Expanding Molecular Coverage in Mass Spectrometry Imaging of Microbial Systems Using Metal-Assisted Laser Desorption/Ionization

Abstract

Mass spectrometry imaging (MSI) is becoming an increasingly popular analytical technique to investigate microbial systems. However, differences in the ionization efficiencies of distinct MSI methods lead to biases in terms of what types and classes of molecules can be detected. Here, we sought to increase the molecular coverage of microbial colonies by employing metal-assisted laser desorption/ionization (MetA-LDI) MSI, and we compared our results to more commonly utilized matrix-assisted laser desorption/ionization MALDI MSI. We found substantial (∼67%) overlap in the molecules detected in our analysis of Bacillus subtilis colony biofilms using both methods, but each ionization technique did lead to the identification of a unique subset of molecular species. MetA-LDI MSI tended to identify more small molecules and neutral lipids, whereas MALDI MSI more readily detected other lipids and surfactin species. Putative annotations were made using METASPACE, Metlin, and the BsubCyc database. These annotations were then confirmed from analyses of replicate bacterial colonies using liquid extraction surface analysis tandem mass spectrometry. Additionally, we analyzed B. subtilis biofilms in a polymer-based emulated soil micromodel using MetA-LDI MSI to better understand bacterial processes and metabolism in a native, soil-like environment. We were able to detect different molecular signatures within the micropore regions of the micromodel. We also show that MetA-LDI MSI can be used to analyze microbial biofilms from electrically insulating material. Overall, this study expands the molecular universe of microbial metabolism that can be visualized by MSI. IMPORTANCE Matrix-assisted laser desorption/ionization mass spectrometry imaging is becoming an important technique to investigate molecular processes within microbial colonies and microbiomes under different environmental conditions. However, this method is limited in terms of the types and classes of molecules that can be detected. In this study, we utilized metal-assisted laser desorption/ionization mass spectrometry imaging, which expanded the range of molecules that could be imaged from microbial samples. One advantage of this technique is that the addition of a metal helps facilitate ionization from electrically nonconductive substrates, which allows for the investigation of biofilms grown in polymer-based devices, like soil-emulating micromodels.

Keywords: Bacillus subtilis; MALDI; MetA-LDI; biofilms; microfluidics; soil microbiome.

FIG 1

Comparison of MetA-LDI MSI and MALDI MSI of a B. subtilis colony grown on agar. (A) Venn diagram illustrating the overlap of annotations found in the two MSI techniques. (B) Bright-field image of the colony analyzed by both techniques after gold sputter coating (MetA-LDI) and DHB matrix deposition (MALDI). (C to H) Representative ion images of (C) corynebactin at m/z 905.2448 [M+Na]⁺, (D) DG (36:4) at m/z 617.5136 [M+H]⁺, (E) Cer (32:3) at m/z 528.4387 [M+Na]⁺, (F) TG (56:3) at m/z 913.8212 [M+H]⁺, (G) PG (32:3) at m/z 755.4260 [M+K]⁺, and (H) surfactin C at m/z 1,036.6904 [M+H]⁺ of the colony analyzed.

FIG 2

MetA-LDI MSI of B. subtilis grown in the emulated soil micromodel. (A) B. subtilis that has formed a biofilm is visualized by fluorescence microscopy prior to lyophilization and gold sputter coating and MetA-LDI MSI analysis. (B) PG (32:0) at m/z 723.5171 [M+H]⁺, (C) PE (34:2) at m/z 716.5225 [M+H]⁺, and (D) DG (36:3) at m/z 619.5296 [M+H]⁺ were able to be visualized throughout the microchannel. The feature outlined in white highlights an area in which an air bubble was present in the microchannel, so minimal lipid signal from B. subtilis was detected in the fluorescence microscopy as well as the MetA-LDI MSI analysis. The feature outlined in a red rectangle highlights an aggregate structure within the micromodel, and Meta-LDI MSI analysis revealed lipids could be clearly detected around the perimeter of this feature.