Studying Plant Specialized Metabolites Using Computational Metabolomics Strategies.

Plant specialized metabolites have diversified vastly over the course of plant evolution, and they are considered key players in complex interactions between plants and their environment. The chemical diversity of these metabolites has been widely explored and utilized in agriculture and crop enhancement, the food industry, and drug development, among other areas. However, the immensity of the plant metabolome can make its exploration challenging. Here we describe a protocol for exploring plant specialized metabolites that combines high-resolution mass spectrometry and computational metabolomics strategies, including molecular networking, identification of structural motifs, as well as prediction of chemical structures and metabolite classes.

Reproducible mass spectrometry data processing and compound annotation in MZmine 3.

Untargeted mass spectrometry (MS) experiments produce complex, multidimensional data that are practically impossible to investigate manually. For this reason, computational pipelines are needed to extract relevant information from raw spectral data and convert it into a more comprehensible format. Depending on the sample type and/or goal of the study, a variety of MS platforms can be used for such analysis. MZmine is an open-source software for the processing of raw spectral data generated by different MS platforms. Examples include liquid chromatography-MS, gas chromatography-MS and MS-imaging. These data might typically be associated with various applications including metabolomics and lipidomics. Moreover, the third version of the software, described herein, supports the processing of ion mobility spectrometry (IMS) data. The present protocol provides three distinct procedures to perform feature detection and annotation of untargeted MS data produced by different instrumental setups: liquid chromatography-(IMS-)MS, gas chromatography-MS and (IMS-)MS imaging. For training purposes, example datasets are provided together with configuration batch files (i.e., list of processing steps and parameters) to allow new users to easily replicate the described workflows. Depending on the number of data files and available computing resources, we anticipate this to take between 2 and 24 h for new MZmine users and nonexperts. Within each procedure, we provide a detailed description for all processing parameters together with instructions/recommendations for their optimization. The main generated outputs are represented by aligned feature tables and fragmentation spectra lists that can be used by other third-party tools for further downstream analysis.

plantMASST - Community-driven chemotaxonomic digitization of plants.

Understanding the distribution of hundreds of thousands of plant metabolites across the plant kingdom presents a challenge. To address this, we curated publicly available LC-MS/MS data from 19,075 plant extracts and developed the plantMASST reference database encompassing 246 botanical families, 1,469 genera, and 2,793 species. This taxonomically focused database facilitates the exploration of plant-derived molecules using tandem mass spectrometry (MS/MS) spectra. This tool will aid in drug discovery, biosynthesis, (chemo)taxonomy, and the evolutionary ecology of herbivore interactions.

Prediction of Collision Cross Section Values: Application to Non-Intentionally Added Substance Identification in Food Contact Materials.

The synthetic chemicals in food contact materials can migrate into food and endanger human health. In this study, the traveling wave collision cross section in nitrogen values of more than 400 chemicals in food contact materials were experimentally derived by traveling wave ion mobility spectrometry. A support vector machine-based collision cross section (CCS) prediction model was developed based on CCS values of food contact chemicals and a series of molecular descriptors. More than 92% of protonated and 81% of sodiated adducts showed a relative deviation below 5%. Median relative errors for protonated and sodiated molecules were 1.50 and 1.82%, respectively. The model was then applied to the structural annotation of oligomers migrating from polyamide adhesives. The identification confidence of 11 oligomers was improved by the direct comparison of the experimental data with the predicted CCS values. Finally, the challenges and opportunities of current machine-learning models on CCS prediction were also discussed.

Critical evaluation of ambient mass spectrometry coupled with chemometrics for the early detection of adulteration scenarios in Origanum vulgare L.

Nowadays, most of the screening methods in food manufacturing are based on spectroscopic techniques. Ambient Mass Spectrometry is a relatively new field of analytical chemistry which has proven to offer similar speed and ease-of-use when compared to other fingerprinting techniques, alongside the advantages of good selectivity, sensitivity and chemical information. Numerous applications have been explored in food authenticity, based either on the target detection of adulteration markers or, less frequently, on the development of multivariate classification models. The aim of the present work was to evaluate and compare the capabilities of Direct Analysis in Real Time (DART) and Atmospheric Solid Analysis Probe (ASAP) Mass Spectrometry (MS) for the high-throughput authenticity screening of commercial herbs and spices products. The gross addition of bulking material to dried Mediterranean oregano was taken as case study. First, a pilot sample set, constituted by authentic dried oregano, olive leaves (a frequently reported adulterant) and mixtures thereof at different levels (i.e. 10, 20, 30 and 50% w/w) was used. Each sample was fingerprinted by both ambient-MS techniques. After appropriate pre-processing, the whole mass spectra were used for the subsequent multivariate data analysis. Soft Independent Modelling of Class Analogy was adopted as classification algorithm and the model was challenged with both new authentic oregano and in-house prepared blends. To the best of our knowledge, this is the first report of DART-MS and ASAP-MS used in full scan mode and coupled to chemometric modelling as rapid fingerprinting approach for food authentication. Although both the techniques provided satisfactory results, ASAP-MS clearly showed greater potential, leading to reproducible, diagnostic feature-rich mass spectra. For this reason, ASAP-MS was further tested under a more convoluted scenario, where the training and validation sets were enlarged with additional authentic oregano samples and a wider range of adulterant species, respectively. Overall good results were achieved, with 93% model predictive accuracy, and screening detection capability estimated between 5-20% (w/w) addition, depending on the adulterant considered with the only exception of majorana. Investigation of Q residuals could highlight the statistically-relevant chemical markers which could be tentatively annotated by coupling the ASAP probe with a high resolution mass analyser. The results from the validation study confirmed the great potential of ASAP-MS in combination with chemometrics as fast MS-based screening solution and demonstrated its feasibility for classification model building.

Exploiting the potential of micropropagated durum wheat organs as modified mycotoxin biofactories: The case of deoxynivalenol.

This study aimed to investigate the potential of in vitro wheat model as biofactory for masked mycotoxin production. Micropropagated durum wheat organs (leaves and roots) were treated during a 14-day time span on a proper medium spiked with deoxynivalenol (DON). After the treatment, DON absorption from culture media was evaluated while roots and leaves were profiled by UHPLC-HRMS to investigate the DON biotransformation products. A total of 10 metabolites have been annotated in both roots and leaves. In particular, 5 phase I metabolites never reported before were putatively identified, suggesting the viability of the model as a tool to investigate the interplay between mycotoxins and wheat. In addition, 5 phase II metabolites previously reported in wheat grown under open field conditions, were identified in both roots and leaves, thus demonstrating the reliability of the cultured organs as model system for wheat plants. An organ-dependent difference in DON uptake and biotransformation was observed, since roots contained a high amount of untransformed DON, while leaves were able to effectively biotransform DON to its glycosylated form and other relevant metabolites. With the perspective of using cultured organs as biofactories for modified mycotoxin production, leaves seemed therefore to offer the best absorption and production yield.

Vibrational Spectroscopy Coupled to a Multivariate Analysis Tiered Approach for Argentinean Honey Provenance Confirmation.

In the present work, the provenance discrimination of Argentinian honeys was used as case study to compare the capabilities of three spectroscopic techniques as fast screening platforms for honey authentication purposes. Multifloral honeys were collected among three main honey-producing regions of Argentina over four harvesting seasons. Each sample was fingerprinted by FT-MIR, NIR and FT-Raman spectroscopy. The spectroscopic platforms were compared on the basis of the classification performance achieved under a supervised chemometric approach. Furthermore, low- mid- and high-level data fusion were attempted in order to enhance the classification results. Finally, the best-performing solution underwent to SIMCA modelling with the purpose of reproducing a food authentication scenario. All the developed classification models underwent to a "year-by-year" validation strategy, enabling a sound assessment of their long-term robustness and excluding any issue of model overfitting. Excellent classification scores were achieved by all the technologies and nearly perfect classification was provided by FT-MIR. All the data fusion strategies provided satisfying outcomes, with the mid- and high-level approaches outperforming the low-level data fusion. However, no significant advantage over the FT-MIR alone was obtained. SIMCA modelling of FT-MIR data produced highly sensitive and specific models and an overall prediction ability improvement was achieved when more harvesting seasons were used for the model calibration (86.7% sensitivity and 91.1% specificity). The results obtained in the present work suggested the major potential of FT-MIR for fingerprinting-based honey authentication and demonstrated that accuracy levels that may be commercially useful can be reached. On the other hand, the combination of multiple vibrational spectroscopic fingerprints represents a choice that should be carefully evaluated from a cost/benefit standpoint within the industrial context.