Michael J. Meehan

Molecular cartography of the human skin surface in 3D

Significance The paper describes the implementation of an approach to study the chemical makeup of human skin surface and correlate it to the microbes that live in the skin. We provide the translation of molecular information in high-spatial resolution 3D to understand the body distribution of skin molecules and bacteria. In addition, we use integrative analysis to interpret, at a molecular level, the large scale of data obtained from human skin samples. Correlations between molecules and microbes can be obtained to further gain insights into the chemical milieu in which these different microbial communities live. The human skin is an organ with a surface area of 1.5–2 m2 that provides our interface with the environment. The molecular composition of this organ is derived from host cells, microbiota, and external molecules. The chemical makeup of the skin surface is largely undefined. Here we advance the technologies needed to explore the topographical distribution of skin molecules, using 3D mapping of mass spectrometry data and microbial 16S rRNA amplicon sequences. Our 3D maps reveal that the molecular composition of skin has diverse distributions and that the composition is defined not only by skin cells and microbes but also by our daily routines, including the application of hygiene products. The technological development of these maps lays a foundation for studying the spatial relationships of human skin with hygiene, the microbiota, and environment, with potential for developing predictive models of skin phenotypes tailored to individual health.

Capturing bacterial metabolic exchange using thin film desorption electrospray ionization-imaging mass spectrometry.

Over 60% of current pharmaceutical drugs have origins in natural products. To expand on current methods allowing one to characterize natural products directly from bacterial culture, herein we describe the use of desorption electrospray ionization (DESI) imaging mass spectrometry in monitoring the exchange of secondary metabolites between Bacillus subtilis and Streptomyces coelicolor using a simple imprinting technique.

Acyltransferase mediated polyketide release from a fungal megasynthase.

LovF is a highly reducing polyketide synthase (HR-PKS) from the filamentous fungus Aspergillus terreus. LovF synthesizes the alpha-S-methylbutyrate side chain that is subsequently transferred to monacolin J to yield the cholesterol-lowering natural product lovastatin. In the report, we expressed the full length LovF and reconstituted the megasynthase activities in vitro. We confirmed the diketide product of LovF is offloaded from the LovF ACP domain by the dissociated acyltransferase LovD. This represents the first example of acyltransferase-mediated release of polyketide products from fungal PKSs. We determined LovD primarily interacts with the ACP domain of LovF and the protein-protein interactions lead to highly efficient transfer of the diketide product. The catalytic efficiency is enhanced nearly 1 x 10(6)-fold when LovF was used as the acyl carrier instead of N-acetylcysteamine. Reconstitution and characterization of the LovF offloading mechanism provide new insights into the functions of fungal HR-PKS.

Connecting Chemotypes and Phenotypes of Cultured Marine Microbial Assemblages by Imaging Mass Spectrometry

From the early days of bacterial culturing over a century ago, microbiologists have known that microorganisms respond to their surroundings. Unicellular organisms rely on metabolic exchange to adapt to environmental stresses, sense colony density, occupy niches within hosts and form biofilms.1-7 For example, Bacillus subtilis utilizes metabolic exchange to lyse neighboring microbes, including siblings, during sporulation.8-10 While other forms of metabolic exchange, such as siderophores, stimulate the growth and development of Streptomyces and uncultured bacteria.11, 12 Despite the importance of chemistry in biology, studies that connect chemotypes and phenotypes to adaptive microbial behavior in Petri-dishes, including signaling and chemical warfare, have largely been disconnected and measured indirectly. To connect the chemotypes and phenotypes in this study, MALDI-based imaging mass spectrometry (IMS)13-16 was utilized to observe the chemical output and metabolic exchange of a marine microbial assemblage in two-dimensions. The capability to monitor the 2-dimensional distribution of a wide array of metabolites simultaneously from a complex mixture of distinct organisms opens the door to comprehensive analyses of interspecies signaling interactions within a microbial assemblage in a spatial fashion. Analyzing the spatial distribution of these metabolites enables one to generate a testable hypothesis, without the immediate need to know the structural characteristics, with respect to functions of the observed chemotypes. IMS provides the ability to correlate the presence of metabolites to phenotypic changes and to detect new chemotypes and/or phenotypes that cannot be observed by the naked eye. As such, IMS allows one to prioritize the molecules to be targeted for identification through proteomic and metabolomic approaches, or to be subjected to mass spectrometry guided isolation and nuclear magnetic resonance based structure elucidation. Understanding these molecular networks and interactions will illuminate how microbes respond to neighboring organisms and in turn influence and alter growth of their neighbors.

A highly efficient, high-throughput lipidomics platform for the quantitative detection of eicosanoids in human whole blood

We have developed an ultra-performance liquid chromatography-multiple reaction monitoring/mass spectrometry (UPLC-MRM/MS)-based, high-content, high-throughput platform that enables simultaneous profiling of multiple lipids produced ex vivo in human whole blood (HWB) on treatment with calcium ionophore and its modulation with pharmacological agents. HWB samples were processed in a 96-well plate format compatible with high-throughput sample processing instrumentation. We employed a scheduled MRM (sMRM) method, with a triple-quadrupole mass spectrometer coupled to a UPLC system, to measure absolute amounts of 122 distinct eicosanoids using deuterated internal standards. In a 6.5-min run, we resolved and detected with high sensitivity (lower limit of quantification in the range of 0.4-460 pg) all targeted analytes from a very small HWB sample (2.5 μl). Approximately 90% of the analytes exhibited a dynamic range exceeding 1000. We also developed a tailored software package that dramatically sped up the overall data quantification and analysis process with superior consistency and accuracy. Matrix effects from HWB and precision of the calibration curve were evaluated using this newly developed automation tool. This platform was successfully applied to the global quantification of changes on all 122 eicosanoids in HWB samples from healthy donors in response to calcium ionophore stimulation.

Molecular Networking As a Drug Discovery, Drug Metabolism, and Precision Medicine Strategy

Molecular networking is a tandem mass spectrometry (MS/MS) data organizational approach that has been recently introduced in the drug discovery, metabolomics, and medical fields. The chemistry of molecules dictates how they will be fragmented by MS/MS in the gas phase and, therefore, two related molecules are likely to display similar fragment ion spectra. Molecular networking organizes the MS/MS data as a relational spectral network thereby mapping the chemistry that was detected in an MS/MS-based metabolomics experiment. Although the wider utility of molecular networking is just beginning to be recognized, in this review we highlight the principles behind molecular networking and its use for the discovery of therapeutic leads, monitoring drug metabolism, clinical diagnostics, and emerging applications in precision medicine.

Biochemical Establishment and Characterization of EncM's Flavin-N5-oxide Cofactor

The ubiquitous flavin-dependent monooxygenases commonly catalyze oxygenation reactions by means of a transient C4a-peroxyflavin. A recent study, however, suggested an unprecedented flavin-oxygenating species, proposed as the flavin-N5-oxide (Fl(N5[O])), as key to an oxidative Favorskii-type rearrangement in the biosynthesis of the bacterial polyketide antibiotic enterocin. This stable superoxidized flavin is covalently tethered to the enzyme EncM and converted into FADH2 (Fl(red)) during substrate turnover. Subsequent reaction of Fl(red) with molecular oxygen restores the postulated Fl(N5[O]) via an unknown pathway. Here, we provide direct evidence for the Fl(N5[O]) species via isotope labeling, proteolytic digestion, and high-resolution tandem mass spectrometry of EncM. We propose that formation of this species occurs by hydrogen-transfer from Fl(red) to molecular oxygen, allowing radical coupling of the formed protonated superoxide and anionic flavin semiquinone at N5, before elimination of water affords the Fl(N5[O]) cofactor. Further biochemical and spectroscopic investigations reveal important features of the Fl(N5[O]) species and the EncM catalytic mechanism. We speculate that flavin-N5-oxides may be intermediates or catalytically active species in other flavoproteins that form the anionic semiquinone and promote access of oxygen to N5.

α,β → β,γ double bond migration in corallopyronin A biosynthesis

In polyketide biosynthesis the reduction of β-carbonyl groups to an alkene usually results in a α,β double bond. However, in a few antibiotics the rare case of such a carbon–carbon double bond in β,γ position is observed. The in vivo active antibiotic corallopyronin A represents such a molecule, whereby a α,β → β,γ double bond migration takes place during the assembly of the molecule. Here we report the in vitro analysis of the enzyme domain responsible for this double bond isomerization. This “shift domain” was heterologously expressed and assayed with its acyl carrier protein bound substrate. To facilitate this analysis the biosynthetic corallopyronin A intermediate was chemically synthesized as a SNAC-derivative. Enzyme activity was analyzed by NMR and high-resolution MS measurements, the latter enabled by performing the assay in deuterated buffer. Mutated enzyme variants gave first experimental evidence for the essential amino acids involved in double bond migration. These results further support the proposed corallopyronin A biosynthesis.

Direct detection of fungal siderophores on bats with white-nose syndrome via fluorescence microscopy-guided ambient ionization mass spectrometry.

White-nose syndrome (WNS) caused by the pathogenic fungus Pseudogymnoascus destructans is decimating the populations of several hibernating North American bat species. Little is known about the molecular interplay between pathogen and host in this disease. Fluorescence microscopy ambient ionization mass spectrometry was used to generate metabolic profiles from the wings of both healthy and diseased bats of the genus Myotis. Fungal siderophores, molecules that scavenge iron from the environment, were detected on the wings of bats with WNS, but not on healthy bats. This work is among the first examples in which microbial molecules are directly detected from an infected host and highlights the ability of atmospheric ionization methodologies to provide direct molecular insight into infection.

Mass Spectrometry-Based Visualization of Molecules Associated with Human Habitats

The cars we drive, the homes we live in, the restaurants we visit, and the laboratories and offices we work in are all a part of the modern human habitat. Remarkably, little is known about the diversity of chemicals present in these environments and to what degree molecules from our bodies influence the built environment that surrounds us and vice versa. We therefore set out to visualize the chemical diversity of five built human habitats together with their occupants, to provide a snapshot of the various molecules to which humans are exposed on a daily basis. The molecular inventory was obtained through untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of samples from each human habitat and from the people that occupy those habitats. Mapping MS-derived data onto 3D models of the environments showed that frequently touched surfaces, such as handles (e.g., door, bicycle), resemble the molecular fingerprint of the human skin more closely than other surfaces that are less frequently in direct contact with humans (e.g., wall, bicycle frame). Approximately 50% of the MS/MS spectra detected were shared between people and the environment. Personal care products, plasticizers, cleaning supplies, food, food additives, and even medications that were found to be a part of the human habitat. The annotations indicate that significant transfer of chemicals takes place between us and our built environment. The workflows applied here will lay the foundation for future studies of molecular distributions in medical, forensic, architectural, space exploration, and environmental applications.