Computational protocol: Changes of Metabolomic Profile in Helianthus annuus under Exposure to Chromium(VI) Studied by capHPLC-ESI-QTOF-MS and MS/MS

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Protocol publication

[…] A general scheme of data analysis is presented in . In the first place, raw capHPLC-ESI-QTOF-MS data acquired for each sample were preprocessed using Bruker DataAnalysis 4.1 which included recalibration of mass accuracy, background subtraction, and finding molecular features (FMF). For FMF, S/N threshold of 3, minimum compound length of 20 spectra, correlation coefficient of 0.7, and smoothing width of 10 were applied. The generated lists were opened in ProfileAnalysis 2.0 (Bruker Daltonics); group attributes were defined as 1 (Cr(VI)-exposed) and 0 (nonexposed controls). The rectangle bucketing was performed using the following settings: the retention time width 60 s and m/z width 1 Da and time range 5.5–44.5 min; sum of buckets option was used for normalization. t-test was carried out comparing exposed and nonexposed plants, and minimum fold change was set at 5 and p value at 0.01; from this analysis and by additional inspection of intensities (higher than 5 · 104 cps), the precursors list was obtained.These same raw data were submitted to XCMS, defining a pairwise job. In the instrument selection, UPLC/Bruker QTOF POS was marked, which automatically activated centWave algorithm for FMF. Parameters used for FMF were mass tolerance of 10 ppm between successive measurements, peak width of 5–20 s, S/N threshold of 6, and obiwarp algorithm for retention time alignment (m/z width 0.9 s, minimum fraction of samples of 0.5 for group validation). To visualize differences between exposed and nonexposed roots, the cloud plot was obtained applying analogous statistical criteria as those used for ProfileAnalysis. Welch t-test was then performed (p < 0.01) yielding the list of precursors.The above two lists of precursors were manually revised leaving only those ions that were obtained by both approaches under criterion of absolute intensity threshold 5 · 104 cps.Once LC-MS/MS data were acquired in a separate analytical run, molecular formulas were generated with the aid of SIRIUS 3.2 plus CSI:FingerID and MS-Finder tools. Few possible molecular structures with relatively high statistical scores were proposed per each molecular formula and searched in biological databases (ChEBI, HMDB, KEGG, KNApSAcK, MeSH, UNPD, and PubChem), accordingly to the first layers of InChIKey. […]

Pipeline specifications

Software tools XCMS, SIRIUS, CSI:FingerID, MS-FINDER
Application MS-based untargeted metabolomics
Organisms Helianthus annuus
Diseases Multiple Sclerosis
Chemicals Benzofurans, Sesquiterpenes