Abstract
Paper spray ionization mass spectrometry (PSI-MS) is a direct MS analysis technique with several reported bacterial metabolomics applications. As with most MS-based bacterial studies, all currently reported PSI-MS bacterial analyses have focused on the chemical signatures of the cellular unit. One dimension of the bacterial metabolome that is often lost in such analyses is the exometabolome (extracellular metabolome), including secreted metabolites, lipids, and peptides. A key component of the bacterial exometabolome that is gaining increased attention in the microbiology and biomedical communities is extracellular vesicles (EVs). These excreted structures, produced by cells in all domains of life, contain a variety of biomolecules responsible for a wide array of cellular functions, thus representing a core component of the bacterial secreted metabolome. Although previously examined using other MS approaches, no reports currently exist for a PSI-MS analysis of bacterial EVs, nor EVs from any other organism (exosomes, ectosomes, etc.). PSI-MS holds unique analytical strengths over other commonly used MS platforms and could thus provide an advantageous approach to EV metabolomics. To address this, we report a novel application representing, to our knowledge, the first PSI-MS analysis of EVs from any organism (using the human gut resident Oxalobacter formigenes as the experimental model, a bacterium whose EVs were never previously investigated). In this report, we show how we isolated and purified EVs from bacterial culture supernatant by EV-specific affinity chromatography, confirmed and characterized these vesicles by nanoparticle tracking analysis, analyzed the EV isolate by PSI-MS, and identified a panel of EV-derived metabolites, lipids, and peptides. This work serves as a pioneering study in the field of MS-based EV analysis and provides a new, rapid, sensitive, and economical approach to EV metabolomics.
Highlights
The extracellular vesicles (EVs) particle size reported in this work should be taken only as a general reference as the effect of different media conditions, biotic and abiotic stressors, and other factors on the O. formigenes EV profile have not been investigated. This serves as the first confirmation that O. formigenes produces these vesicles, and further work is needed to understand the specifics of their biological nature
We demonstrated a novel application of Paper spray ionization mass spectrometry (PSI-MS) to the analysis of EVs by examining a bacterial EV isolate from O. formigenes culture supernatant, a bacterium whose EVs had never previously been investigated nor confirmed
Our future endeavors to build upon the results from this investigation will mainly focus on the following: (1) confirming putative identifications assigned to EV features, which will require use of MS2 /MSn and comparison of fragmentation spectra to pure standards, and
Summary
PSI-MS offers several advantages over conventional liquid chromatography (LC)-MS approaches, including minimal sample volume, reduced or eliminated dependence on extraction or other sample preparation, and shortened analysis time [2]. In most microbiology-focused metabolomics experiments, bacterial cells are often separated from their conditioned medium matrix and washed prior to MS analysis [19,20] Such experiments provide a chemical characterization of the cellular unit, but information regarding the secreted metabolome, including all extracellular bacterium-derived metabolites, lipids, and peptides, is lost. This dimension of the molecular profile, often referred to as the metabolic footprint [21], is valuable to a bacterial metabolomics experiment as many microbes produce compounds that are predominantly secreted that may not be detected and identified in a strictly cellular analysis [22]. The bacterial exometabolome is a major factor of the microbiome-derived exposome [24], making its characterization imperative to understand both the bacterium itself and its host-microbe biochemical relationship
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