Stable isotope phosphate labelling of diverse metabolites is enabled by a family of 18O-phosphoramidites (original) (raw)
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Pure and Applied Chemistry, 2017
Procedures are proposed for the naming of individual atoms, P, O, F, N, and S in phosphate esters, amidates, thiophosphates, polyphosphates, their mimics, and analogues of transition states for enzyme-catalyzed phosphoryl transfer reactions. Their purpose is to enable scientists in very different fields, e.g. biochemistry, biophysics, chemistry, computational chemistry, crystallography, and molecular biology, to share standard protocols for the labelling of individual atoms in complex molecules. This will facilitate clear and unambiguous descriptions of structural results, as well as scientific intercommunication concerning them. At the present time, perusal of the Protein Data Bank (PDB) and other sources shows that there is a limited degree of commonality in nomenclature, but a large measure of irregularity in more complex structures. The recommendations described here adhere to established practice as closely as possible, in particular to IUPAC and IUBMB recommendations and to “b...
Analytical and Bioanalytical Chemistry, 2012
A new method was here developed for determination of 18 O labeling ratios in metabolic oligophosphates, such as ATP, at different phosphoryl moieties (α-, β-, and γ-ATP) using sensitive and rapid electrospray ionization mass spectrometry (ESI-MS). The ESI-MS based method for monitoring of 18 O/ 16 O exchange was validated with GC-MS and 2D 31 P NMR correlation spectroscopy, the current standard methods in labeling studies. Significant correlation was found between isotopomer selective 2D 31 P NMR spectroscopy and isotopomer less selective ESI-MS method. Results demonstrate that ESI-MS provides a robust analytical platform for simultaneous determination of levels, 18 O-labeling kinetics and turnover rates of α-, β-, and γphosphoryls in ATP molecule. Such method is advantageous for large scale dynamic phosphometabolomic profiling of metabolic networks and acquiring information on the status of probed cellular energetic system.
Phosphorus NMR and Its Application to Metabolomics
Analytical Chemistry
Stable isotopes are routinely employed by NMR metabolomics to highlight specific metabolic processes and to monitor pathway flux. 13 C-carbon and 15 N-nitrogen labeled nutrients are convenient sources of isotope tracers and are commonly added as supplements to a variety of biological systems ranging from cell cultures to animal models. Unlike 13 C and 15 N, 31 Pphosphorus is a naturally abundant and NMR active isotope that does not require an external supplemental source. To date, 31 P NMR has seen limited usage in metabolomics because of a lack of reference spectra, difficulties in sample preparation, and an absence of two-dimensional (2D) NMR experiments, but 31 P NMR has the potential of expanding the coverage of the metabolome by detecting phosphorus-containing metabolites. Phosphorylated metabolites regulate key cellular processes, serve as a surrogate for intracellular pH conditions, and provide a measure of a cell's metabolic energy and redox state, among other processes. Thus, incorporating 31 P NMR into a metabolomics investigation will enable the detection of these key cellular processes. To facilitate the application of 31 P NMR in metabolomics, we present a unified protocol that allows for the simultaneous and efficient detection of
Journal of proteome research, 2018
Existing hydrophilic interaction liquid chromatography (HILIC) methods, considered individually, each exhibit poor chromatographic performance for a substantial fraction of polar metabolites. In addition to limiting metabolome coverage, such deficiencies also complicate automated data processing. Here we show that some of these analytical challenges can be addressed for the ZIC-pHILIC, a zwitterionic stationary phase commonly used in metabolomics, with the addition of trace levels of phosphate. Specifically, micromolar phosphate extended metabolome coverage by hundreds of credentialed features, improved peak shapes, and reduced peak-detection errors during informatic processing. Although the addition of high levels of phosphate (millimolar) as a HILIC mobile phase buffer has been explored previously, such concentrations interfere with mass spectrometric (MS) detection. We show that using phosphate as a trace additive at micromolar concentrations improves analysis by electrospray MS,...
Journal of Chromatography B, 2018
The phosphometabolome is comprised of all phosphorylated metabolites including the major metabolite classes sugar phosphates and nucleoside phosphates. Phosphometabolites are invaluable in any cell as a part of primary-and energy-metabolism, and as building blocks in the biosynthesis of macromolecules. Here, we report quantitative profiling of the phosphometabolome by applying capillary ion chromatography-tandem mass spectrometry (capIC-MS/MS), ensuring improved chromatographic separation, robustness and quantitative precision. Baseline separation was achieved for six out of eight tested hexose phosphates. Quantitative precision and reproducibility was improved by introducing a fully uniformly (U) 13 C-labeled biological extract and applying an isotope dilution (ID) correction strategy. A 13 C-labeled biological extract does in principle contain internal standards (IS) for all metabolites, but low abundant metabolites pose a challenge, and solutions to this are discussed. The extreme reproducibility and reliability of this capIC-MS/MS method was demonstrated by running the instrumentation continuously for ten days.
Journal of Biomolecular Nmr, 2000
Intramolecular correlations among the 18O-labels of metabolic oligophosphates, mapped by J-decoupled 31P NMR 2D chemical shift correlation spectroscopy, impart stringent constraints to the 18O-isotope distributions over the whole oligophosphate moiety. The multiple deduced correlations of isotopic labels enable determination of site-specific fractional isotope enrichments and unravel the isotopologue statistics. This approach ensures accurate determination of 18O-labeling rates of phosphometabolites, critical
Analytical chemistry, 2015
Phosphoinositides, the phosphorylated derivatives of phosphatidylinositol (PtdIns), are key regulators of many fundamental biological processes, including cell growth, proliferation, and motility. Here, we present a novel method for rapid, sensitive, and simultaneous profiling of phosphatidylinositol trisphosphate (PtdInsP3), phosphatidylinositol bisphosphate (PtdInsP2), and phosphatidylinositol phosphate (PtdInsP) of different fatty acid compositions. This method is based on a technique called "charged diacylglycerol fragment ion-specific multiple precursor ion scanning" (DAG(+)-specific MPIS), coupled with prior phosphate methylation. Using DAG(+)-specific MPIS, we were able to identify 32 PtdIns, 28 PtdInsP, 30 PtdInsP2, and 3 PtdInsP3 molecular species from bovine brain extracts or prostatic cancer cell lines in an efficient and time-saving manner. Our analysis revealed a large range of fatty acyl compositions in phosphoinositides not obtained previously from mammalian...
Journal of the American Chemical Society, 1983
Tetrasodium pyrophosphate with I8O enrichment in the bridge position has been prepared from HzLBO in an overall yield of 32%. 31P NMR analysis at 97.3 MHz has given the percentages of unlabeled, lS0-nonbridge, and lS0-bridge species as 13%, 11%, and 76%, respectively. Resonances corresponding to I80-nonbridge and 180-bridge pyrophosphate are shifted upfield by 0.01 1 and 0.019 ppm, respectively, from the resonance corresponding to the unlabeled species. This material has, in turn, been used to synthesize 180-labeled ATP with unlabeled, I80-nonbridge, and @,y-Is0-bridge species at the @-phosphorus position of 18%, 6%, and 76%, respectively. The @-phosphorus resonances corresponding to "0-nonbridge and Is0-bridge label appear 0.026 and 0.016 ppm upfield from those corresponding to unlabeled material. 31P NMR analysis at 97.3 MHz has shown that carbamoyl-phosphate synthetase from Escherichia coli catalyzes the bicarbonate-dependent @,y-bridge to 8-nonbridge positional oxygen exchange in &y-I80-bridge ATP. The ratio of micromoles of ATP exchanged to micromoles of ADP produced was 1.2 i 0.1 in the presence of bicarbonate and the absence of glutamine and ornithine. By the same procedures, pyrophosphate and ATP with 170 enrichments in the bridge and @,y-bridge were prepared from H2I7O (containing 33.6% I6O, 39.1% I7O, and 27.3% lag). The presence of I8O in this sample of ATP enabled the determination of positional oxygen exchange with carbamoyl-phosphate synthetase by 31P NMR at 97.3 MHz as described above. The ratio of micromoles of ATP exchanged to micromoles of ADP produced was found in this case to be 1.3 f 0.1. In addition, positional oxygen exchange could be observed by 31P NMR at 40.5 MHz as an increase in the intensity of the P, doublet due to movement of I7O from the P,y-bridge to 8-nonbridge position. The significance of this observation lies in the ability to observe positional oxygen exchange of this type on virtually any lower frequency NMR instrument capable of observing phosphorus.
Phospho-iTRAQ: assessing isobaric labels for the large-scale study of phosphopeptide stoichiometry
Journal of proteome research, 2015
The ability to distinguish between phosphopeptides of high and low stoichiometry is essential to discover the true extent of protein phosphorylation. We here extend the strategy whereby a peptide sample is briefly split in two identical parts and differentially labeled preceding the phosphatase treatment of one part. Our use of isobaric tags for relative and absolute quantitation (iTRAQ) marks the first time that isobaric tags have been applied for the large-scale analysis of phosphopeptides. Our Phospho-iTRAQ method focuses on the unmodified counterparts of phosphorylated peptides, which thus circumvents the ionization, fragmentation, and phospho-enrichment difficulties that hamper quantitation of stoichiometry in most common phosphoproteomics methods. Since iTRAQ enables multiplexing, simultaneous (phospho)proteome comparison between internal replicates and multiple samples is possible. The technique was validated on multiple instrument platforms by adding internal standards of hi...
Physiological Genomics, 2012
Next-generation screening of disease-related metabolomic phenotypes requires monitoring of both metabolite levels and turnover rates. Stable isotope18O-assisted31P nuclear magnetic resonance (NMR) and mass spectrometry uniquely allows simultaneous measurement of phosphometabolite levels and turnover rates in tissue and blood samples. The18O labeling procedure is based on the incorporation of one18O into Pifrom [18O]H2O with each act of ATP hydrolysis and the distribution of18O-labeled phosphoryls among phosphate-carrying molecules. This enables simultaneous recording of ATP synthesis and utilization, phosphotransfer fluxes through adenylate kinase, creatine kinase, and glycolytic pathways, as well as mitochondrial substrate shuttle, urea and Krebs cycle activity, glycogen turnover, and intracellular energetic communication. Application of expanded18O-labeling procedures has revealed significant differences in the dynamics of G-6-P[18O] (glycolysis), G-3-P[18O] (substrate shuttle), a...