bioRxiv preprint doi: https://doi.org/10.1101/824888; this version posted October 30, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 1 Title: Native Electrospray-based Metabolomics Enables the Detection of Metal-binding 2 Compounds 3 4 Allegra Aron1,2, Daniel Petras1,2,3, Robin Schmid4, Julia M. Gauglitz,1,2 Isabell Büttel5, Luis 5 Antelo6, Hui Zhi7, Christina C. Saak10, Kien P. Malarney10, Eckhard Thines5,6, Rachel J. 6 Dutton9,10, Manuela Raffatellu7,8,9, and Pieter C. Dorrestein1,3,9,11 7 8 Affiliations: 9 1Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San 10 Diego, La Jolla, CA 92093, USA 11 2Collaborative Mass Spectrometry Innovation Center, University of California, San Diego, La 12 Jolla, CA 92093, USA 13 3Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA 14 92093, USA 15 4Institute of Inorganic and Analytical Chemistry, University of Münster, Corrensstr. 30, 16 Münster, D-48149, Germany 17 5Institute of Molecular Physiology, Microbiology and Wine Research, Johannes Gutenberg- 18 University Mainz, Johann-Joachim-Becherweg 15, Mainz, D-55128, Germany 19 6Institute of Biotechnology and Drug Research (IBWF gGmbH), Erwin-Schrödinger-Str. 56, 20 Kaiserslautern, D-67663, Germany 21 7Division of Host-Microbe Systems & Therapeutics, Department of Pediatrics, University of 22 California San Diego, La Jolla, CA 92093, USA 23 8Chiba University-University of California San Diego Center for Mucosal Immunology, 24 Allergy, and Vaccines (CU-UCSD cMAV), La Jolla, CA 92093, United States of America 25 9Center for Microbiome Innovation, University of California San Diego, La Jolla, CA 92093, 26 USA 27 10Division of Biological Sciences, University of California, San Diego, 95000 Gilman Dr, La 28 Jolla, CA 92093, USA 29 30 Author contributions: 31 ATA, DP, RS, and PCD developed the concept. 32 ATA, JMG, DP ran mass spectrometry experiments. 33 RS wrote code and provided feedback. 34 JMG, IB, LA, ET, HZ, CCS, KPM, RJD and MR cultured organisms and prepared samples. 35 ATA, DP and PCD wrote and edited the manuscript. 36 All authors edited and approved the manuscript 37 38 Abstract 39 Metals are essential for the molecular machineries of life, and microbes have evolved a 40 variety of small molecules to acquire, compete for, and utilize metals. Systematic methods 41 for the discovery of metal-small molecule complexes from biological samples are limited. 42 Here we describe a two-step native electrospray ionization mass spectrometry method, in 43 which double-barrel post-column metal-infusion and pH adjustment is combined with ion 44 identity molecular networking, a rule-based informatics workflow. This method can be used 45 to identify metal-binding compounds in complex samples based on defined mass (m/z) 46 offsets of ion features with the same chromatographic profiles. As this native metal 47 metabolomics approach can be easily implemented on any liquid chromatography-based bioRxiv preprint doi: https://doi.org/10.1101/824888; this version posted October 30, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 48 mass spectrometry system, this method has the potential to become a key strategy for 49 elucidating and understanding the role of metal-binding molecules in biology. 50 51 Main 52 Life, as we know it, cannot exist without metals. Metals are essential cofactors in 53 important biochemical reactions such as DNA replication and repair (iron and manganese), 54 respiration (iron and copper), photosynthesis (iron and manganese), and biosynthesis of 55 countless primary and secondary metabolites (iron, zinc, vanadium, molybdenum, 56 magnesium, calcium, etc.). One common strategy for metal acquisition in microorganisms is 57 through the production and secretion of small molecule ionophores that bind and form non- 58 covalent metal complexes. These complexes bind to specific ionophore receptors for their 59 uptake and release of metal into the cytoplasm. Siderophores are high affinity chelators of 60 ferric iron (Fe3+) and include ferrioxamines (i.e. desferrioxamines B and E)2-5, catecholates 61 (i.e. enterobactin)2-4, and carboxylates (i.e. rhizoferrin and aerobactin)2-5, while chalkophores 62 such as methanobactins and SF2768 (a diisonitrile natural product) play the analogous role 63 in binding cuprous copper (Cu+)6,7, and a number of zincophores have also been recently 64 elucidated8-11. In addition to small molecule ionophores that play a role in microbial metal 65 transport and homeostasis, metals can also serve as cofactors essential for the function of 66 other small molecules. Examples include iron in heme and magnesium in chlorophyll, 67 calcium and magnesium in metal-dependent antibiotics12-14, and cobalt in the vitamin 68 cobalamin (vitamin B12). 69 Although metal-binding small molecules have a variety of biological functions and 70 many potential biomedical applications, it is currently challenging to find metal-binding 71 compounds present in complex mixtures such as microbial culture extracts15, dissolved 72 organic matter16, or fecal extracts17, and it is additionally challenging to assess metal-binding 73 preferences. Predictions of small molecule structures using genome mining strategies have 74 improved tremendously in recent years18,19; however, it is still not possible to predict the 75 selectivity and affinity of metal coordination sites, and it is even difficult to predict whether a 76 small molecule contains a metal-binding site20,21. This is because small molecule metal- 77 binding sites are diverse and not conserved; as such, metal binding must be experimentally 78 established. Various analytical methods including inductively coupled plasma mass 79 spectrometry (ICP-MS)22, atomic absorption spectroscopy (AAS)23, x-ray fluorescence 80 spectroscopy (XRF)24, UV-visible (UV-vis) absorption spectroscopy and nuclear magnetic 81 resonance (NMR) spectroscopy in addition to multimodal approaches such as high- 82 performance matrix-assisted laser desorption/ionization Fourier transform ion cyclotron 83 resonance imaging mass spectrometry (MALDI FT-ICR IMS)25 can be used to analyze metal 84 content and/or metal coordination. With each of these methods, it is still difficult to 85 understand which molecules form metal complexes within a biological matrix that contains a 86 pool of candidate metal ions, especially when the identities of metal-binding species are not 87 known ahead of time. To address this shortcoming, we set out to develop a non-targeted 88 liquid chromatography-tandem mass spectrometry (LC-MS/MS) based approach. 89 Screening for metal-binding compounds from complex biological matrices by non- 90 targeted LC-MS/MS requires specific conditions to be met. Typical conditions used during 91 sample extraction, preparation, and chromatography for non-targeted mass spectrometry 92 involve low pH and high percentages of organic solvent, which both disfavor metal 93 complexation. Even when a metal-bound species is observed, it commonly elutes at a 94 different retention time than the apo (unbound) species and typically differs significantly in its 95 MS/MS fragmentation behavior than the apo species. This inherent problem of conserving bioRxiv preprint doi: https://doi.org/10.1101/824888; this version posted October 30, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 96 metal-ionophore complexes, in addition to differing retention times and differing gas phase 97 fragmentation behavior (MS/MS) between apo- and metal-bound forms make it difficult to 98 connect these forms to identify a given (metal specific) mass (m/z) offset. In order to 99 overcome these limitations, we developed a two-step native electrospray ionization (ESI) 100 MS/MS workflow, in which post-column metal infusion is coupled with pH adjustment via a 101 double barrel syringe pump (Figure 1a) to directly detect candidate small molecules that 102 bind to metals. 103 After the implementation of post-column metal infusion that re-forms metal-ionophore 104 complexes, one of the key challenges in developing this method was how to identify these 105 metal-binding complexes within large datasets and/or complex mixtures that typically contain 106 thousands of features. To overcome this limitation, a rule-based informatic workflow called 107 ion identity molecular networking (IIN) was utilized (Figure 1b). Native ESI metal infusion 108 coupled with IIN has enabled the systematic detection of candidate metal-binding molecules 109 and metal-binding properties in different samples, such as bacterial and fungal culture 110 extracts. 111 112 Figure 1. Overview of the native spray small molecule binding experiment. (a) The two-step 113 native ESI-MS/MS workflow utilizes a post-column infusion of ammonium hydroxide solution followed 114 by infusion of metal salt solution. (b) Data can be analyzed using a computational ion identity 115 molecular networking (IIN) workflow in MZmine and GNPS. bioRxiv preprint doi: https://doi.org/10.1101/824888;