iMet-Q: A User-Friendly Tool for Label-Free Metabolomics Quantitation Using Dynamic Peak-Width Determination

<div><p>Efficient and accurate quantitation of metabolites from LC-MS data has become an important topic. Here we present an automated tool, called iMet-Q (<u>i</u>ntelligent <u>Met</u>abolomic <u>Q</u>uantitation), for label-free metabolomics quantitation from high-throughput MS1 data. By performing peak detection and peak alignment, iMet-Q provides a summary of quantitation results and reports ion abundance at both replicate level and sample level. Furthermore, it gives the charge states and isotope ratios of detected metabolite peaks to facilitate metabolite identification. An in-house standard mixture and a public Arabidopsis metabolome data set were analyzed by iMet-Q. Three public quantitation tools, including XCMS, MetAlign, and MZmine 2, were used for performance comparison. From the mixture data set, seven standard metabolites were detected by the four quantitation tools, for which iMet-Q had a smaller quantitation error of 12% in both profile and centroid data sets. Our tool also correctly determined the charge states of seven standard metabolites. By searching the mass values for those standard metabolites against Human Metabolome Database, we obtained a total of 183 metabolite candidates. With the isotope ratios calculated by iMet-Q, 49% (89 out of 183) metabolite candidates were filtered out. From the public Arabidopsis data set reported with two internal standards and 167 elucidated metabolites, iMet-Q detected all of the peaks corresponding to the internal standards and 167 metabolites. Meanwhile, our tool had small abundance variation (≤0.19) when quantifying the two internal standards and had higher abundance correlation (≥0.92) when quantifying the 167 metabolites. iMet-Q provides user-friendly interfaces and is publicly available for download at <a href="http://ms.iis.sinica.edu.tw/comics/Software_iMet-Q.html" target="_blank">http://ms.iis.sinica.edu.tw/comics/Software_iMet-Q.html</a>.</p></div

Hierarchical clustering by using the quantitation results of iMet-Q, XCMS, MetAlign, and MZmine 2.

<p>Each entry in the tree leaves of a dendrogram represents a replicate. For each tool, we first combined its quantitation results of positive- and negative-ion modes. Colors were assigned to each replicate in the combined quantitation results according to the plant classes which the replicates originated from as follows: orange for cotyledon, red for stem, green for leaf, blue for flower, light blue for shoot apex, yellow for root, pink for seed, and gray for silique. Next, the figure was produced using MATLAB <i>dendrogram</i> function with PMMCC as the abundance correlation measure between any two replicates in the combined quantitation results.</p

The main graphical user interface of Metab-Q.

<p>The Arabidopsis data from positive-ion mode is used as an example. After processing the data, Metab-Q lists the detected peaks in the summary table where the peaks are sorted according to their retention time. When users select peaks of interest in the summary table, the abundances of the selected peaks in different samples are plotted in the sample abundance plot and the detailed information of the selected peak in the technical replicates of a sample is listed in the panel below the summary table. The left panel is the quantitation parameter explorer that lists the parameters of a quantitation. Users can use the provided filter function to narrow down the number of peaks in the summary table.</p

The box plot of abundance correlations between 19 verified metabolites and their in-source fragments detected in the public Arabidopsis data.

<p>The box plot of abundance correlations between 19 verified metabolites and their in-source fragments detected in the public Arabidopsis data.</p

The box plot of abundance correlation of 167 elucidated metabolites across replicates in the public Arabidopsis data detected by the four quantitation tools.

<p>The box plot of abundance correlation of 167 elucidated metabolites across replicates in the public Arabidopsis data detected by the four quantitation tools.</p

The reproducibility (Rep.) and normalized abundance (Abund.) of two internal standards detected by four quantitation tools.

<p>The reproducibility (Rep.) and normalized abundance (Abund.) of two internal standards detected by four quantitation tools.</p

A cartoon for the illustration of constructing extracted ion chromatograms.

<p>The blue straight lines represent the clustered signals, <i>w</i> and <i>t</i> are the FWHM and retention time of , respectively. Signal A and B are determined as the boundaries of the EIC, and the area in light blue color is the abundance.</p

The quantitation error (%) of seven standard metabolites calculated by iMet-Q, XCMS, MetAlign, and MZmine 2.

<p>The quantitation error (%) of seven standard metabolites calculated by iMet-Q, XCMS, MetAlign, and MZmine 2.</p

Schematic depiction of iMet-Q workflow for peak detection and peak alignment.

<p>Schematic depiction of iMet-Q workflow for peak detection and peak alignment.</p