Primer on Agar-Based Microbial Imaging Mass Spectrometry

Abstract

Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) imaging mass spectrometry (IMS) applied directly to microbes on agar-based medium captures global information about microbial molecules, allowing for direct correlation of chemotypes to phenotypes. This tool was developed to investigate metabolic exchange factors of intraspecies, interspecies, and polymicrobial interactions. Based on our experience of the thousands of images we have generated in the laboratory, we present five steps of microbial IMS: culturing, matrix application, dehydration of the sample, data acquisition, and data analysis/interpretation. We also address the common challenges encountered during sample preparation, matrix selection and application, and sample adherence to the MALDI target plate. With the practical guidelines described herein, microbial IMS use can be extended to bio-based agricultural, biofuel, diagnostic, and therapeutic discovery applications.

Utilizing a Robotic Sprayer for High Lateral and Mass Resolution MALDI FT-ICR MSI of Microbial Cultures

“…V isualizing microbial interactions with mass spectrometry imaging (MSI) has provided insights into many important biological processes, where it has captured molecular snapshots of metabolic exchange, antibiotic resistance, microbial competition, and more [1][2][3][4][5][6][7]. There are a number of different MSI methods reported for chemical mapping of microbial systems [3,4,6].…”

Nostopeptolide plays a governing role during cellular differentiation of the symbiotic cyanobacterium Nostoc punctiforme

“…Thin-layer BG11 and BG11 0 agar plates of N. punctiforme wild-type and pks2 − mutant monocultures or bacterial interactions were inoculated from fresh liquid cultures and incubated for 10 d (29). Sample preparation, matrix application, and dehydration were conducted as previously described (30). For MALDI imaging experiments of plant cyanobacteria symbiosis, G. manicata plants harboring the symbiotic glands were cut into thin slices.…”

Homogeneous Matrix Deposition on Dried Agar for MALDI Imaging Mass Spectrometry of Microbial Cultures

“…The matrix was applied to wet agar by sieving through a 53 μm stainless steel sieve until the surface was covered with matrix powder as previously described [12]. A second round of matrix was applied after 1 h of drying at 40°C followed by a final drying step of 2 h at 40°C.…”

“…Consequently, several sample preparation techniques may be used, mainly based on methods established for tissue imaging [9]. Following the approach of applying dry matrix on a tissue sample [10,11], sieving matrix powder onto wet agar samples has become a common sample preparation technique for microbial IMS in our lab [12]. This sieving method has been proven to be useful in most studies and certainly benefits from its simple and straight-forward procedure [12].…”

“…Following the approach of applying dry matrix on a tissue sample [10,11], sieving matrix powder onto wet agar samples has become a common sample preparation technique for microbial IMS in our lab [12]. This sieving method has been proven to be useful in most studies and certainly benefits from its simple and straight-forward procedure [12]. Vergeiner et al have recently shown a modification to this approach by spraying a matrix suspension onto wet agar, which resulted in a more homogeneous matrix coverage and better sensitivity for a specific metabolite [13].…”