CAMERA: An Integrated Strategy for Compound Spectra Extraction and Annotation of Liquid Chromatography/Mass Spectrometry Data Sets

Liquid chromatography coupled to mass spectrometry is routinely used for metabolomics experiments. In contrast to the fairly routine and automated data acquisition steps, subsequent compound annotation and identification require extensive manual analysis and thus form a major bottleneck in data interpretation. Here we present CAMERA, a Bioconductor package integrating algorithms to extract compound spectra, annotate isotope and adduct peaks, and propose the accurate compound mass even in highly complex data. To evaluate the algorithms, we compared the annotation of CAMERA against a manually defined annotation for a mixture of known compounds spiked into a complex matrix at different concentrations. CAMERA successfully extracted accurate masses for 89.7% and 90.3% of the annotatable compounds in positive and negative ion modes, respectively. Furthermore, we present a novel annotation approach that combines spectral information of data acquired in opposite ion modes to further improve the annotation rate. We demonstrate the utility of CAMERA in two different, easily adoptable plant metabolomics experiments, where the application of CAMERA drastically reduced the amount of manual analysis

Data_Sheet_2_Detailed Phytochemical Analysis of High- and Low Artemisinin-Producing Chemotypes of Artemisia annua.XLSX

<p>Chemical derivatives of artemisinin, a sesquiterpene lactone produced by Artemisia annua, are the active ingredient in the most effective treatment for malaria. Comprehensive phytochemical analysis of two contrasting chemotypes of A. annua resulted in the characterization of over 80 natural products by NMR, more than 20 of which are novel and described here for the first time. Analysis of high- and low-artemisinin producing (HAP and LAP) chemotypes of A. annua confirmed the latter to have a low level of DBR2 (artemisinic aldehyde Δ¹¹⁽¹³⁾ reductase) gene expression. Here we show that the LAP chemotype accumulates high levels of artemisinic acid, arteannuin B, epi-deoxyarteannuin B and other amorpha-4,11-diene derived sesquiterpenes which are unsaturated at the 11,13-position. By contrast, the HAP chemotype is rich in sesquiterpenes saturated at the 11,13-position (dihydroartemisinic acid, artemisinin and dihydro-epi-deoxyarteannunin B), which is consistent with higher expression levels of DBR2, and also with the presence of a HAP-chemotype version of CYP71AV1 (amorpha-4,11-diene C-12 oxidase). Our results indicate that the conversion steps from artemisinic acid to arteannuin B, epi-deoxyarteannuin B and artemisitene in the LAP chemotype are non-enzymatic and parallel the non-enzymatic conversion of DHAA to artemisinin and dihyro-epi-deoxyarteannuin B in the HAP chemotype. Interestingly, artemisinic acid in the LAP chemotype preferentially converts to arteannuin B rather than the endoperoxide bridge containing artemisitene. In contrast, in the HAP chemotype, DHAA preferentially converts to artemisinin. Broader metabolomic and transcriptomic profiling revealed significantly different terpenoid profiles and related terpenoid gene expression in these two morphologically distinct chemotypes.</p

Acyl-CoA levels in developing castor endosperm.

<p>The percentage of the total acyl-CoA peak area represented by specified acyl-CoAs are shown for castor endosperm stages during seed development. Analysis of fluorescent acyl-CoA derivatives was as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030100#pone.0030100-Larson1" target="_blank">[24]</a> and average values from three extractions and analyses for each stage are shown, together with the standard error in brackets (<i>n</i> = 3).</p>a<p>Endosperm samples were staged according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030100#pone.0030100-Greenwood1" target="_blank">[25]</a>.</p

TLC analysis of neutral lipids from castor developing male flowers and pollen.

<p>Lipid extracts were applied to a silica TLC plate which was developed with hexane/diethyl ether/acetic acid (70∶30∶1) before iodine-staining. The position of lipid standard components (lane N) and proposed nature of resolved sample lipids are shown. Lipid X and proposed sterol esters were purified for further analysis.</p

MS analysis of purified neutral lipids from pollen.

<p>Purified neutral lipids from pollen were analysed by TLC and used for electrospray MS analysis in the presence of lithium (panel A). Ions between 830 and 900 amu in the lipid X fraction (highlighted in red and Panel B) were selected for fragmentation and the diagnostic fragments produced, such as those in Panel C from the 858 mass ion, allowed determination of fatty acid composition of proposed TAG species (Panel D).</p

Candidate genes that may be important in tri-ricinolein synthesis based on RNA-Seq data.

<p>Candidate genes that may be important in tri-ricinolein synthesis based on RNA-Seq data.</p

Acyl-CoA analysis of developing castor endosperm.

<p>A chromatogram of fluorescent acyl-CoA derivatives from developing castor endosperm stage III is shown - dark trace. Results from analysis of synthesised 18:1-OH-CoA (green line) and other acyl-CoA standards (red trace) are superimposed to confirm the indicated peak identities in the endosperm sample.</p

Pathways of triacylglycerol biosynthesis.

<p>Reactions in the formation of hydroxylated TAGs via the acyl-CoA dependent (solid arrows) and independent (dashed arrows) pathways are shown. Lipid substrates are abbreviated: 18:1, oleic acid; 18:1-OH, ricinoleic acid; LPC, lysophosphatidylcholine; DAG, diacylglycerol. Enzyme abbreviations are: Δ12-OHase, oleate-12-hydroxylase; LPCAT, 1-acylglycerol-3-phosphocholine acyltransferase; PL-A₂, phospholipase A₂; LACS, long chain acyl-CoA synthetase; GPAT, glycerol-3-phosphate acyltransferase; LPAT, lysophosphatidic acid acyltransferase; PAP, phosphatidic acid phosphatase; DGAT, diacylglycerol acyltransferase; CPT, CDP-choline:diacylglycerol cholinephosphotransferase; PL-C, phospholipase C; PDAT, phosphatidylcholine diacylglycerol acyltransferase.</p

Fatty acid analysis of castor tissues and purified lipid X.

<p>Average peak-area percentages of fatty acid methyl ester derivatives are listed. The number of separate sample extractions and analyses are listed in brackets, except for lipid X, where the result from two GC injections of the purified fraction is shown.</p>a<p>Italicised entries are probable fatty acid designations, although the species did not exactly co-chromatograph with standards.</p>b<p>Indicates the identity of the molecular species is not known but likely wax or hydroxylated fatty acid derivatives from pollen protective layers.</p

Data_Sheet_1_Detailed Phytochemical Analysis of High- and Low Artemisinin-Producing Chemotypes of Artemisia annua.DOCX

<p>Chemical derivatives of artemisinin, a sesquiterpene lactone produced by Artemisia annua, are the active ingredient in the most effective treatment for malaria. Comprehensive phytochemical analysis of two contrasting chemotypes of A. annua resulted in the characterization of over 80 natural products by NMR, more than 20 of which are novel and described here for the first time. Analysis of high- and low-artemisinin producing (HAP and LAP) chemotypes of A. annua confirmed the latter to have a low level of DBR2 (artemisinic aldehyde Δ¹¹⁽¹³⁾ reductase) gene expression. Here we show that the LAP chemotype accumulates high levels of artemisinic acid, arteannuin B, epi-deoxyarteannuin B and other amorpha-4,11-diene derived sesquiterpenes which are unsaturated at the 11,13-position. By contrast, the HAP chemotype is rich in sesquiterpenes saturated at the 11,13-position (dihydroartemisinic acid, artemisinin and dihydro-epi-deoxyarteannunin B), which is consistent with higher expression levels of DBR2, and also with the presence of a HAP-chemotype version of CYP71AV1 (amorpha-4,11-diene C-12 oxidase). Our results indicate that the conversion steps from artemisinic acid to arteannuin B, epi-deoxyarteannuin B and artemisitene in the LAP chemotype are non-enzymatic and parallel the non-enzymatic conversion of DHAA to artemisinin and dihyro-epi-deoxyarteannuin B in the HAP chemotype. Interestingly, artemisinic acid in the LAP chemotype preferentially converts to arteannuin B rather than the endoperoxide bridge containing artemisitene. In contrast, in the HAP chemotype, DHAA preferentially converts to artemisinin. Broader metabolomic and transcriptomic profiling revealed significantly different terpenoid profiles and related terpenoid gene expression in these two morphologically distinct chemotypes.</p