope labelling is state-of-the-art in quantitative mass spectrometry, yet often accessing the required standards is cumbersome and very expensive. Here, a unifying synthetic concept for 18O-labelled phosphates is presented, based on a family of modified 18O2-phosphoramidite reagents. This toolbox offers access to major classes of biologically highly relevant phosphorylated metabolites as their isotopologues including nucleotides, inositol phosphates, -pyrophosphates, and inorganic polyphosphates. 18O-enrichment ratios >95 % and good yields are obtained consistently in gram-scale reactions, while enabling late-stage labelling. We demonstrate the utility of the 18O-labelled inositol phosphates and pyrophosphates by assignment of these metabolites from different biological matrices. We demonstrate that phosphate neutral loss is negligible in an analytical setup employing capillary electrophoresis electrospray ionisation triple quadrupole mass spectrometry.
Highlights
Isotopologues are molecular entities that differ only in isotopic composition.[1]
We demonstrate the utility of phosphate-labelled inositol phosphates in capillary electrophoresis mass-spectrometry-based (CE-ESI-QqQ) analytics
We have recently reported the use of 13C-labelled inositol phosphates and pyrophosphates as internal references for quantitative mass spectrometry.[50]
Summary
Isotopologues are molecular entities that differ only in isotopic composition.[1]. Their chemical properties are almost identical, yet they can be readily distinguished by several analytical methods. Zhou et al used 18O4-ATP and MS/MS to identify the phosphorylation sites within substrates of a human tyrosine/ serine kinase.[23] Analogously, Sulbaran et al characterised kinase phosphorylation sites on myosin regulatory light chains.[24] Fu et al reported a kinase assay based on 18O4ATP, to determine effectiveness and specificity of kinase inhibitors.[25] Molden et al demonstrated the application of g18O4-ATP in nucleo to follow protein phosphorylation rates.[26] several proteomics-based assays with 18Ox-ATP (x = 2–4) were developed, identifying substrates of specific kinases of interest.[27,28,29,30,31] These various applications underline the tremendous potential of 18O-labelled phosphates to serve as tool compounds for addressing chemical and biological questions. Our CE-ESI-QqQ setup enables accurate assignment of analytes without neutral loss from diverse biological matrices
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