Introduction: Lichens contain unique metabolites that most often need to be characterized from a limited amount of material. While thin layer chromatography is still the preferred analysis method for most lichenologists, liquid chromatography gives a deeper insight in the lichen metabolome, but an extractive step is needed before any analysis. Therefore, ambient ionization mass spectrometry (MS) analysis of lichen samples using Atmospheric Solid Analysis Probe (ASAP) and Direct Acquisition in Real Time (DART) techniques is evaluated.Objective: We looked for a faster method to screen the metabolome by disrupting the classical workflow of analysis.Methods: Four lichens selected for their metabolic diversity were analyzed with MS; namely Evernia prunastri, Lichina pygmaea, Parmelia saxatilis, and Roccella fuciformis.ASAP and DART analyses were compared against the reference electrospray ionization with a bioinformatic process including Van Krevelen diagrams as well as the multivariate comparison of the ionization methods in positive and negative modes.Results: Metabolite profiles obtained from DART and ASAP analyses of lichen samples are consistent with classical analyses of lichen extracts. Through an easy and rapid experiment and without any extraction solvent, a large and informative profile of lichen metabolites is obtained when using complementary ionization modes of these high resolution mass spectrometry methods.Conclusion: ASAP-MS and DART-MS are two ancillary methods that provide a comprehensive evaluation of the lichen metabolome.

Introduction:In recent years, LC-MS has become the gold standard for metabolomic studies. Indeed, liquid chromatography is relatively easy to couple with the soft electrospray ionization. As a consequence, many tools have been developed for the structural annotation of tandem mass spectra. However, it is sometimes difficult to do Data Dependent Acquisition (DDA), especially when developing new methods that stray from the classical LC-MS workflow.Objective: An old tool from petroleomics that has recently gained popularity in metabolomics, the Van Krevelen (VK) diagram, is adapted for an overview of the molecular diversity profile in lichens through HR-MS.Methods: A new method is benchmarked against the state-of-the-art classification tool ClassyFire using a database containing most known lichen metabolites (n ≈ 2,000). Four lichens known for their contrasted chemical composition were selected, and extractions with apolar, aprotic polar and protic polar solvents were performed to cover a wide range of polarities. Extracts were analyzed with Direct Infusion ElectroSpray Ionization Mass Spectrometry (DI-ESI-MS) and Atmospheric Solids Analysis Probe Mass Spectrometry (ASAP-MS) techniques to be compared with the chemical composition described in the literature. Results:The most common lichen metabolites were efficiently classified, with more than 90% of the molecules in some classes being matched with ClassyFire. Results from this method are consistent with the various extraction protocols in the present case study. Conclusion:This approach is a rapid and efficient tool to gain structural insight regarding lichen metabolites analyzed by high resolution mass spectrometry without relying on DDA by LC-MS/MS analysis. It may notably be of use during the development phase of novel MS-based