What are we imaging? Software tools and experimental strategies for annotation and identification of small molecules in mass spectrometry imaging.

Mass spectrometry imaging (MSI) has become a widespread analytical technique to perform nonlabeled spatial molecular identification. The Achilles' heel of MSI is the annotation and identification of molecular species due to intrinsic limitations of the technique (lack of chromatographic separation and the difficulty to apply tandem MS). Successful strategies to perform annotation and identification combine extra analytical steps, like using orthogonal analytical techniques to identify compounds; with algorithms that integrate the spectral and spatial information. In this review, we discuss different experimental strategies and bioinformatics tools to annotate and identify compounds in MSI experiments. We target strategies and tools for small molecule applications, such as lipidomics and metabolomics. First, we explain how sample preparation and the acquisition process influences annotation and identification, from sample preservation to the use of orthogonal techniques. Then, we review twelve software tools for annotation and identification in MSI. Finally, we offer perspectives on two current needs of the MSI community: the adaptation of guidelines for communicating confidence levels in identifications; and the creation of a standard format to store and exchange annotations and identifications in MSI.

Lipidomics Reveals Myocardial Lipid Composition in a Murine Model of Insulin Resistance Induced by a High-Fat Diet.

Ectopic fat accumulation in non-adipose tissues is closely related to diabetes-related myocardial dysfunction. Nevertheless, the complete picture of the lipid metabolites involved in the metabolic-related myocardial alterations is not fully characterized. The aim of this study was to characterize the specific lipid profile in hearts in an animal model of obesity/insulin resistance induced by a high-fat diet (HFD). The cardiac lipidome profiles were assessed via liquid chromatography-mass spectrometry (LC-MS)/MS-MS and laser desorption/ionization-mass spectrometry (LDI-MS) tissue imaging in hearts from C57BL/6J mice fed with an HFD or standard-diet (STD) for 12 weeks. Targeted lipidome analysis identified a total of 63 lipids (i.e., 48 triacylglycerols (TG), 5 diacylglycerols (DG), 1 sphingomyelin (SM), 3 phosphatidylcholines (PC), 1 DihydroPC, and 5 carnitines) modified in hearts from HFD-fed mice compared to animals fed with STD. Whereas most of the TG were up-regulated in hearts from animals fed with an HFD, most of the carnitines were down-regulated, thereby suggesting a reduction in the mitochondrial ß-oxidation. Roughly 30% of the identified metabolites were oxidated, pointing to an increase in lipid peroxidation. Cardiac lipidome was associated with a specific biochemical profile and a specific liver TG pattern. Overall, our study reveals a specific cardiac lipid fingerprint associated with metabolic alterations induced by HFD.

Rapid Screening of Polyol Polyketides from Marine Dinoflagellates.

Dinoflagellate-derived polyketides are typically large molecules (>1000 Da) with complex structures, potent bioactivities, and high toxicities. Their discovery suffers three major bottlenecks: insufficient bioavailability, low-yield cultivation of producer organisms, and production of multiple highly related analogues by a single strain. Consequently, the biotechnological production of therapeutics or toxicological standards of dinoflagellate-derived polyketides is also hampered. Strategies based on sensitive and selective techniques for chemical prospection of dinoflagellate extracts could aid in overcoming these limitations, as it allows selecting the most interesting candidates for discovery and exploitation programs according to the biosynthetic potential. In this work, we assess the combination of data-dependent liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-HRMS2) and molecular networking to screen polyol polyketides. To demonstrate the power of this approach, we selected dinoflagellate Amphidinium carterae since it is commonly used as a biotechnological model and produces amphidinols, a family of polyol-polyene compounds with antifungal and antimycoplasmal activity. First, we screened families of compounds with multiple hydroxyl groups by examining MS2 profiles that contain sequential neutral losses of water. Then, we clustered MS2 spectra by molecular networking to facilitate the dereplication and discovery of amphidinols. Finally, we used the MS2 fragmentation behavior of well-characterized luteophanol D as a model to propose a structural hypothesis of nine novel amphidinols. We envision that this strategy is a valuable approach to rapidly monitoring toxin production of known and unknown polyol polyketides in dinoflagellates, even in small culture volumes, and distinguishing strains according to their toxin profiles.

SALDI-MS and SERS Multimodal Imaging: One Nanostructured Substrate to Rule Them Both.

Imaging techniques based on mass spectrometry or spectroscopy methods inform in situ about the chemical composition of biological tissues or organisms, but they are sometimes limited by their specificity, sensitivity, or spatial resolution. Multimodal imaging addresses these limitations by combining several imaging modalities; however, measuring the same sample with the same preparation using multiple imaging techniques is still uncommon due to the incompatibility between substrates, sample preparation protocols, and data formats. We present a multimodal imaging approach that employs a gold-coated nanostructured silicon substrate to couple surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) and surface-enhanced Raman spectroscopy (SERS). Our approach integrates both imaging modalities by using the same substrate, sample preparation, and data analysis software on the same sample, allowing the coregistration of both images. We transferred molecules from clean fingertips and fingertips covered with plasticine modeling clay onto our nanostructure and analyzed their chemical composition and distribution by SALDI-MS and SERS. Multimodal analysis located the traces of plasticine on fingermarks and provided chemical information on the composition of the clay. Our multimodal approach effectively combines the advantages of mass spectrometry and vibrational spectroscopy with the signal enhancing abilities of our nanostructured substrate.

Perspective on Multimodal Imaging Techniques Coupling Mass Spectrometry and Vibrational Spectroscopy: Picturing the Best of Both Worlds.

Studies on complex biological phenomena often combine two or more imaging techniques to collect high-quality comprehensive data directly in situ, preserving the biological context. Mass spectrometry imaging (MSI) and vibrational spectroscopy imaging (VSI) complement each other in terms of spatial resolution and molecular information. In the past decade, several combinations of such multimodal strategies arose in research fields as diverse as microbiology, cancer, and forensics, overcoming many challenges toward the unification of these techniques. Here we focus on presenting the advantages and challenges of multimodal imaging from the point of view of studying biological samples as well as giving a perspective on the upcoming trends regarding this topic. The latest efforts in the field are discussed, highlighting the purpose of the technique for clinical applications.

Multimodal Molecular Imaging and Identification of Bacterial Toxins Causing Mushroom Soft Rot and Cavity Disease.

Soft rot disease of edible mushrooms leads to rapid degeneration of fungal tissue and thus severely affects farming productivity worldwide. The bacterial mushroom pathogen Burkholderia gladioli pv. agaricicola has been identified as the cause. Yet, little is known about the molecular basis of the infection, the spatial distribution and the biological role of antifungal agents and toxins involved in this infectious disease. We combine genome mining, metabolic profiling, MALDI-Imaging and UV Raman spectroscopy, to detect, identify and visualize a complex of chemical mediators and toxins produced by the pathogen during the infection process, including toxoflavin, caryoynencin, and sinapigladioside. Furthermore, targeted gene knockouts and in vitro assays link antifungal agents to prevalent symptoms of soft rot, mushroom browning, and impaired mycelium growth. Comparisons of related pathogenic, mutualistic and environmental Burkholderia spp. indicate that the arsenal of antifungal agents may have paved the way for ancestral bacteria to colonize niches where frequent, antagonistic interactions with fungi occur. Our findings not only demonstrate the power of label-free, in vivo detection of polyyne virulence factors by Raman imaging, but may also inspire new approaches to disease control.

rMSIproc: an R package for mass spectrometry imaging data processing.

SUMMARY: Mass spectrometry imaging (MSI) can reveal biochemical information directly from a tissue section. MSI generates a large quantity of complex spectral data which is still challenging to translate into relevant biochemical information. Here, we present rMSIproc, an open-source R package that implements a full data processing workflow for MSI experiments performed using TOF or FT-based mass spectrometers. The package provides a novel strategy for spectral alignment and recalibration, which allows to process multiple datasets simultaneously. This enables to perform a confident statistical analysis with multiple datasets from one or several experiments. rMSIproc is designed to work with files larger than the computer memory capacity and the algorithms are implemented using a multi-threading strategy. rMSIproc is a powerful tool able to take full advantage of modern computer systems to completely develop the whole MSI potential. AVAILABILITY AND IMPLEMENTATION: rMSIproc is freely available at https://github.com/prafols/rMSIproc. CONTACT: pere.rafols@urv.cat. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Synergistic activity of cosecreted natural products from amoebae-associated bacteria.

Investigating microbial interactions from an ecological perspective is a particularly fruitful approach to unveil both new chemistry and bioactivity. Microbial predator-prey interactions in particular rely on natural products as signal or defense molecules. In this context, we identified a grazing-resistant Pseudomonas strain, isolated from the bacterivorous amoeba Dictyostelium discoideum. Genome analysis of this bacterium revealed the presence of two biosynthetic gene clusters that were found adjacent to each other on a contiguous stretch of the bacterial genome. Although one cluster codes for the polyketide synthase producing the known antibiotic mupirocin, the other cluster encodes a nonribosomal peptide synthetase leading to the unreported cyclic lipopeptide jessenipeptin. We describe its complete structure elucidation, as well as its synergistic activity against methicillin-resistant Staphylococcus aureus, when in combination with mupirocin. Both biosynthetic gene clusters are regulated by quorum-sensing systems, with 3-oxo-decanoyl homoserine lactone (3-oxo-C10-AHL) and hexanoyl homoserine lactone (C6-AHL) being the respective signal molecules. This study highlights the regulation, richness, and complex interplay of bacterial natural products that emerge in the context of microbial competition.

Raman2imzML converts Raman imaging data into the standard mass spectrometry imaging format.

BACKGROUND: Multimodal imaging that combines mass spectrometry imaging (MSI) with Raman imaging is a rapidly developing multidisciplinary analytical method used by a growing number of research groups. Computational tools that can visualize and aid the analysis of datasets by both techniques are in demand. RESULTS: Raman2imzML was developed as an open-source converter that transforms Raman imaging data into imzML, a standardized common data format created and adopted by the mass spectrometry community. We successfully converted Raman datasets to imzML and visualized Raman images using open-source software designed for MSI applications. CONCLUSION: Raman2imzML enables both MSI and Raman images to be visualized using the same file format and the same software for a straightforward exploratory imaging analysis.

Injury-Triggered Blueing Reactions of Psilocybe "Magic" Mushrooms.

Upon injury, psychotropic psilocybin-producing mushrooms instantly develop an intense blue color, the chemical basis and mode of formation of which has remained elusive. We report two enzymes from Psilocybe cubensis that carry out a two-step cascade to prepare psilocybin for oxidative oligomerization that leads to blue products. The phosphatase PsiP removes the 4-O-phosphate group to yield psilocin, while PsiL oxidizes its 4-hydroxy group. The PsiL reaction was monitored by in situ 13 C NMR spectroscopy, which indicated that oxidative coupling of psilocyl residues occurs primarily via C-5. MS and IR spectroscopy indicated the formation of a heterogeneous mixture of preferentially psilocyl 3- to 13-mers and suggest multiple oligomerization routes, depending on oxidative power and substrate concentration. The results also imply that phosphate ester of psilocybin serves a reversible protective function.

A Pair of Bacterial Siderophores Releases and Traps an Intercellular Signal Molecule: An Unusual Case of Natural Nitrone Bioconjugation.

In microbial interactions bacteria employ diverse molecules with specific functions, such as sensing the environment, communication with other microbes or hosts, and conferring virulence. Insights into the molecular basis of bacterial communication are thus of high relevance for ecology and medicine. Targeted gene activation and in vitro studies revealed that the cell-to-cell signaling molecule and disease mediator IQS (aeruginaldehyde) of the human pathogen Pseudomonas aeruginosa and related bacteria derives from the siderophore pyochelin. Addition of IQS to bacterial cultures (Burkholderia thailandensis) showed that the signaling molecule is captured by a congener of another siderophore family, malleobactin, to form a nitrone conjugate (malleonitrone) that is active against the IQS-producer. This study uncovers complex communication processes with derailed siderophore functions, a novel nitrone bioconjugation, and a new type of antibiotic against Gram-negative bacteria.

Induced Chemical Defense of a Mushroom by a Double-Bond-Shifting Polyene Synthase.

The antilarval mushroom polyenes 18-methyl-19-oxoicosaoctaenoic acid and 20-methyl-21-oxodocosanonaenoic acid appear in response to injury of the mycelium of the stereaceous mushroom BY1. We identified a polyketide synthase (PPS1) which belongs to a hitherto completely uncharacterized clade of polyketide synthases. Expression of the PPS1 gene is massively upregulated following mycelial damage. The synthesis of the above polyenes was reconstituted in the mold Aspergillus niger as a heterologous host. This demonstrates that PPS1 1) synchronously produces branched-chain polyketides of varied lengths, and 2) catalyzes the unprecedented shift of eight or nine double bonds. This study represents the first characterization of a reducing polyketide synthase from a mushroom. We also show that injury-induced de novo synthesis of polyketides is a fungal response strategy.

The novel ovatoxin-g and isobaric palytoxin (so far referred to as putative palytoxin) from Ostreopsis cf. ovata (NW Mediterranean Sea): structural insights by LC-high resolution MS(n.).

Blooms of the benthic dinoflagellate Ostreopsis cf. ovata are a concern in the Mediterranean Sea, since this species produces a wide range of palytoxin-like compounds listed among the most potent marine toxins. This study focused on two analogs of palytoxin found in cultures of six strains of O. cf. ovata isolated from the south of Catalonia (NW Mediterranean Sea). In addition to some already known ovatoxins, our strains produced two minor compounds, ovatoxin-g and the so far called putative palytoxin, whose structures had not been elucidated before. Insufficient quantity of these compounds impeded a full nuclear magnetic resonance (NMR)-based structural elucidation; thus, we studied their structure in crude algal extracts through liquid chromatography-electrospray ionization high-resolution mass spectrometry(n) (LC-ESI-HRMS(n)) in positive ion mode. Under the used MS conditions, the molecules underwent fragmentation at many sites of their backbone and a large number of diagnostic fragment ions were identified. As a result, tentative structures were assigned to both ovatoxin-g and the putative palytoxin, the latter being identified as a palytoxin isomer and re-named as  isobaric palytoxin.

Confirmation of pinnatoxins and spirolides in shellfish and passive samplers from Catalonia (Spain) by liquid chromatography coupled with triple quadrupole and high-resolution hybrid tandem mass spectrometry.

Cyclic imines are lipophilic marine toxins that bioaccumulate in seafood. Their structure comprises a cyclic-imino moiety, responsible for acute neurotoxicity in mice. Cyclic imines have not been linked yet to human poisonings and are not regulated in Europe, although the European Food Safety Authority requires more data to perform a conclusive risk assessment for consumers. This work presents the first detection of pinnatoxin G (PnTX-G) in Spain and 13-desmethyl spirolide C (SPX-1) in shellfish from Catalonia (Spain, NW Mediterranean Sea). Cyclic imines were found at low concentrations (2 to 60 µg/kg) in 13 samples of mussels and oysters (22 samples analyzed). Pinnatoxin G has been also detected in 17 seawater samples (out of 34) using solid phase adsorption toxin tracking devices (0.3 to 0.9 µg/kg-resin). Pinnatoxin G and SPX-1 were confirmed with both low and high resolution (<2 ppm) mass spectrometry by comparison of the response with that from reference standards. For other analogs without reference standards, we applied a strategy combining low resolution MS with a triple quadrupole mass analyzer for a fast and reliable screening, and high resolution MS LTQ Orbitrap® for unambiguous confirmation. The advantages and limitations of using high resolution MS without reference standards were discussed.

rMSIfragment: improving MALDI-MSI lipidomics through automated in-source fragment annotation.

Matrix-Assisted Laser Desorption Ionization Mass Spectrometry Imaging (MALDI-MSI) spatially resolves the chemical composition of tissues. Lipids are of particular interest, as they influence important biological processes in health and disease. However, the identification of lipids in MALDI-MSI remains a challenge due to the lack of chromatographic separation or untargeted tandem mass spectrometry. Recent studies have proposed the use of MALDI in-source fragmentation to infer structural information and aid identification. Here we present rMSIfragment, an open-source R package that exploits known adducts and fragmentation pathways to confidently annotate lipids in MALDI-MSI. The annotations are ranked using a novel score that demonstrates an area under the curve of 0.7 in ROC analyses using HPLC-MS and Target-Decoy validations. rMSIfragment applies to multiple MALDI-MSI sample types and experimental setups. Finally, we demonstrate that overlooking in-source fragments increases the number of incorrect annotations. Annotation workflows should consider in-source fragmentation tools such as rMSIfragment to increase annotation confidence and reduce the number of false positives.

Hypothalamic JNK1-hepatic fatty acid synthase axis mediates a metabolic rewiring that prevents hepatic steatosis in male mice treated with olanzapine via intraperitoneal: Additional effects of PTP1B inhibition.

Olanzapine (OLA), a widely used second-generation antipsychotic (SGA), causes weight gain and metabolic alterations when administered orally to patients. Recently, we demonstrated that, contrarily to the oral treatment which induces weight gain, OLA administered via intraperitoneal (i.p.) in male mice resulted in body weight loss. This protection was due to an increase in energy expenditure (EE) through a mechanism involving the modulation of hypothalamic AMPK activation by higher OLA levels reaching this brain region compared to those of the oral treatment. Since clinical studies have shown hepatic steatosis upon chronic treatment with OLA, herein we further investigated the role of the hypothalamus-liver interactome upon OLA administration in wild-type (WT) and protein tyrosine phosphatase 1B knockout (PTP1B-KO) mice, a preclinical model protected against metabolic syndrome. WT and PTP1B-KO male mice were fed an OLA-supplemented diet or treated via i.p. Mechanistically, we found that OLA i.p. treatment induces mild oxidative stress and inflammation in the hypothalamus in a JNK1-independent and dependent manner, respectively, without features of cell dead. Hypothalamic JNK activation up-regulated lipogenic gene expression in the liver though the vagus nerve. This effect concurred with an unexpected metabolic rewiring in the liver in which ATP depletion resulted in increased AMPK/ACC phosphorylation. This starvation-like signature prevented steatosis. By contrast, intrahepatic lipid accumulation was observed in WT mice treated orally with OLA; this effect being absent in PTP1B-KO mice. We also demonstrated an additional benefit of PTP1B inhibition against hypothalamic JNK activation, oxidative stress and inflammation induced by chronic OLA i.p. treatment, thereby preventing hepatic lipogenesis. The protection conferred by PTP1B deficiency against hepatic steatosis in the oral OLA treatment or against oxidative stress and neuroinflammation in the i.p. treatment strongly suggests that targeting PTP1B might be also a therapeutic strategy to prevent metabolic comorbidities in patients under OLA treatment in a personalized manner.

rMSIannotation: A peak annotation tool for mass spectrometry imaging based on the analysis of isotopic intensity ratios.

Mass spectrometry imaging (MSI) consist of spatially located spectra with thousands of peaks. Only a fraction of these peaks corresponds to unique monoisotopic peaks, as mass spectra include isotopes, adducts and fragments of compounds. Current peak annotation solutions depend on matching MS features to compounds libraries. We present rMSIannotation, a peak annotation algorithm to annotate carbon isotopes and adducts in metabolomics and lipidomics imaging mass spectrometry datasets without using supporting libraries. rMSIannotation measures and evaluates the intensity ratio between carbon isotopic peaks and models their distribution across the m/z axis of the compounds in the Human Metabolome Database. Monoisotopic peak selection is based on the isotopic likelihood score (ILS) made of three components: image morphology correlation, validation of isotopic intensity ratios, and peak centroid mass deviation. rMSIannotation proposes pairs of peaks that can be adducts based on three scores: isotopic pattern coherence, image correlation and mass error. We validated rMSIannotation with three MALDI-MSI datasets which were manually annotated by experts, and compared the annotations obtained with rMSIannotation and with the METASPACE annotation platform. rMSIannotation replicated more than 90% of the manual annotation reported in FT-ICR datasets and expanded the list of annotated compounds with additional monoisotopic peaks and neutral masses. Finally, we evaluated isotopic peak annotation as a data reduction method for MSI by comparing the results of PCA and k-means segmentation before and after removing non-monoisotopic peaks. The results show that monoisotopic peaks retain most of the biologic variance in the dataset.

Toxin profile of Ostreopsis cf. ovata from Portuguese continental coast and Selvagens Islands (Madeira, Portugal).

The toxigenic dinoflagellate Ostreopsis cf. ovata is known to produce a range of palytoxin (PLTX) - related compounds named ovatoxins (OVTX). O. cf. ovata presents a wide variability in toxin production and its toxic profile is strain-specific. Several OVTXs, denominated from -a to -h and -l have been reported from different strains of this benthic microalgae up to now, mainly in Mediterranean isolates. However, less is known about the toxin profile of the strains present in the Atlantic coasts of Europe. In this work, strains of O. cf. ovata isolated from the South coast of Portugal mainland (Algarve) and Selvagens Island (Madeira, Portugal) were cultured and tested for toxicity by hemolytic assay. Toxin profiles were qualitatively elucidated by Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS). The strain from Algarve presented lower toxic potency than the strain from Selvagens island (12.3 against 54.8 pg of PLTX equivalents per cell) showing in both cases the characteristic toxin profile of Mediterranean strains. The major component, OVTX-a, was concomitant with OVTX from -b to -g and isobaric PLTX. Regarding the morphological characteristics of these strains, as well as their toxin fingerprint, it is likely they are closely related to strains from Mediterranean coasts. The present study reports for the first time the occurrence of several OVTX congeners and iso-PLTX in O. cf. ovata from Portuguese waters. This study provides valuable information to characterize the risk of OVTXs-related outbreaks in Portugal.

Opisthobranch grazing results in mobilisation of spherulous cells and re-allocation of secondary metabolites in the sponge Aplysina aerophoba.

Sponges thrive in marine benthic communities due to their specific and diverse chemical arsenal against predators and competitors. Yet, some animals specifically overcome these defences and use sponges as food and home. Most research on sponge chemical ecology has characterised crude extracts and investigated defences against generalist predators like fish. Consequently, we know little about chemical dynamics in the tissue and responses to specialist grazers. Here, we studied the response of the sponge Aplysina aerophoba to grazing by the opisthobranch Tylodina perversa, in comparison to mechanical damage, at the cellular (via microscopy) and chemical level (via matrix-assisted laser desorption/ionization imaging mass spectrometry, MALDI-imaging MS). We characterised the distribution of two major brominated alkaloids in A. aerophoba, aerophobin-2 and aeroplysinin-1, and identified a generalised wounding response that was similar in both wounding treatments: (i) brominated compound-carrying cells (spherulous cells) accumulated at the wound and (ii) secondary metabolites reallocated to the sponge surface. Upon mechanical damage, the wound turned dark due to oxidised compounds, causing T. perversa deterrence. During grazing, T. perversa's way of feeding prevented oxidation. Thus, the sponge has not evolved a specific response to this specialist predator, but rather relies on rapid regeneration and flexible allocation of constitutive defences.

Gold Nanoparticle-Assisted Black Silicon Substrates for Mass Spectrometry Imaging Applications.

Mass spectrometry imaging (MSI) based on matrix-assisted laser desorption ionization (MALDI) is widely used in proteomics. However, matrix-free technologies are gaining popularity for detecting low molecular mass compounds. Small molecules were analyzed with nanostructured materials as ionization promoters, which produce low-to-no background signal, and facilitate enhanced specificity and sensitivity through functionalization. We investigated the fabrication and the use of black silicon and gold-coated black silicon substrates for surface-assisted laser desorption/ionization mass spectrometry imaging (SALDI-MSI) of animal tissues and human fingerprints. Black silicon was created using dry etching, while gold nanoparticles were deposited by sputtering. Both methods are safe for the user. Physicochemical characterization and MSI measurements revealed the optimal properties of the substrates for SALDI applications. The gold-coated black silicon worked considerably better than black silicon as the LDI-MSI substrate. The substrate was also compatible with imprinting, as a sample-simplification method that allows efficient transference of metabolites from the tissues to the substrate surface, without compound delocalization. Moreover, by modifying the surface with hydrophilic and hydrophobic groups, specific interactions were stimulated between surface and sample, leading to a selective analysis of molecules. Thus, our substrate facilitates targeted and/or untargeted in situ metabolomics studies for various fields such as clinical, environmental, forensics, and pharmaceutical research.