“Replica-Extraction-Transfer” Nanostructure-Initiator
Mass Spectrometry Imaging of Acoustically Printed Bacteria
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Abstract
Traditionally,
microbes are studied under controlled laboratory
conditions as isolates in planktonic culture. However, this is a vast
extrapolation from their natural state; development of new techniques
is required to decipher the largely unknown world of microbial chemical
interactions in more realistic environments. The field of mass spectrometry
imaging has made significant progress in localizing metabolites in
and around bacterial colonies, primarily by using MALDI and ESI-based
techniques that interrogate the top surface of the sample. Unfortunately,
surface-based laser-desorption techniques, such as nanostructure-initiator
mass spectrometry (NIMS), which has advantages in detection of small
metabolite compounds and low background, has not been suitable for
direct microbe imaging because desorption/ionization occurs on the
bottom of the sample. Here, we describe a “replica-extraction-transfer”
(REX) technique that overcomes this barrier by transferring biomolecules
from agar cultures of spatially arrayed bacterial colonies onto NIMS
surfaces; further, we demonstrate that acoustic printing of bacteria
can be used to create complex colony geometries to probe microbial
interactions with NIMS imaging. REX uses a solvent-laden semisolid
(e.g., gel) to first extract metabolites from a microbial sample,
such as a biofilm or agar culture; the metabolites are then replica
“stamped” onto the NIMS surface. Using analytical standards
we show that REX–NIMS effectively transfers and detects a range
of small molecule compounds including amino acids and polyamines.
This approach is then used to analyze the metabolite composition of
streaked Shewanella oneidensis MR1
and Pseudomonas stutzeri RCH2 colonies
and further resolve complex patterns produced by acoustic printing
of liquid microbial cultures. Applying multivariate statistical analysis
of the NIMS imaging data identified ions that were localized to different
regions between and within colonies, as well as to the agar gel. Subsequent
high-resolution tandem mass spectrometry was used to characterize
two species-specific lipids that correlated with the spatial location
of each microbial species and were found to be highly abundant in
cell extracts. Overall, the use of acoustic printing of bacteria with
REX–NIMS imaging will extend the range of analytical capabilities
available for characterization of microbial interactions with mass
spectrometry