Native Electrospray-based Metabolomics Enables the Detection of Metal-binding Compounds

Abstract

38Metals are essential for the molecular machineries of life, and microbes have evolved a 39 variety of small molecules to acquire, compete for, and utilize metals. Systematic methods 40for the discovery of metal-small molecule complexes from biological samples are limited. 41Here we describe a two-step native electrospray ionization mass spectrometry method, in 42 which double-barrel post-column metal-infusion and pH adjustment is combined with ion 43 identity molecular networking, a rule-based informatics workflow. This method can be used 44to identify metal-binding compounds in complex samples based on defined mass (m/z) 45 offsets of ion features with the same chromatographic profiles. As this native metal 46 metabolomics approach can be easily implemented on any liquid chromatography-based 47

Ion Identity Molecular Networking in the GNPS Environment

“…Ion identity molecular networking can be used in combination with native ESI-based metabolomics 12 to find biologically-relevant metal-binding compounds or to elucidate metalbinding preferences of known or novel metal-binding molecules (Zhi, H. et al, submitted). One recent example in which IIMN was instrumental in understanding metal-binding and selectivity is yersiniabactin.…”

“…Here, the application of IIMN to identify post-column induced ion species can be particularly useful for the screening of biologically-relevant metal-binding compounds. In a native ESI-based metabolomics study, IIMN specifically revealed that the known siderophore yersiniabactin also acts as a zincophore (Supplementary Figure 3) 12 . In a dataset with 88 animal bile acid extracts, multiple smaller networks and unconnected nodes were combined to a large network of free bile acids and those conjugated to amino acids or sulfate, resulting in higher connectivity in the network (Supplementary Figure 4).…”

“…An exciting application of IIMN is the expansion of spectral libraries by (re)-processing public datasets and propagating spectral library annotations to create library entries of connected ion identities. The identification of ion adducts can reveal novel ionophores, some of which will be biologically relevant and are still underappreciated in the function of small molecules 12,13 . The integration into FBMN and the GNPS environment provided a platform to utilize IIMN in other related bioinformatics tools, e.g., SIRIUS 14 and CANOPUS 15 .…”

Siderophore-mediated zinc acquisition enhances enterobacterial colonization of the inflamed gut

“…To identify whether the ybt gene cluster produces a zincophore, we cultured EcN wildtype and the irp2 mutant in M9 minimal media and collected culture pellets and supernatants to then run ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). In addition to running UHPLC-MS/MS metabolomics on these samples, we performed experiments using post-liquid chromatography (LC) pH adjustment to 6.8 and infusion of a zinc acetate solution, followed by mass spectrometry, in order to assess whether any of the metabolites produced were capable of binding zinc 34 . This native spray metabolomics strategy is then combined with ion identity-based molecular networking, a new computational and data visualization strategy that allows for the discovery of mass spectrometry features with the same retention time and predictable mass offsets 34 ; mass spectrometry features with the same retention time and a mass difference resulting from zinc binding can be discovered directly from complex metabolomics samples.…”

“…After a genome search did not yield any promising candidate transporters, we hypothesized that EcN produces and secretes an unknown zincophore. Using our recently developed native spray metabolomics approach 34 , we subsequently discovered that EcN produces the siderophore yersiniabactin (Ybt), which is capable of binding zinc. Moreover, we demonstrate that EcN utilizes Ybt, in addition to the zinc transporters ZnuABC and ZupT, to effectively acquire zinc in vitro, to resist the antimicrobial activity of CP, and to colonize the inflamed gut.…”