<i>mzGroupAnalyzer</i>-Predicting Pathways and Novel Chemical Structures from Untargeted High-Throughput Metabolomics Data

<div><p>The metabolome is a highly dynamic entity and the final readout of the genotype x environment x phenotype (GxExP) relationship of an organism. Monitoring metabolite dynamics over time thus theoretically encrypts the whole range of possible chemical and biochemical transformations of small molecules involved in metabolism. The bottleneck is, however, the sheer number of unidentified structures in these samples. This represents the next challenge for metabolomics technology and is comparable with genome sequencing 30 years ago. At the same time it is impossible to handle the amount of data involved in a metabolomics analysis manually. Algorithms are therefore imperative to allow for automated <i>m/z</i> feature extraction and subsequent structure or pathway assignment. Here we provide an automated pathway inference strategy comprising measurements of metabolome time series using LC- MS with high resolution and high mass accuracy. An algorithm was developed, called <i>mzGroupAnalyzer</i>, to automatically explore the metabolome for the detection of metabolite transformations caused by biochemical or chemical modifications. Pathways are extracted directly from the data and putative novel structures can be identified. The detected <i>m/z</i> features can be mapped on a van Krevelen diagram according to their H/C and O/C ratios for pattern recognition and to visualize oxidative processes and biochemical transformations. This method was applied to <i>Arabidopsis thaliana</i> treated simultaneously with cold and high light. Due to a protective antioxidant response the plants turn from green to purple color via the accumulation of flavonoid structures. The detection of potential biochemical pathways resulted in 15 putatively new compounds involved in the flavonoid-pathway. These compounds were further validated by product ion spectra from the same data. The <i>mzGroupAnalyzer</i> is implemented in the graphical user interface (GUI) of the metabolomics toolbox COVAIN (Sun & Weckwerth, 2012, Metabolomics 8: 81–93). The strategy can be extended to any biological system.</p></div

A proposed network of the detected anthocyanin family featuring putatively novel compounds as well as known structures including the KEGG pathway of anthocyanin biosynthesis [38], [44].

<p>Only compounds from the KEGG anthocyanin pathway are depicted, for which a suitable precursor mass was found in the data. Exact masses, sum formulas, and main MS² fragments of the new compounds are compiled in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096188#pone-0096188-t001" target="_blank">Table 1</a>; reconstructed structures together with MS² scans are in the supporting information. The network was created with VANTED <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096188#pone.0096188-Junker1" target="_blank">[45]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096188#pone.0096188-Rohn1" target="_blank">[46]</a>.</p

Relative changes of metabolic clusters under light and extended night conditions.

<p>Bars indicate the ratios of metabolic clusters from Col-0 under extended night and light conditions. Clusters are named according to the description in the main text (<i>Results)</i> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092299#pone.0092299.s002" target="_blank">Table S1</a>.</p

Putative compounds including their <i>mzGroupAnalyzer</i>- predicted sum formulas, the corresponding exact mass as well as dominant MS² product ion fragments.

<p>The nomenclature is according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096188#pone.0096188-Tohge1" target="_blank">[33]</a>. Compounds <i>m/z</i> 1125, 1197 and 1211 were found in <i>Matthiola incana</i> by <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096188#pone.0096188-Saito1" target="_blank">[39]</a>.</p

Structure validation of <i>m/z</i> 1121 by MS³ product ion scans.

<p>Both MS² fragments <i>m/z</i> 535 and 873 result in the core cyanidin structure by undergoing MS³ fragmentation. <i>m/z</i> 1077, the putatively decarboxylized form of 1121, yields <i>m/z</i> 491, as observed in the MS² spectrum already, by scission of the 3-O-glycosidic bond. Fragment <i>m/z</i> 873 arises again from the breaking of the glycosidic bond at 5-O. <i>m/z</i> 1017 would comply with the complete removal of the rest of the former malonyl group together with a water loss (−60 u). A putative structure is given.</p

The hydrophobic patch and its interaction with active site pocket.

<p><b>(A)</b> The hydrophobic patch (Pro225-Arg237) in Alr_<i>Tt</i> (PDB 4Y2W) is colored in gray, the active site pocket is shown in orange, the substrate and key amino acids mediating the interactions are shown in sticks, the hydrogen bonding interactions are indicated. The hydrophobic patch and active site pocket of the other three bacterial alanine racemases are shown in same view: <b>(B)</b> the PLP-D-Ala complex of Alr_<i>Bst</i> (PDB 1L6G). <b>(C)</b> PLP complex of Alr_<i>Cd</i> (PDB 4LUS), <b>(D)</b> PLP and DLY complex of DadX_<i>pao</i> (PDB 1RCQ).</p

After oxidative stress the <i>Arabidopsis thaliana</i> plants turn from green into purple indicating a dramatic shift in metabolism, specifically elevated flavonoid biosynthesis involved in oxidative stress protection [6].

<p><b>A</b> Plants turns from green to purple under high light and cold temperature treatment. <b>B</b> Van Krevelen diagram of the most abundant <i>m/z</i> values of unstressed (green dots) and 20-day cold stressed (purple dots) Arabidopsis plants. A clear shift of metabolism in the stressed plants is visible.</p

Scheme of the <i>mzGroupAnalyzer</i> and <i>Pathway Viewer</i> algorithm and GUI implementation.

<p>The program reads the <i>m/z</i> features which are extracted from Xcalibur, as well as the user predefined reaction rules. Then it finds transformations between all pairs of <i>m/z</i> features, and reports the frequency of transformations for the listed and not listed but potentially existing rules. Next, the program starts searching pathways inside the <i>m/z</i> features' network. A shorter path existing in other longer paths is removed, thereby non-redundant pathways are obtained. Then, <i>mzGroupAnalyzer</i> opens the Pathway Viewer, in which pathways satisfying user-defined filtering options will be displayed on the panel. The pathway diagram, which consists of reaction rules, <i>m/z</i> feature names, compositions and time points, can be plotted by clicking the table. Finally, all the results, including the frequency table of transformations, the interconnected network visualization file (in Pajek's format), the inferred pathways and a Matlab workspace (suffixed with mzStruct.mat) containing all results, will be exported to the user-specified folder.</p

Relative activity of the pyruvate dehydrogenase complex in Col-0 under conditions of light and extended night.

<p>Enzyme activity is given in arbitrary units which are normalised to gram fresh weight. The blue bar shows relative activity under normal light condition, the red bar shows activity under condition of extended darkness. The difference of relative activity is significant (p<0.05) and bars represent means ± SD (n  = 5).</p

Structural based sequence alignment of Alr_<i>Tt</i>, DadX_<i>Tt</i> and other three representative bacterial alanine racemases.

<p>Amino acid sequences of alanine racemase from a gram positive bacteria <i>Bacillus stearothermophilus</i> (Alr_<i>Bst</i>), a gram negative bacteria <i>Pseudomonas aeruginosa</i> (DadX_<i>pao</i>), and <i>Clostridium difficile</i> strain 630 (Alr_<i>Cd</i>) are aligned with Alr_<i>Tt</i> and DadX_<i>Tt</i> from <i>T</i>. <i>tengcongensis</i> MB4. Amino acids are numbered and secondary structures are labeled, strictly conserved amino acids are highlighted in yellow box. Amino acids form the substrate entryway are colored in blue (middle layer) and magenta (inner layer), key catalytic residues mediating the phosphate group and L-Ala binding are colored in red, residues necessary for hydrogen bonding interactions for PLP-binding are colored in green. Two key catalytic residues Lys40 and Tyr268’ are marked with a star. The hydrophobic patch (Pro225-Arg337) in Alr_<i>Tt</i> is indicated by a red box.</p