Computational protocol: Metabolomics and molecular marker analysis to explore pepper (Capsicum sp.) biodiversity

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Protocol publication

[…] Volatile and semi-polar metabolite profiles derived using the SPME–GC–MS and the LC–PDA–QTOF–MS platforms, respectively, were processed as described by Tikunov et al. (, ). Both datasets were processed independently by the MetAlign software package ( for baseline correction, noise estimation, and ion-wise mass spectral alignment. The MetAlign outputs for both the GC–MS and LC–MS data were processed separately with MSClust software for data reduction and compounds mass spectra extraction (Tikunov et al. ). [...] The identification of semi-polar metabolites was carried out by means of their UV spectra, exact molecular weight and MSn fragmentation pattern. Putative identification of semi-polar metabolites was obtained using different metabolite databases such as Dictionary of Natural Products (, KNApSAcK ( and in-house metabolite databases, and using previous results on pepper described by Marin et al. () and Wahyuni et al. ().Putative identification of volatile metabolites was performed by automatic matching of their mass spectra extracted by MSClust with the National Institute of Standards and Technology (NIST) mass spectral library entries, using the NIST MS Search v2.0 software ( The compound hit that showed the highest matching factor (MF) value (≥600) and the lowest deviation from the retention index (RI) value was used for the putative metabolite identity. Additional manual spectral matching was performed for selected metabolites with low MF and high RI deviations by deconvoluting the chromatographic peak at the corresponding retention time using AMDIS (version 2.64) ( followed by matching the resulting spectra with those in the NIST library database. […]

Pipeline specifications

Software tools MetAlign, MSClust, NIST MS Search, AMDIS
Application MS-based untargeted metabolomics
Organisms Capsicum annuum, Capsicum frutescens
Chemicals Flavonoids, Quercetin, Apigenin, Luteolin