Stable Isotope Labeling of Prodiginines and Serratamolides Produced by Serratia marcescens Directly on Agar and Simultaneous Visualization by Matrix-Assisted Laser Desorption/Ionization Imaging High-Resolution Mass Spectrometry

Abstract

Matrix-assisted laser desorption/ionization imaging high-resolution mass spectrometry (MALDI-imaging-HRMS) is an important technique for visualizing the spatial distribution of compounds directly on the surface of organisms such as microorganisms, insects, plants, animals, and human tissues. However, MALDI-imaging-HRMS and the stable isotope labeling approach have never been combined for the detection and simultaneous visualization of labeled and unlabeled compounds, their analogues and derivatives, as well as their precursors. Herein, we present a methodology that labels microbial secondary metabolites directly on agar with stable isotopes and allows concurrent spatial distribution analyses by MALDIimaging-HRMS. Using a thin film of labeled agar supplemented with [1-13 C]-L-proline, [methyl-D 3 ]-L-methionine, 15 NH 4 Cl, or [ 15 N]-L-serine overlaid on unlabeled agar, we demonstrate the incorporation of labeled precursors into prodiginines and serratamolides produced by an endophytic bacterium, Serratia marcescens, by MALDI-imaging-HRMS and HPLC−HRMS. Further, we show the incorporation of CD 3 into prodigiosin as well as its characteristic fragments directly by MALDI-imaging-HRMS 2 . Our methodology has several advantages over currently existing techniques. First, both labeled and unlabeled compounds can be visualized simultaneously in high spatial resolution along with their labeled and unlabeled precursors. Second, by using a thin film of labeled agar, we utilize minimum amounts of expensive labeled compounds (1−3 mg) ensuring a cost-effective method for investigating biosynthetic pathways. Finally, our method allows in situ visualization and identification of target and nontarget compounds without the need of isolating the compounds. This is important for compounds that are produced by microorganisms in low, physiologically, or ecologically relevant concentrations.

Image to insight: exploring natural products through mass spectrometry imaging

“…A labelled precursor is usually administered to the organism to conrm the presence of certain precursor-derived metabolites detected by MSI according to their labelling patterns and spatial distribution. 88,89 It can be also applied in an untargeted manner to potentially detect all precursor-derived metabolites. For instance, Feldberg et al developed a novel approach that integrates in vivo dual tracer-based SIL with LCMS and MALDI imaging (termed DLEMMA-MALDI-imaging) for metabolite identication and metabolic network reconstruction.…”

Applications of stable isotopes in MALDI imaging: current approaches and an eye on the future

“…Using a stable isotope labelling by amino acids (SILAC) MALDI-IMS approach, the production of prodiginines and serratamolides, which can mediate the motility of bacterial colonies, has been studied in Serratia marcescens, an endophytic bacterium commonly found in soil and water (Fig. 4) [78]. By supplementing the agar with 13 C-L-proline, methyl-D 3 -L-methionine, and 15 N-L-serine, newly synthesised compounds that incorporated the isotopic labels, alongside the older, unlabelled compounds, were simultaneously observed and localised to specific regions of the bacterial colonies.…”

“…In addition to natural supplementation strategies, SIL compounds have also been introduced into biological systems via experimental interventions, such as intravenous injections [82,83] or intraperitoneal [71,84] administration. While these studies are also designed to monitor metabolic processes [78], Copyright (2018), with permission from the American Chemical Society (ACS). This figure is sourced from https://pubs.acs.org/doi/10.…”

Local metabolism in preclinical disease models studied with mass spectrometry imaging

“…While MALDI-MSI dramatically increases the specificity of metabolites detection [221], it is, due to its invasiveness, limited to providing a single and static snapshot of highly dynamic systems in which molecules are constantly synthesized and consumed [81]. Using stable isotope labeling of compounds such as lipids [123,494], amino acids [490,491,495] and glucose [496,497] in MALDI-MSI combined with longitudinal animal studies allows tracing metabolic processes in which the labeled molecules are involved in a time-resolved manner. This is achieved by detecting the mass-shifted signals containing the isotopic tracer or the products of its metabolic conversion, and thus adds an extra dimension to the understanding of both normal metabolic processes and pathological processes [221].…”

“…This is achieved by detecting the mass-shifted signals containing the isotopic tracer or the products of its metabolic conversion, and thus adds an extra dimension to the understanding of both normal metabolic processes and pathological processes [221]. MALDI-MSI has been used to map the uptake and metabolism of stable isotope labeled nutrients and other compounds in endophytic bacterium [495], radish plants [498], cultured hippocampal neurons [499], mouse lungs [494], mouse spleen [500], mouse liver [491], mouse brain [497,501], non-small cell lung carcinoma in mice [490], atherosclerotic mouse aorta [123], acute myocardial ischaemic mouse heart [497], and normal bovine lenses [496].…”