Reproducible molecular networking of untargeted mass spectrometry data using GNPS.

Global Natural Product Social Molecular Networking (GNPS) is an interactive online small molecule-focused tandem mass spectrometry (MS2) data curation and analysis infrastructure. It is intended to provide as much chemical insight as possible into an untargeted MS2 dataset and to connect this chemical insight to the user's underlying biological questions. This can be performed within one liquid chromatography (LC)-MS2 experiment or at the repository scale. GNPS-MassIVE is a public data repository for untargeted MS2 data with sample information (metadata) and annotated MS2 spectra. These publicly accessible data can be annotated and updated with the GNPS infrastructure keeping a continuous record of all changes. This knowledge is disseminated across all public data; it is a living dataset. Molecular networking-one of the main analysis tools used within the GNPS platform-creates a structured data table that reflects the molecular diversity captured in tandem mass spectrometry experiments by computing the relationships of the MS2 spectra as spectral similarity. This protocol provides step-by-step instructions for creating reproducible, high-quality molecular networks. For training purposes, the reader is led through a 90- to 120-min procedure that starts by recalling an example public dataset and its sample information and proceeds to creating and interpreting a molecular network. Each data analysis job can be shared or cloned to disseminate the knowledge gained, thus propagating information that can lead to the discovery of molecules, metabolic pathways, and ecosystem/community interactions.

Deciphering the influence of column chemistry and mass spectrometry settings for the analyses of geometrical isomers of L-chicoric acid.

Resolving the chemo-diversity of plant extract samples is an essential step for in-depth analyses of natural products which often exhibit promising biological activities. One of the challenges in this endeavor has been the confident differentiation of geometrical isomers. In this study, we investigated these aspects in chromatography (column chemistry and mobile phase composition) and mass spectrometry settings with regards to better differentiation of geometrical isomers. A standard of a hydroxycinnamic acid (HCA) derivative, L-chicoric acid (L-CA) - a di-acylated caffeoyltartaric acid ester found in a number of plant families - was used. Geometrical isomers of L-CA were formed by exposing the compound to ultraviolet (UV) radiation, to mimic the natural environment. The high performance liquid chromatography photo-diode array (HPLC-PDA) and ultra-high performance liquid chromatography mass spectrometry (UHPLC-MS) platforms were used to analyze the trans and cis geometrical isomers of L-CA. The HPLC-PDA results confirmed the generation of two cis geometrical isomers following UV exposure of the authentic trans-L-CA standard. Furthermore, the HPLC-PDA analyses demonstrated that the changes in both column chemistry (reverse-phase: C18, biphenyl, phenyl-hexyl and pentafluorophenyl propyl) and mobile phase composition (aqueous acetonitrile and aqueous methanol) affect the chromatographic elution profiles of the L-CA isomers. The MS results, on the other hand, revealed undisputed fragmentation differences between the geometrical isomers of L-CA. Thus, this study demonstrates that the identification of the L-CA isomers can be achieved more efficiently and confidently with good chromatography coupled to well-optimized mass spectrometry conditions, a requirement which has been proven impossible with other types of HCA derivatives. Moreover, differences in the binding modes of L-CA geometrical isomers to the HIV type 1 integrase enzyme were observed, suggesting a synergistic anti-HIV-1 activity of these isomers.

Simultaneous analysis of defense-related phytohormones in Arabidopsis thaliana responding to fungal infection.

PREMISE OF THE STUDY: Simultaneous analysis of defense-related phytohormones can provide insights into underlying biochemical interactions. Ultra-high-performance liquid chromatographic (UHPLC) techniques hyphenated to electrospray ionization mass spectrometry (ESI-MS) are powerful analytical platforms, suitable for quantitative profiling of multiple classes of metabolites. METHODS: An efficient and simplified extraction method was designed followed by reverse-phase UHPLC for separation of seven phytohormones: salicylic acid, methyl salicylate, jasmonic acid, methyl jasmonate, absiscic acid, indole acetic acid, and the ethylene precursor 1-aminocyclopropane-1-carboxylic acid. A triple quadrupole multiple reaction monitoring (MRM) method was developed for MS quantification. The methods were applied to analyze phytohormones in Arabidopsis leaf tissue responding to biotic stresses. RESULTS: Under the optimized conditions, the phytohormones were separated within 15 min, with good linearities and high sensitivity. Repeatable results were obtained, with the limits of detection and quantification around 0.01 ng/µL (∼9 ng/g tissue). The method was validated and applied to monitor, quantify, and compare the temporal changes of the phytohormones under biotic stress. DISCUSSION: Quantitative changes indicate increased production of defense phytohormones from the various classes. The analytical method was useful and suitable to distinguish distinctive variations in the phytohormonal profiles and balance in A. thaliana leaves resulting from pathogen attack.