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

Qiu, Danye; Wilson, Miranda S. C.; Eisenbeis, Verena B.; Harmel, Robert K.; Riemer, Esther; Haas, Thomas; Wittwer, Christopher; Jork, Nikolaus; Gu, Chunfang; Shears, Stephen B.; Schaaf, Gabriel; Kammerer, Bernd; Fiedler, Dorothea; Saiardi, Adolfo; Jessen, Henning J.

doi:10.1038/s41467-020-19928-x

Cited by 85 publications

(109 citation statements)

References 48 publications

(83 reference statements)

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“…While an ESI-QToF system produced substantial neutral loss (ca. 10%), [50] this was not the case in an ESI-QqQ system, a common MS analyser for quantitative studies, where we found less than 0.3% of neutral phosphate loss. Figure 3 shows electropherograms of several analysed samples spiked with heavy isotopologues.…”

Section: Determination Of Insps In Biological Samples By Ce-mscontrasting

confidence: 75%

Stable isotope phosphate labelling of diverse metabolites is enabled by a family of 18O-phosphoramidites

Haas

Mundinger

Qiu

et al. 2021

Preprint

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Stable isotope labelling is state-of-the-art in quantitative mass spectrometry, yet often accessing the required standards is cumbersome and very expensive. As 18O can be derived from heavy water (H218O), it is comparably cheap and particularly suited for labelling of phosphorylated compounds, provided the introduction is straight-forward and phosphate neutral loss in the ion source can be avoided. Here, a unifying synthetic concept for 18O-labelled phosphates is presented, based on a family of modified 18O2‑phosphoramidite reagents. This flexible toolbox offers access to major classes of biologically highly relevant phosphorylated metabolites as their isotopologues including - but not limited to - 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, such as mammalian cell lysates, slime mold and plant samples. We demonstrate that phosphate neutral loss is negligible in an analytical setup employing capillary electrophoresis electrospray ionization triple quadrupole mass spectrometry.

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Section: Determination Of Insps In Biological Samples By Ce-mscontrasting

confidence: 75%

Stable isotope phosphate labelling of diverse metabolites is enabled by a family of 18O-phosphoramidites

Haas

Mundinger

Qiu

et al. 2021

Preprint

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“…Since 32 P labeling does not provide a mass assay of the inositol species, and PAGE is not suited to the analysis of lower inositol phosphates (InsPs), alternative approaches are still required to obtain a complete picture of P i -dependent metabolism of InsPs and PP-InsPs in plants. The recent development of a capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS) method for ultrasensitive analysis of inositol (pyro)phosphates ( Qiu et al., 2020 ) offers a unique opportunity to perform isomer identification and quantitation in different plant tissues.…”

Section: Introductionmentioning

confidence: 99%

ITPK1 is an InsP6/ADP phosphotransferase that controls phosphate signaling in Arabidopsis

et al. 2021

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In plants, phosphate (P i ) homeostasis is regulated by the interaction of PHR transcription factors with stand-alone SPX proteins, which act as sensors for inositol pyrophosphates. In this study, we combined different methods to obtain a comprehensive picture of how inositol (pyro)phosphate metabolism is regulated by P i and dependent on the inositol phosphate kinase ITPK1. We found that inositol pyrophosphates are more responsive to P i than lower inositol phosphates, a response conserved across kingdoms. Using the capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS) we could separate different InsP 7 isomers in Arabidopsis and rice, and identify 4/6-InsP 7 and a PP-InsP 4 isomer hitherto not reported in plants. We found that the inositol pyrophosphates 1/3-InsP 7 , 5-InsP 7 , and InsP 8 increase several fold in shoots after P i resupply and that tissue-specific accumulation of inositol pyrophosphates relies on ITPK1 activities and MRP5-dependent InsP 6 compartmentalization. Notably, ITPK1 is critical for P i -dependent 5-InsP 7 and InsP 8 synthesis in planta and its activity regulates P i starvation responses in a PHRdependent manner. Furthermore, we demonstrated that ITPK1-mediated conversion of InsP 6 to 5-InsP 7 requires high ATP concentrations and that Arabidopsis ITPK1 has an ADP phosphotransferase activity to dephosphorylate specifically 5-InsP 7 under low ATP. Collectively, our study provides new insights into P i -dependent changes in nutritional and energetic states with the synthesis of regulatory inositol pyrophosphates.

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“…Thanks to its isomerization to inositol-3P carried out by the inositol phosphate synthase (IPS) called ISYNA1 in human or Ino1 in yeast ( Desfougeres et al, 2019 ; Donahue and Henry, 1981 ). However, recent findings demonstrating the ability of isyna1 −/− null cell to synthesize inositol suggest the existence of ISYNA1 independent pathway of inositol synthesis ( Qiu et al, 2020 ).…”

Section: Resultsmentioning

confidence: 99%

“…The metabolic complexity of IPs network and the absence of a chromophore facilitating their detection are the major challenges associated with studying the cellular function of these molecules. Therefore, a major focus in the field is to establish novel methods allowing us to map the subcellular concentration as well as the molecular identity of different IPs ( Qiu et al, 2020 ; Harmel et al, 2019 ; Ito et al, 2018 ; Losito et al, 2009 ; Wilson et al, 2015 ).…”

Section: Introductionmentioning

confidence: 99%

Inositol phosphate kinases in the eukaryote landscape

Laha

Portela-Torres

Desfougères

et al. 2021

Advances in Biological Regulation

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Inositol phosphate encompasses a large multifaceted family of signalling molecules that originate from the combinatorial attachment of phosphate groups to the inositol ring. To date, four distinct inositol kinases have been identified, namely, IPK, ITPK, IPPK (IP5–2K), and PPIP5K. Although, ITPKs have recently been identified in archaea, eukaryotes have taken advantage of these enzymes to create a sophisticated signalling network based on inositol phosphates. However, it remains largely elusive what fundamental biochemical principles control the signalling cascade. Here, we present an evolutionary approach to understand the development of the ‘inositol phosphate code’ in eukaryotes. Distribution analyses of these four inositol kinase groups throughout the eukaryotic landscape reveal the loss of either ITPK, or of PPIP5K proteins in several species. Surprisingly, the loss of IPPK, an enzyme thought to catalyse the rate limiting step of IP 6 (phytic acid) synthesis, was also recorded. Furthermore, this study highlights a noteworthy difference between animal (metazoan) and plant (archaeplastida) lineages. While metazoan appears to have a substantial amplification of IPK enzymes, archaeplastida genomes show a considerable increase in ITPK members. Differential evolution of IPK and ITPK between plant and animal lineage is likely reflective of converging functional adaptation of these two types of inositol kinases. Since, the IPK family comprises three sub-types IPMK, IP6K, and IP3–3K each with dedicated enzymatic specificity in metazoan, we propose that the amplified ITPK group in plant could be classified in sub-types with distinct enzymology.

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

Cited by 85 publications

References 48 publications

Stable isotope phosphate labelling of diverse metabolites is enabled by a family of 18O-phosphoramidites

Stable isotope phosphate labelling of diverse metabolites is enabled by a family of 18O-phosphoramidites

ITPK1 is an InsP6/ADP phosphotransferase that controls phosphate signaling in Arabidopsis

Inositol phosphate kinases in the eukaryote landscape

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