Profiling of Stable Isotope Enrichment in Specialized Metabolites Using Liquid Chromatography and Multiplexed Nonselective Collision-Induced Dissociation

Engineering of specialized metabolites in plants, microbes, and other organisms is hindered by significant knowledge gaps about metabolic pathways responsible for metabolite accumulation and degradation. While isotopic tracers have provided important information about metabolic fluxes in central metabolism, limitations of mass spectrometric strategies for quantifying stable isotope incorporation into both intact metabolites and specific substructures have slowed extension of these techniques to large specialized metabolites. This report describes the application of electrospray ionization with data-independent multiplexed nonselective collision induced dissociation (CID) on a time-of-flight mass spectrometer. This strategy yields quasi-simultaneous collection, on the chromatographic time scale, of mass spectra with different degrees of fragment ion formation without biases introduced by precursor mass selection or selective ion activation and provides measurements of stable isotope enrichments in intact metabolites and individual substructures. The utility and precision of these analyses is demonstrated by labeling acylsugar metabolites in glandular trichomes of tomato (<i>Solanum lycopersicum</i>) using ¹³CO₂ and analyzing ¹³C enrichments in acylsugar specialized metabolites. The high precision and avoidance of mass bias provide a promising tool for extending metabolic flux analyses to complex specialized metabolites in a wide range of organisms

Phylogenetic tree of tomato CYP71BN1 and other functionally characterized terpene-modifying P450s.

<p>Neighbor-joining phylogenetic tree analysis using amino acid sequences was performed by MEGA 5 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119302#pone.0119302.ref040" target="_blank">40</a>]. Bootstrap values were performed with 1000 replications (values shown next to branches). LsGAO1, <i>Lactuca sativa</i> germacrene A oxidase (GAO) 1 (ADF32078.1); CiGAO2, <i>Cichorium intybus</i> (ADF43080.1); HaGAO4, <i>Helianthus annuus</i> (ADF43082.1); ScGAO3, <i>Saussurea costus</i> (ADF43081.1); AaAMO1, <i>Artemisia annua</i> amorpha-4, 11-diene monooxygenase (Q1PS23.1); BsGAO5, <i>Barnadesia spinosa</i> (ADF43083.1); HmHPO, <i>Hyoscyamus muticus</i> premnaspirodiene oxygenase (HPO) (A6YIH8.1); Nt-CYP71D20, <i>Nicotiana tabacum</i> 5-epiaristolochene dihydroxylase (Q94FM7.2); Ms-CYP71D18, <i>Mentha spicata</i> (-)-(4<i>S</i>)-limonene-6-hydroxylase (Q9XHE8.1); Mp-CYP71D13, <i>Mentha x piperita</i> (-)-(4<i>S</i>)-limonene-3-hydroxylase (Q9XHE7.1); Mp-CYP71D15, <i>Mentha x piperita</i> (-)-(4<i>S</i>)-limonene-3-hydroxylase (Q9XHE6.1); Zz-CYP71BA1, <i>Zingiber zerumbet</i> α-humulene oxidase (E3W9C4.1); AtKO, <i>Arabidopsis thaliana ent</i>-kaurene oxidase (KO) (NM_122491); OsKO2, <i>Oryza sativa</i> (BAF19823); OsKO4, <i>Oryza sativa</i> (BAF19823).</p

Ratios of lycosantalene levels to β-phellandrene and β-caryophyllene levels in petiolule with and without trichomes, and in trichomes of transgenic <i>S</i>. <i>lycopersicum</i> plants overexpressing <i>CPT2</i>.

<p>Terpenes were extracted with hexane and analyzed by GC-MA as described in Materials and Methods.</p><p>Ratios of lycosantalene levels to β-phellandrene and β-caryophyllene levels in petiolule with and without trichomes, and in trichomes of transgenic <i>S</i>. <i>lycopersicum</i> plants overexpressing <i>CPT2</i>.</p

Lycosantalonol biosynthesis in <i>Solanum lycopersicum</i>.

<p>(<b>A</b>) The terpene gene cluster on the tip of chromosome 8. (<b>B</b>) The biosynthetic pathway to lycosantanolol. AOX, alcohol oxidase; TPS, terpene synthase; CPT, <i>cis</i>-prenyl transferase; NDPS1, neryl diphosphate synthase 1; CYP, cytochrome P450; AAT, alcohol acyltransferase; DMAPP, dimethylallyl diphosphate; IPP, isopentenyl diphosphate; NNPP, nerylneryl diphosphate. Genes that are not functional because of deletions or insertions are shown with a “ψ” symbol.</p

qRT-PCR analysis of <i>CPT2</i>, <i>TPS21</i>, <i>CYP71BN1</i> transcripts in petiolules.

<p>RNA was isolated from whole petiolule, petiolules from which trichomes have been removed, and from the trichomes. Error bars represent SE. Values are from four biological replicates with three technical replicates of each.</p

GC-MS analysis of diterpenes from hexane extracts of whole petiolules.

<p>(<b>A</b>) Non-transformed <i>S</i>. <i>lycopersicum</i>, (<b>B</b>–<b>D</b>) three individual plants of <i>S</i>. <i>lycopersicum</i> transformed with the <i>35S-</i>CPT2 gene construct. Chromatograms of the selected ion of <i>m/z</i> 109 are shown here. (<b>E</b>) Four combined chromatographs of authentic standards. 1, nerylnerol; 2, lycosantalene: 3, epoxy-lycosantalene; 4, lycosantalonol. Mass Spectra for all peaks is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119302#pone.0119302.s002" target="_blank">S2 Fig.</a> Whole petiolules were ground and extracted with hexane as described in Materials and Methods.</p

Characterization of Model Peptide Adducts with Reactive Metabolites of Naphthalene by Mass Spectrometry

<div><p>Naphthalene is a volatile polycyclic aromatic hydrocarbon generated during combustion and is a ubiquitous chemical in the environment. Short term exposures of rodents to air concentrations less than the current OSHA standard yielded necrotic lesions in the airways and nasal epithelium of the mouse, and in the nasal epithelium of the rat. The cytotoxic effects of naphthalene have been correlated with the formation of covalent protein adducts after the generation of reactive metabolites, but there is little information about the specific sites of adduction or on the amino acid targets of these metabolites. To better understand the chemical species produced when naphthalene metabolites react with proteins and peptides, we studied the formation and structure of the resulting adducts from the incubation of model peptides with naphthalene epoxide, naphthalene diol epoxide, 1,2-naphthoquinone, and 1,4-naphthoquinone using high resolution mass spectrometry. Identification of the binding sites, relative rates of depletion of the unadducted peptide, and selectivity of binding to amino acid residues were determined. Adduction occurred on the cysteine, lysine, and histidine residues, and on the N-terminus. Monoadduct formation occurred in 39 of the 48 reactions. In reactions with the naphthoquinones, diadducts were observed, and in one case, a triadduct was detected. The results from this model peptide study will assist in data interpretation from ongoing work to detect peptide adducts <em>in vivo</em> as markers of biologic effect.</p> </div

Sites of Adduction.

<p>Sites of adduction deduced for each peptide.</p>*<p>represents an undetermined site of adduction. Diadducts and triadduct formed during reactions conducted at pH 8.5 were also formed at pH 7.4.</p

NNPS activity of CPT2.

<p>NNPS activity of CPT2.</p

qRT-PCR analyses of <i>CPT2</i>, <i>TPS21</i> and <i>CYP71BN1</i> transcripts in different tissues of <i>S</i>. <i>lycopersicum</i>.

<p>Total RNA was isolated from various tomato tissues. Leaflets and petiolules were prepared from four different developmental compound leaf stages. Error bars represent SE. Values are from three biological and three technical replicates.</p