Mass spectrometry imaging of secondary metabolites directly on fungal cultures

Abstract

Desorption electrospray ionization mass spectrometry (DESI-MS) is an ambient ionization technique that enables imaging experiments directly on fungal cultures. Much information can be gained by examining an organism directly from culture, rather than through an extraction process, as the regional and temporal distribution of bioactive compounds can give a better understanding of interactions in the environment. However, this technique has been underutilized towards the direct analysis of fungal cultures. A major challenge of fungal culture analysis with DESI-MS is the requirement of a firm, flat surface for effective ionization. The media upon which fungi grow can be easily deformed by the pressures from the solvent spray and gas, and the fungal topography is naturally uneven, often containing mycelium and spores that move freely. Furthermore, DESI-MS imaging can only analyse the surface of a sample, thus internal compounds remain undetected. This project first sought to overcome these issues, and then, apply the newly developed methodology to explore the chemical interactions between two distinct fungi. To test the methodology, a fungus that produces antifungal metabolites was grown against a mycotoxin producing fungus, both of the phylum Ascomycota. By comparing the spatial and temporal distribution of secondary metabolites between both isolated cultures and co-cultures, the chemical exchanges that took place were visualized.

Acetophenone derivatives from a freshwater fungal isolate of recently described Lindgomyces madisonensis (G416)

“…This result was not surprising, as it is well known that variation of media conditions can alter the biosynthesis of secondary metabolites in fungi (Du et al, 2012, Vandermolen et al, 2013. An alternative hypothesis could be that compound 7 was located inside the mycelium, rather than on the surface (Sica et al, 2014). Overall, the heat profile (Fig.…”

Mass spectrometry tools and workflows for revealing microbial chemistry

“…DESI generates charged droplets that are projected with gas jet onto a sample placed on a surface 105 . The extracted analytes, such as proteins, peptides and small molecules, are directed into the adjacent mass spectrometer, enabling the analysis of native surface at the microbial community level 95, 105, 116, 117 . DESI’s spraying mechanism, however, causes secondary droplets to splash onto the surrounding area, while too high back pressure results in the creation of the holes in the agar 118, 119 .…”

Review and perspectives on the applications of mass spectrometry imaging under ambient conditions

“…Unlike nano‐DESI, DESI avoids these problems although the high gas and spray pressure of DESI create deformations on the microbial media surface that likewise alter the consistency of the analysis. To overcome these impediments before inoculation with fungal culture, Sica et al used several microporous, chemically inert inserts on top of agar, such as cheesecloth, filter paper, cardboard, and balsa wood, which not only allowed fungus to grow uniformly on them, but also provided a firm surface necessary for effective direct DESI‐MS analysis . Cardboard was most effective for the ionization of bacterial metabolites and offered an ideal and uniform surface for the fungus to grow on top of the substrate.…”

“…To access the inner layers of microbial cultures, the agar culture has been cryosectioned directly by making a perpendicular cut using a razor blade prior to flash freezing the excised agar piece by placing the culture on the ventral side . Sica et al dried the frozen agar block using a vacuum desiccator just prior to the analysis of the cross‐sectional area for fungal secondary metabolites by DESI‐MS imaging . Other approaches to 3D profile bacterial and fungal cultures involved extraction by a surface sampling probe (flow‐probe) that uses a pneumatic nebulizer prior to performing electrospray ionization as reported by Hsu and coworkers .…”