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

Abstract

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 *

Synergizing the potential of bacterial genomics and metabolomics to find novel antibiotics

“… 48 A study that involves future utility of DESI-MSI for natural product research shows antibiotics production during predation by Myxococcus xanthus bacteria. 49 Although the study pinpoints a number of molecular features belonging to natural products associated with bacterial predation, isolation of those compounds remains a very challenging task due to the limited scalability of 2D cultivations on surfaces.…”

Accelerating Strain Engineering using Desorption Electrospray Ionization-Imaging Mass Spectrometry and Untargeted Molecular Analysis of Intact Microbial Colonies

“…26 A previously developed microporous membrane scaffold method was adapted to sample a mixed culture of engineered E. coli strains (figure 1). 15 Briefly, microorganisms were grown on a membrane scaffold placed on top of an agar substrate, which facilitated the uptake of nutrients to support microbial growth and production, while retaining microorganisms and their excreted biosynthetic products on the scaffold surface. At desired timepoints, the membrane was removed from the agar and affixed to a glass microscope slide to provide a solid support on which DESI can be performed directly with no further sample pretreatment.…”

“…We have recently described an aligned DESI-MS strategy operated in an imaging MS (IMS) mode for rapid and reproducible analysis of microbially produced metabolites and chemicals in situ. 15 Building on this approach, we have tailored this sampling method for the simultaneous analysis of many bacterial colonies including multiple strains and biosynthetic products in a spatially-resolved manner. Herein, we describe the application of DESI-IMS to untargeted metabolomic characterization of engineered microorganisms.…”

Integrating ion mobility and imaging mass spectrometry for comprehensive analysis of biological tissues: A brief review and perspective

“…In particular, TWIMS has proven to lend itself to analysis of large proteins and protein complexes, even becoming popular for use in native MS. [71][72][73] Upon commercialization, TWIMS has also been incorporated into many IMS workflows for increasing the chemical information obtained from a pixel and applied to various analyte classes in an imaging context. 60,74,75 For example, TWIMS has been coupled to many ion sources including MALDI, 36,[76][77][78][79][80] DESI, 60,74,80,81 LAESI, 82,83 and LESA (Figure 2A-D). 48,61,84,85 is a point of much discussion in the scientific community.…”