Dereplicating and Spatial Mapping of Secondary Metabolites
from Fungal Cultures <i>in Situ</i>
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Abstract
Ambient ionization mass spectrometry
techniques have recently become
prevalent in natural product research due to their ability to examine
secondary metabolites <i>in situ</i>. These techniques retain
invaluable spatial and temporal details that are lost through traditional
extraction processes. However, most ambient ionization techniques
do not collect mutually supportive data, such as chromatographic retention
times and/or UV/vis spectra, and this can limit the ability to identify
certain metabolites, such as differentiating isomers. To overcome
this, the droplet–liquid microjunction–surface sampling
probe (droplet–LMJ–SSP) was coupled with UPLC–PDA–HRMS–MS/MS,
thus providing separation, retention times, MS data, and UV/vis data
used in traditional dereplication protocols. By capturing these mutually
supportive data, the identity of secondary metabolites can be confidently
and rapidly assigned <i>in situ</i>. Using the droplet–LMJ–SSP,
a protocol was constructed to analyze the secondary metabolite profile
of fungal cultures without any sample preparation. The results demonstrate
that fungal cultures can be dereplicated from the Petri dish, thus
identifying secondary metabolites, including isomers, and confirming
them against reference standards. Furthermore, heat maps, similar
to mass spectrometry imaging, can be used to ascertain the location
and relative concentration of secondary metabolites directly on the
surface and/or surroundings of a fungal culture