Spatiochemically Profiling Microbial Interactions with Membrane Scaffolded Desorption Electrospray Ionization-Ion Mobility-Imaging Mass Spectrometry and Unsupervised Segmentation

Imaging
the inventory of microbial small molecule interactions
provides important insights into microbial chemical ecology and human
medicine. Herein we demonstrate a new method for enhanced detection
and analysis of metabolites present in interspecies interactions of
microorganisms on surfaces. We demonstrate that desorption electrospray
ionization-imaging mass spectrometry (DESI-IMS) using microporous
membrane scaffolds (MMS) enables enhanced spatiochemical analyses
of interacting microbes among tested sample preparation techniques.
Membrane scaffolded DESI-IMS has inherent advantages compared to matrix-assisted
laser desorption ionization (MALDI) and other IMS methods through
direct IMS analyses of microbial chemistry in situ. This rapid imaging method yields sensitive MS analyses with unique m/z measurements when compared to liquid
chromatography–electrospray ionization-mass spectrometry (LC–ESI-MS)
via unmediated sampling by MMS DESI-IMS. Unsupervised segmentation
imaging analysis of acquired DESI-IMS data reveals distinct chemical
regions corresponding to intermicrobial phenomenon such as predation
and communication. We validate the method by linking Myxovirescin
A and DKxanthene-560 to their known biological roles of predation
and phase variation, respectively. In addition to providing the first
topographic locations of known natural products, we prioritize 54
unknown features using segmentation within the region of predation.
Thus, DESI-IMS and unsupervised segmentation spatially annotates the
known biology of myxobacteria and provides functional exploration
of newly uncharacterized small molecules.