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Synthesis and Use of Stable-Isotope-Labeled Internal Standards for Quantification of Phosphorylated Metabolites by LC−MS/MS Stéphanie Arrivault,*,† Manuela Guenther,† Stephen C. Fry,‡ Maximilian M. F. F. Fuenfgeld,† Daniel Veyel,† Tabea Mettler-Altmann,†,§ Mark Stitt,† and John E. Lunn† †

Max Planck Institute of Molecular Plant Physiology, Am Muehlenberg 1, 14476 Potsdam-Golm, Germany The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, School of Biological Sciences, The University of Edinburgh, Edinburgh, EH9 3BF U.K.

‡

S Supporting Information *

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-isotopelabeled 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.

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Phospholipids contribute to ion suppression during analysis of blood plasma samples,13,16 and chloride ions from the culture medium caused ion suppression during analysis of Chlamydomonas reinhardtii cell extracts.15 However, the source of interference is usually unknown. Matrix effects can be overcome by avoiding or minimizing the interference and by using methods to quantify and correct for the interference. To reduce interference (reviewed in ref 17), changes can be made to either the source of the biological material (e.g., by using a different cell culture medium15) or the analytical technique itself. Potential analytical modifications include (i) decreasing the volume or concentration of the extract injected; (ii) partially purifying the sample prior to analysis (e.g., by selective precipitation of interfering compounds, two-phase (liquid−liquid or liquid−solid) extraction,16 or membrane filtration2); (iii) reducing the flow rate during the LC phase;18 (iv) changing the type of column matrix, eluent composition, or eluent gradient to separate the analyte of interest from the interfering compound(s);16 (v) combining

iquid chromatography coupled to tandem mass spectrometry (LC−MS/MS) has become the method of choice for measurement of plant metabolites,1 including phosphorylated intermediates,2−4 lipids,5 and secondary compounds,6−8 and is also widely used for metabolomics in other organisms, including bacteria9 and yeast.10 Like other MS-based analytical techniques, LC−MS/MS is susceptible to matrix effects that can reduce the accuracy and reliability of the analysis.11 Matrix effects occur when sample components coelute with the analyte of interest and interfere with the ionization process. The most common matrix effect is ionization suppression, which leads to underestimation of the affected analyte, but enhancement of ionization, leading to overestimation, can also occur. While the exact mechanisms are still not fully understood, it has been postulated that coeluting compounds interfere with the charging or desolvation of analytes at the interface between the LC and the MS system (the ion source).12,13 Electrospray ionization (ESI), the most widely used ion source, is more susceptible to matrix effects than atmospheric pressure photoionization (APPI) and atmospheric pressure chemical ionization (APCI).13,14 The degree of interference also depends on the chemical nature of the analytes12 and on the biological source of the sample.15 © 2015 American Chemical Society

Received: April 14, 2015 Accepted: May 25, 2015 Published: May 26, 2015 6896

DOI: 10.1021/acs.analchem.5b01387 Anal. Chem. 2015, 87, 6896−6904

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Analytical Chemistry

Arrivault and colleagues3 developed a reverse-phase LC− MS/MS protocol to measure 27 metabolites involved in photosynthetic or respiratory metabolism in plants. Commercially available isotopomers of six of these metabolites were used as SIL-IS: [2,3,3-2H3]glutamate, [2,3,3-2H3]aspartate, [2,3,3-2H3]glycerate, [U-13C]succinate, [2,3,3-2H3]malate, and [1,2,3,4-13C4]2-oxoglutarate, and a deuterated form of glucose 6-phosphate, [6,6-2H2]G6P, was enzymatically synthesized from commercially available [6,6-2H2]glucose. Here we discuss what factors need to be considered when selecting or designing an SIL-IS and report the enzymatic synthesis of isotopomers of eight further phosphorylated metabolites and their validation for use as SIL-IS in LC−MS/MS analysis. SIL-IS can also be used to assess the recovery of metabolites during extraction, but here we focus on their use to correct for matrix effects in LC− MS/MS analysis.

two different types of chromatographic separation in series (LC−LC);19 and (vi) using a different ion source.20 Some of these options might not be suitable for low abundance metabolites, due to poor recoveries from preanalytical cleanup or inadequate machine sensitivity, and most of these approaches increase the technical complexity and often the cost of the analysis. Despite efforts to minimize matrix effects, it is often not possible to eliminate them altogether. Therefore, methods to quantify and compensate for the interference are required for metabolite measurements to be accurate and reproducible.21,22,15 Standard addition23 and matrix-matched calibration24 attempt to compensate for the interference by mixing standards with extract or interfering compounds to generate sample-specific calibration curves. However, both of these approaches increase the workload and time needed for analysis. Furthermore, unless applied to each individual sample, correction using these methods is still prone to error arising from variation in matrix effects between different biological samples. These techniques have largely been superseded by use of stable-isotope-labeled internal standards (SIL-IS), which allow correction for matrix effects in each individual sample with minimal addition to the workload and analysis time.23 A SIL-IS is an isotopically labeled form of the metabolite of interest, in which two or more atoms have been substituted by nonradioactive isotopes. The most commonly used isotopes are 2H, 13C, and, to a lesser extent, 15N and 17O. The inclusion of stable isotopes has no significant effect on most of the physicochemical properties of the molecule, so both the labeled and unlabeled forms behave in the same way during sample extraction and chemical derivatization. Labeled and unlabeled forms usually coelute during chromatography, although heavily deuterated isotopomers sometimes show a shift in retention time.25 The unlabeled and labeled isotopomers have essentially identical ionization characteristics. However, following ionization, the resulting ions have different m/z values and so can be readily distinguished by MS. To correct for matrix effects, standard mixes and samples are spiked with a known amount of the SIL-IS before analysis. The labeled and unlabeled forms of the metabolite of interest coelute from the LC and are exposed to identical ionization conditions, including any interference by other molecules, before entering the MS. There are several ways to use SIL-IS to correct for matrix effects. A simple approach is to compare the signals from a known amount of the SIL-IS analyzed on its own (i.e., no matrix effects) and when spiked into a sample. The ratio between the signals can then be used as a correction factor to normalize the signal from the unlabeled compound in the sample. Some types of software used for analysis of LC−MS/MS chromatograms allow more automated approaches. For example, mixtures of unlabeled standards can be spiked with a known amount of the SIL-IS to set up calibration curves, and then the ratio of the signals from the labeled and unlabeled metabolite in an SIL-IS-spiked sample is compared with the calibration standards to calculate the amount of unlabeled metabolite in the sample. Ideally, SILIS should be used for each measured metabolite, but with few exceptions,26 this goal is rarely achieved, or even approached, due to the limited commercial availability of suitable labeled isotopomers. In fact in many studies, the issue of matrix effects is simply, but ill-advisedly, ignored. For molecules of particular importance, effort may be made to synthesize a suitable labeled isotopomer for use as an internal standard.2

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EXPERIMENTAL SECTION Materials (Chemicals). [2,3,3- 2 H 3 ]Aspartic acid, [2,3,3-2H3]glutamic acid, and [1,2,3,4-13C4]2-oxoglutarate (2OG) were obtained from Cambridge Isotope Laboratories (http://www.isotope.com). [2,3,3-2H3]Malic acid, [U-13C]succinic acid, [2,3,3-2H3]glyceric acid, [6,6-2H2]glucose, [1,6-13C2]fructose, [13C10,14N5]ATP, [13C10,14N5]UTP, and ATP were obtained from Sigma-Aldrich (http://www. sigmaaldrich.com). [U-13C]FBP (fructose 1,6-bisphosphate), [U-13C]sucrose, and [2,3,4,5-13C4]ribose were from Campro (http://www.campro.eu). All enzymes were from SigmaAldrich, with the exception of hexokinase, inorganic pyrophosphatase, and aldolase (Roche Diagnostics GmbH, Mannheim, Germany, http://www.roche.de) and Escherichia coli ribokinase (kindly provided by Dr Roman Esipov, Shemyakin−Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia). The sources of plant, bacterial, and animal material and sampling procedures are described in detail in the Supporting Information. Synthesis of Isotopically Labeled Compounds. Unless stated otherwise, reaction mixtures (1 mL final volume) were incubated at 30 °C for 3 h, and then the reaction was stopped by heating at 99 °C for 2 min. Precipitated proteins were removed by centrifugation (18 000g, 2 min), and the labeled product was purified by high-voltage paper electrophoresis (HVPE) of the supernatant. The composition of specific labeling reactions was as follows. [6,6-2H2]G6P and [1,6-13C2]F6P (fructose 6-phosphate). Ten micromoles of [6,6-2H2]glucose or 10 μmol of [1,6-13C2]fructose, respectively, 12 μmol of ATP, 5 U of hexokinase (EC 2.7.7.1), 5 mM Tris-HCl (pH 7.5), and 1 mM MgCl2 were used. [U-13C]G1P (glucose 1-phosphate). Ten micromoles of [U-13C]sucrose, 10 U of sucrose phosphorylase (EC 2.4.1.7), 50 μmol of K2HPO4−KOH (pH 7.0), 5 mM Tris-HCl (pH 7.0), and 1 mM MgCl2 were used. As the supplied sucrose phosphorylase contained sucrose as stabilizer, 125 U of the enzyme was dissolved in 1.5 mL of 20 mM MES−NaOH (pH 7.0) and desalted using a 20-mL centrifugal concentrator according the manufacturer’s recommendations (10 kDa cutoff; Amicon Ultra, Merck Millipore, http://merckmillipore.com). [13C10,14N5]UDPG (UDP glucose). Twelve micromoles of 13 [ C10,14N5]UTP, 10 μmol of G1P, 5 U of UGPase (EC 2.7.7.9), 5 U of inorganic pyrophosphatase (EC 3.6.1.1), 5 mM Tris-HCl (pH 7.5), and 1 mM MgCl2 were used. 6897


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Analytical Chemistry Table 1. Isotope Distribution for Parent Ionsa abundance (%)

a

compounds

parent ion ([M − H]−) formula

mass (M)

M

M+1

M+2

M+3

M+4

M+5

M+6

G6P/F6P/G1P UDPG R5P/Ru5P/Xu5P DHAP AMP FBP 2-OG malate glycerate succinate glutamate aspartate

C6H12O9P − C15H23N2O17P2− C5H10O8P− C3H6O6P− C10H13N5O7P − C6H13O12P2− C4CH5O5− C4H5O5− C3H5O4− C4H5O4− C5H8NO4− C4H6NO4−

259 565 229 169 346.1 339 145 133 105 117 146 132

100 100 100 100 100 100 100 100 100 100 100 100

6.9703 17.8664 5.8276 3.5423 13.0585 7.0961 5.6558 4.5743 3.4546 4.5362 6.0176 4.9130

2.0562 4.9997 1.7838 1.2778 2.2285 2.6812 1.1580 1.1085 0.8639 0.9013 0.9720 0.9176

0.1307 0.7009 0.0965 0.0424 0.2150 0.1766 0.0584 0.0460 0.0267 0.0356 0.0487 0.0386

0.0184 0.1114 0.0137 0.0063 0.0193 0.0322 0.0054 0.0042 0.0025 0.0025 0.0025 0.0025

n/ab 0.0113 n/a n/a n/a 0.0018 n/a n/a n/a n/a n/a n/a

n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a

Abundances were obtained using an online calculator34 (http://www.chemcalc.org/main). bn/a: not applicable.

[2,3,4,5-13C4]R5P (ribose 5-phosphate). Ten micromoles of [2,3,4,5-13C4]ribose and 12 μmol of ATP, with 5 U ribokinase (EC 2.7.1.15), 5 mM Tris-HCl (pH 7.9), 1 mM MgCl2, and 30 mM KCl (the presence of potassium and magnesium ions increase ribokinase activity27), were combined and incubated at 37 °C for 4 h. After stopping the reaction by heating at 100 °C for 2 min, ATP and AMP were quantitatively hydrolyzed to AMP by addition of 5 U nucleoside-triphosphate phosphatase (apyrase 1, EC 3.6.1.19) and 5 U nucleoside-diphosphate phosphatase (apyrase 2, EC 3 6.1.6) and incubation at 37 °C for 2 h. The apyrase reactions were stopped by heating at 100 °C for 2 min. [2,3,4,5- 1 3 C 4 ]Ru5P (ribulose 5-phosphate) and [2,3,4,5-13C4]Xu5P (xylulose 5-phosphate). Ten micromoles of [2,3,4,5-13C4]ribose and 12 μmol of ATP, with 5 U of ribokinase, 5 U of ribose-5-phosphate isomerase (EC 5.3.1.6), 5 U of ribulose-phosphate 3-epimerase (EC 5.1.3.1), 5 mM TrisHCl (pH 7.9), 1 mM MgCl2, and 30 mM KCl, were combined. After incubation at 37 °C for 2 h, a further 5 U of ribokinase was added and the reaction incubated at 37 °C for another 2 h. After stopping the reaction by heating at 100 °C for 2 min, ATP and ADP were removed by treatment with apyrase 1 and apyrase 2 as described above. [U-13C]DHAP (dihydroxy-acetone-phosphate). Ten micromoles of [U-13C]FBP, 2 U of aldolase (EC 4.1.2.13), 3 U of TPI (EC 5.3.1.1), 5 mM Tris-HCl (pH 7.9), and 1 mM MgCl2 were used. [13C10,15N5]AMP (adenosine monophosphate). Twelve micromoles of [13C10,15N5]ATP, 5 U of apyrase 1, 5 U of apyrase 2, 5 mM MES−NaOH (pH 6.0), and 1 mM CaCl2 were used. Separation by HVPE. Preparative HVPE was performed as described in refs 28 and 29. Standards and reaction mixtures were applied to Whatman No. 1 paper (42 × 57 cm), along an origin line 9 cm from the edge of the paper. Authentic standards were loaded at 50 μg per spot except DHAP (25 μg) and Pi and PPi (15 μg each). Five micrograms of the visible mobile marker orange G was mixed with the sample or loaded as a series of spots alternating with the samples. After sample loading, the paper was wetted with a volatile electrophoresis buffer of acetic acid/pyridine/H2O (10:1:189 by volume) at pH 3.5 or of acetic acid/pyridine/H2O (1:33:300 by volume) at pH 6.5. In preliminary trials to identify the optimal electrophoresis conditions for separation, a volatile electrophoresis buffer at pH 2.0 (formic acid/acetic acid/H2O; 1:4:45 by volume) was also

tested. The paper was suspended in the HVPE apparatus, consisting of a glass tank fitted with platinum electrodes at the top and bottom. The edge of the paper closest to the origin was placed uppermost, dipping into a glass trough containing electrophoresis buffer (cathode), while the lower end of the paper dipped into a layer of electrophoresis buffer at the bottom (anode) of the tank. The remainder of the tank was filled with a nonpolar solvent (white spirit for pH 2.0 and 3.5, toluene/pyridine 20:1 by volume for pH 6.5) that was cooled by circulation of cold tap water through cooling coils suspended from the lid of the apparatus. The paper was electrophoresed at 4.5 kV for 20−40 min (as indicated for individual experiments). Following electrophoresis and drying to remove the volatile buffers and coolant, standards were visualized by UV absorbance (nucleotides, including UDPG) or staining with molybdate reagent or AgNO3.29 Using the appropriate standard as a guide, a strip containing the labeled compound of interest was excised from the preparative zone, and the compound was eluted with water using the syringe-barrel method.29 The eluted compound was lyophilized and redissolved in 250 or 500 μL of deionized water (Millipore, http://www.merckmillipore.com) before use. Quantification of Metabolites. Metabolite analysis was performed by ion pair reverse-phase chromatography coupled to tandem mass spectrometry using a Thermo triple quadrupole MS as previously described,3 with slight modifications of the LC gradient.30 Aliquots of frozen tissue powder from Arabidopsis, maize, wheat, and tobacco leaves (15 mg FW) and green and red tomato fruits, potato tuber, and mouse liver (20 mg FW) were extracted with chloroform−methanol as described.3 C. reinhardtii cells were extracted as described.31,30 Yeast and E. coli were extracted by addition of 1 mL of 90% (v/ v) methanol cooled to −20 °C to the frozen cells on liquid nitrogen, thawed on ice, refrozen, and sonicated in a bath of iced-water for 10 min. The extract was centrifuged at 21 000g (0 °C) for 10 min. The supernatant (900 μL) was transferred to a fresh tube and 260 μL of chloroform was added to each methanolic extract, with subsequent phase partitioning and lyophilization as previously described.3 After lyophilization, extracts of mouse liver, C. reinhardtii, yeast, and E. coli cells were dissolved in 0.5 vol (150 μL) of water before analysis. Synthesis reaction mixtures and purified SIL-IS were diluted 1/ 150 with deionized water prior to analysis. Fragmentation products for SIL-IS are listed in Table S1 (Supporting Information). A concentrated SIL-IS mixture was prepared 6898


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Figure 1. Synthesis of SIL-IS: (A) [6,6-2H2]G6P, (B) [1,6-13C2]F6P, (C) [U-13C]G1P, (D) [13C11,15N2]UDPG, (E) [2,3,4,5-13C4]R5P/Xu5P/Ru5P, (F) [U-13C]DHAP, and (G) [13C10,15N5]AMP. The isotopically labeled elements are indicated in red. Structures were made with ACD/ChemSketch (http://www.acdlabs.com/resources/freeware/chemsketch/).

and 5 μL, containing the amounts shown in Table S1 (Supporting Information), was added to each sample prior to measurement. ATP, fructose, and sucrose were measured enzymatically using a Sigma-22 dual-wavelength photometer.32

metabolites of interest with estimates of the natural abundance of isotopomers with m/z values of +1 to +6 above the most abundant naturally occurring form expressed as a percentage of the latter. It should be noted that these calculations might overstate the observed isotopic interference because they do not take into account the fragmentation used in MS/MS.35 Nevertheless, this is a simple approach to avoid selection of inappropriate SIL-IS that might be subject to isotopic interference. For example, putative UDPG SIL-IS labeled with one or two 2H or 13C atoms would have non-negligible overlap with naturally occurring isotopomers with m/z values of M + 1 (18% abundance) and M + 2 (5% abundance), respectively. Therefore, for UDPG, an SIL-IS with an m/z value of M + 3 or higher would be preferred. Following this principle, we selected the following commercially available isotopomers for use as SIL-IS: [1,2,3,4-13C4]2-OG, [2,3,3-2H3]malate, [2,3,3-2H3]glycerate, [U13C]succinate, [2,3,3-2H3]glutamate, and [2,3,3-2H3]aspartate. The estimated signal contribution from M + 3 and M + 4 isotopomers of these compounds was 7%).

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RESULTS AND DISCUSSION Criteria for Selection of SIL-IS. The stable isotopes most commonly used for labeling of biological molecules also occur naturally. The most abundant is 13C, which comprises 1.1% of total C on a global scale. Other stable isotopes are less abundant2H, 0.0156%; 17O, 0.0373%; and 15N, 0.364%but can be higher (or lower) in biological materials due to environmental variation or biological discrimination.33 The presence of stable isotopes in molecules of biological origin can interfere with the signal of the corresponding SIL-IS in LC− MS/MS assays if the m/z value of the selected SIL-IS overlaps with naturally occurring isotopomers in the sample. We used the ChemCalc (http://www.chemcalc.org/main)34 tool to calculate the abundance of naturally occurring isotopomers of the metabolites of interest. Calculations were based on the monovalent anionic form of metabolites of interest ([M − H]−), as our established LC−MS/MS protocols2,3 operate in negative ESI mode. Table 1 lists 6899


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Figure 2. Preparative HVPE for substrates, products, and reaction mixtures. Separation of compounds involved in the synthesis of [6,6-2H2]G6P and [1,6-13C2]F6P (A), [U-13C]G1P (B), [13C11,15N2]UDPG (C), [2,3,4,5-13C4]R5P (D), [U-13C]DHAP (E), and [13C10,15N5]AMP (F). Compounds run are listed along the origin of each electrophoretogram. HVPE were performed at pH 3.5 for panels B−D and at pH 6.5 for panels A, E, and F. Electrophoreses were conducted at 4.5 kV for 30 min except for panel E (40 min). The colored marker Orange G (orange dots) was added into each sample before electrophoresis except in panel A, where it was loaded alternatively with the samples. Phosphate-containing compounds were stained with molybdate reagent. Sucrose and fructose in panel B were stained with AgNO3. Some of the fainter spots were manually outlined with dotted lines, with the synthesized IS shown in red. The origin of each electrophoretogram is indicated by a solid gray line.

Previously we had synthesized [2,2-2H2]G6P from [2,2-2H2]glucose using hexokinase (Figure 1A), for use as a SIL-IS for this metabolite.3 M + 2 isotopomers were predicted to have a natural abundance of 2% (Table 1). The potential error from isotopic interference by G6P (M + 2) in G6P measurements would be in the same range as pipetting error, so use of [2,2-2H2]G6P as a SIL-IS was judged to be acceptable. No retention time shift was observed for this deuterated SIL-IS. There were no commercially available stable isotopomers for the other metabolites of interest. Therefore, we designed strategies for synthesizing suitable SIL-IS enzymatically, based on the availability of potential precursors and enzymes, and using the data in Table 1 to decide the minimum degree of labeling necessary to avoid isotopic interference. Synthesis of 13C-Labeled Hexose-Phosphates. Hexosephosphates are respiratory intermediates in most organisms, and in plants they are also important as intermediates of the Calvin−Benson cycle (F6P) and sucrose and starch synthesis. We synthesized [1,6-13C2]F6P from [1,6-13C2]fructose in a one-step reaction catalyzed by hexokinase (Figure 1B), with a stoichiometric excess of ATP to ensure quantitative conversion of [1,6-13C2]fructose to [1,6-13C2]F6P. ADP is the major byproduct of the reaction, although AMP can also be produced if the hexokinase is contaminated with adenylate kinase (myokinase). To avoid interference with measurements of ADP and AMP in samples, it was essential to remove these byproducts from the preparation of [1,6-13C2]F6P to be used as

a SIL-IS. [U-13C]G1P was synthesized by phosphorolytic cleavage of [U-13C]sucrose using sucrose phosphorylase (SPase; Figure 1C) and a stoichiometric excess of orthophosphate (Pi), with fructose as a byproduct.36 Two methods offered the necessary resolution and capacity for purification of the synthesized compounds: high-performance liquid chromatography (HPLC) and HVPE. However, typical HPLC eluents (e.g., sodium hydroxide) would be incompatible with LC−MS/MS analysis, whereas HVPE uses volatile solvents (formic + acetic acids for electrophoresis at pH 2.0 and pyridinium acetate buffers for electrophoresis at pH 3.5 or 6.529) that can easily be removed by drying the paper before elution of the labeled compound. Therefore, HVPE was chosen for purification of the SIL-IS. Low concentrations of buffering agents and cofactors (e.g., MgCl2) were used for the enzymatic synthesis reactions, to minimize the ionic load in the HVPE purification step. The optimal pH for separation of the labeled compound from potentially interfering contaminants was selected on the basis of previous knowledge or preliminary analyses of authentic standards. Figure 2A shows an HVPE electrophoretogram at pH 6.5, of hexose-phosphates and adenine nucleotide standards, showing clear separation of G6P, F6P, and G1P from AMP, ADP, and ATP. Results obtained at pH 2.0 and 3.5 are shown in Figure S1 (Supporting Information). At pH 2, hexose-phosphates were not separated from ATP, and at pH 3.5, they partially overlapped with ATP and ADP. Therefore, [2,2-2H2]G6P and 6900


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ribose-5-phosphate isomerase39 and ribulose-phosphate-3-epimerase40 (Figure 1E). The pentose-phosphates could be separated from ADP, the byproduct of the initial ribokinase reaction, by HVPE at pH 3.5 but not from unreacted ATP (Figure 2D). Therefore, the reaction mixtures were incubated with nucleoside-triphosphate phosphatase (apyrase 1) and nucleoside-diphosphate phosphatase (apyrase 2) to hydrolyze unreacted ATP and the ADP byproduct to AMP and Pi, which were well-resolved from the pentose-phosphates by HVPE at pH 3.5 (Figure 2D). As an example, a preparative electrophoretogram for purification of [2,3,4,5- 13 C 4 ]R5P is shown in Figure S2 (Supporting Information). Although the initial ribokinase reaction gave 79% conversion of [2,3,4,5-13C4]ribose to [2,3,4,5-13C4]R5P, after HVPE purification the final yield of the latter was only 24%, while the final yield of the mixed epimers, [2,3,4,5-13C4]Ru5P and [2,3,4,5-13C4]Xu5P, was 53% (Table S2, Supporting Information). All of the purified pentosephosphate isotopomers are four mass units greater than the unlabeled forms, so isotopic interference from the naturally isotopomers is expected to be negligible (95% [U-13C]DHAP,41 which was separated from unreacted FBP and GAP by HVPE at pH 6.5 (Figure 2E). The final yield of [U-13C]DHAP after HVPE purification was 37%, mainly reflecting the inefficiency of the aldolase reaction (Table S2, Supporting Information). Isotopic interference from the naturally occurring M + 3 isotopomer will be negligible (95% for [1,6- 13 C 2 ]F6P and [U- 13 C]G1P (Table S2, Supporting Information). In biological samples, naturally occurring M + 2 isotopomers will represent approximately 2% of the total F6P (Table 1), therefore, isotopic interference with the [1,6-13C2]F6P SIL-IS will be in a similar range to pipetting error, and as discussed above for G6P, this was judged to be acceptable. Isotopic interference from G1P (M + 6) should be negligible (Table 1). Synthesis of [13C11,15N2]UDPG. UDPG is the glucosyl donor for synthesis of sucrose and cellulose in plants and the precursor of glycogen in animals. As noted above, potential for isotopic interference was particularly high for UDPG, making it desirable to synthesize an isotopomer with an m/z of at least M + 3 (