title: Synthesis and Use of Stable-Isotope-Labeled Internal Standards for Quantification of Phosphorylated Metabolites by LC-MS/MS.
abstract: Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is a highly specific and sensitive technique for measuring metabolites. However, coeluting components in tissue extracts can interfere with ionization at the interface of the LC and MS/MS phases, causing under- or overestimation of metabolite concentrations. Spiking of samples with known amounts of stable-isotope-labeled internal standards (SIL-IS) allows measurements of the corresponding metabolites to be corrected for such matrix effects. We describe criteria for selection of suitable SIL-IS and report the enzymatic synthesis and purification of nine SIL-IS for hexose-, pentose-, and triose-phosphates, UDP-glucose, and adenosine monophosphate (AMP). Along with commercially available SIL-IS for seven other metabolites, these were validated by LC-MS/MS analyses of extracts from leaves, nonphotosynthetic plant tissues, mouse liver, and cells of Chlamydomonas reinhardtii, Escherichia coli and baker's yeast (Saccharomyces cerevisiae). With only a few exceptions, spiking with SIL-IS significantly improved the reproducibility of LC-MS/MS-based metabolite measurements across a wide range of extract dilutions, indicating effective correction for matrix effects by this approach. With use of SIL-IS to correct for matrix effects, LC-MS/MS offers unprecedented scope for reliable determination of photosynthetic and respiratory intermediates in a diverse range of organisms.
authors.affiliation: †Max Planck Institute of Molecular Plant Physiology, Am Muehlenberg 1, 14476 Potsdam-Golm, Germany.
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Arrivault
authors.affiliation: †Max Planck Institute of Molecular Plant Physiology, Am Muehlenberg 1, 14476 Potsdam-Golm, Germany.
authors.forename: Manuela
authors.initials: M
authors.lastname: Guenther
authors.affiliation: ‡The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, School of Biological Sciences, The University of Edinburgh, Edinburgh, EH9 3BF U.K.
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Fry
authors.affiliation: †Max Planck Institute of Molecular Plant Physiology, Am Muehlenberg 1, 14476 Potsdam-Golm, Germany.
authors.forename: Maximilian M F F
authors.initials: MM
authors.lastname: Fuenfgeld
authors.affiliation: †Max Planck Institute of Molecular Plant Physiology, Am Muehlenberg 1, 14476 Potsdam-Golm, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Veyel
authors.affiliation: †Max Planck Institute of Molecular Plant Physiology, Am Muehlenberg 1, 14476 Potsdam-Golm, Germany.
authors.forename: Tabea
authors.initials: T
authors.lastname: Mettler-Altmann
authors.affiliation: †Max Planck Institute of Molecular Plant Physiology, Am Muehlenberg 1, 14476 Potsdam-Golm, Germany.
authors.forename: Mark
authors.initials: M
authors.lastname: Stitt
authors.affiliation: †Max Planck Institute of Molecular Plant Physiology, Am Muehlenberg 1, 14476 Potsdam-Golm, Germany.
authors.forename: John E
authors.initials: JE
authors.lastname: Lunn
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D007553:Isotope Labeling; D010766:Phosphorylation; D012015:Reference Standards; D053719:Tandem Mass Spectrometry / Q000379:methods*