Peter Verhaert

Contributions that advance precisely this capacity, uniting mass spectrometry imaging (MSI) and complementary multimodal imaging approaches in service of spatially resolved microbial science.The four articles assembled here span the breadth of this emerging field, from foundational method development to clinical application. Situating these methodological and clinical advances within a broader biological framework, Meyers et al. provide a comprehensive review of the microbiome's role in cancer metastasis, surveying how specific bacterial taxa, including Fusobacterium nucleatum, Bacteroides fragilis, and Helicobacter pylori, may influence four key steps of the metastatic cascade through direct invasion, toxin secretion, and immune modulation. Critically, the review argues that resolving the microbiome's contribution to metastatic progression requires moving beyond compositional profiling toward spatially-resolved approaches. This includes MALDI-MSI, DESI-MSI, NanoSIMS, imaging mass cytometry, and spatial transcriptomics workflows that can map microbial communities, their metabolites, and their interactions with host cells directly within the tumor microenvironment.Taken together, these four contributions articulate a unifying conviction at the heart of this Research Topic: that the biology of microorganisms is inseparable from their spatial and molecular context, and that imaging technologies, specificically MSI and complementary multimodal imaging, are the instruments that make that context achievable. Whether the objective is to map a metabolite to a bacterial niche in the gut, to capture the metabolic signature of a pathogen in a living host cell, to identify an organism from a clinical isolate, or to chart the microbiome's influence across the stages of metastasis, each paper demonstrates that spatial resolution and molecular specificity are not technical luxuries but scientific necessities. This Research Topic reflects a broader shift in approaching the Biology of Microbes: from cataloguing the microbiome "who is present?", to understanding "where everybody is", "what they are doing", and, ultimately, "how microbial activities shape the biological systems and environment they inhabit".

We describe a method that allows high-resolution mass spectrometry (HRMS) imaging of metabolites in tissue sections from formaldehyde-fixed, paraffin-embedded (FFPE) biobanks. This top-down variant of MS imaging expands the molecular scope of mass spectrometry histochemistry (MSHC) from peptidomics to metabolomics. The method makes the vast archives of FFPE biobanks accessible for MSHC-based biomarker discovery research of not only small endogenous peptides but also (a subset of) metabolites. FFPE biobank tissues include well-documented clinical samples representing diseases with a high medical need and often presently not clinically diagnosable and/or curable. Our protocol starts with FFPE tissue sections prepared from samples procured from biobanks. We describe how to remove paraffin and coat the section with MALDI matrix while maximally reducing analyte delocalization or washout. We detail appropriate programming of the MSHC data acquisition and illustrate a way to process MSHC data (including conversion to the generic imzML format) and browse MSHC datasets. Finally, we show options to present the data in the form of annotated MSHC images.

Quercetin is known to reduce blood pressure (BP); however, its acute effects are unclear. We investigated the acute effects of quercetin on BP, aortic mechanical properties and vascular reactivity in female Sprague–Dawley (SD) rats. Hypertension was induced using L‐NAME (40 mg/kg/day). Quercetin (4.5 mg/kg) was administered intravenously. Mechanical properties of the aortae were measured by echo‐tracking in normotensive and hypertensive rats. L‐NAME and quercetin quantities in the aorta were determined using AP‐MALDI‐MSI. Vascular reactivity was performed in mesenteric and renal arteries. L‐NAME increased BP and PWVβ while decreasing strain. Quercetin decreased BP and ameliorated PWVβ in L‐NAME‐induced hypertensive rats. Ex vivo, the acetylcholine (ACh)‐induced increase in tension at 100 μM was reduced in renal arteries when exposed to quercetin while phenylephrine (Phe)‐induced contractile response was augmented. In quiescent rings of renal arteries incubated with L‐NAME (10 μM) and TRAM‐34 (1 μM), the ACh‐induced vasoconstrictions were inhibited by quercetin. Quercetin resulted in concentration‐dependent vasodilation in mesenteric arteries and increased its sensitivity to ACh‐induced relaxations. Quercetin lowered BP in L‐NAME‐induced hypertensive rats, likely due to changes in aortic mechanical properties and relaxation of resistance arteries. Further research is warranted to clarify the acute effects of quercetin on renal arteries in this hypertensive model.

Using our earlier developed protocol, mass spectrometry imaging of small endogenous peptides (and a selection of small metabolites) can be successfully performed directly in tissue sections of formaldehyde-fixed paraffin-embedded (FFPE) samples, such as those available in Homo sapiens biobanks. In analogy with immunohistochemistry (IHC) which employs antibodies as detection probes, this method was designated mass spectrometry histochemistry (MSHC) as it solely relies on (top-down) mass spectrometry for analyte detection. We demonstrate that MSHC enables the localization of cystine-containing neuropeptides in histological sections of human FFPE biobanked tissue and illustrate this on pituitary adenomas and non-diseased pituitary tissues archived for several years in an academic hospital pathology biobank. The instrumental setup consists of high-resolution mass spectrometers (several orbitrap systems and one dedicated hybrid TOF instrument) fitted with atmospheric pressure (AP) scanning matrix-assisted laser desorption/ionization MALDI. Currently, the best spatial resolution routinely achievable with such (MALDI) apparatus is 5m. The high mass spectrometric resolution obtained allows revealing the full isotope envelopes of the peptides. As such both reduced and oxidized cysteine-containing proteoforms of e.g., the neurosecretory nonapeptides vasopressin and oxytocin can be visualized in biobanked FFPE tissue, demonstrating yet a novel application of MSHC.

Formalin-fixed neuroendocrine tissues from American cockroaches (Periplaneta americana) embedded in paraffin more than 30 years ago were recently analyzed by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), to reveal the histological localization of more than 20 peptide ions. These represented protonated, and other cationic species of, at least, 14 known neuropeptides. The characterization of peptides in such historical samples was made possible by a novel sample preparation protocol rendering the endogenous peptides readily amenable to MSI analysis. The protocol comprises brief deparaffinization steps involving xylene and ethanol, and is further devoid of conventional aqueous washing, buffer incubations, or antigen retrieval steps. Endogenous secretory peptides that are typically highly soluble are therefore retained in-tissue with this protocol. The method is fully ‘top-down’, i.e., without laborious in situ enzymatic digestion that typically disturbs the detection of low-abundance endogenous peptides by MSI. Peptide identifications were supported by accurate mass, on-tissue tandem MS analyses, and by earlier MALDI-MSI results reported for freshly prepared P. americana samples. In contrast to earlier literature accounts stating that MALDI-MSI detection of endogenous peptides is possible only in fresh or freshly frozen tissues, or exceptionally in formalin-fixed, paraffin-embedded (FFPE) material of less than 1 year old, we demonstrate that MALDI-MSI works for endogenous peptides in FFPE tissue of up to 30 years old. Our findings put forward a useful method for digestion-free, high-throughput analysis of endogenous peptides from FFPE samples, and offer the potential for re-investigating archived and historically interesting FFPE material, such as those stored in hospital biobanks.

L‐fucose is a constituent of glycoconjugates in different organisms. Fucosidases catalyze the removal of fucose residues, and have been correlated to different physiological and pathological processes, such as fertilization, cancer, fucosidosis, and digestion in molluscs and ticks. An α‐l‐fucosidase sequence was identified from the transcriptome and proteome from the midgut diverticula of the synanthropic spider Nephilingis cruentata. In this article, we describe the isolation of this α‐l‐fucosidase and the characterization of its activity using substrates and inhibitors demonstrating different specificities among fucosidases. The enzyme had a K_m of 32 and 400 μM for 4‐methylumbelliferyl α‐l‐fucopyranoside and 4‐nitrophenyl α‐l‐fucopyranoside, respectively; and was unable to hydrolyze fucoidan. Nephilingis cruentata α‐l‐fucosidase was inhibited competitively by fucose and fuconojyrimycin. The fucosidase had two distinct pH optima even in the isolated form, due to oligomerization dependent on pH, as previously described to other fucosidases. Alignment and molecular homology modeling of the protein sequence with other fucosidases indicated that the active sites and catalytic residues were different, including residues involved in acid/base catalysis. Phylogenetic analysis showed, for the first time, gene‐duplication events for fucosidases in Arachnida species. All these data reveal that studies on fucosidases in organisms distinct from bacteria, fungi, and humans are important.

In this final chapter I project my personal perspective on the future of peptidomics. A bird’s eye view is shed on the discipline and a bid is made to frame it in the broader arena of the life sciences of tomorrow. Inferring from its present state-of-the-art and from the general direction of some evolutionary trends which are to be discerned, a case is made that peptidomics enjoys full ripeness as a young branch of science today, from which a bright future for the discipline can be predicted.

Venom and toxin samples derived from animal origins are a rich source of bioactive peptides. A high proportion of bioactive peptides that have been identified in venom contain one or more disulfide bridges, which are thought to stabilize tertiary structure, and therefore influence the peptides’ specificity and activity. In this chapter, we describe a label-free mass spectrometry-based screening workflow specifically to detect peptides that contain inter- and intramolecular disulfide bonds, followed by elucidation of their primary structure. This method is based on the determination of the normalized isotope shift (NIS) and the normalized mass defect (NMD) of peptides, two parameters which are heavily influenced by the presence of sulfur in a peptide, where cysteines are the main contributing residues. Using ant defensive secretions as an example, we describe the initial fractionation of the venom on strong cation exchange followed by nanoflow HPLC and mass spectrometry. High resolution zoom scan spectra of high-abundance peptides are acquired, allowing an accurate determination of both monoisotopic and average mass, which are essential for calculation of NMD and NIS. Candidate peptides exhibiting relative low NMD and high NIS values are selected for targeted de novo sequencing. By fine-tuning the collision energy for optimal fragmentation of each selected precursor ions, the full sequence of several novel inter- and intramolecular disulfide bond containing ant defensive peptides can be established.

Methanol is generally metabolized through a pathway initiated by a cobalamine-containing methanol methyltransferase by anaerobic methylotrophs (such as methanogens and acetogens), or through oxidation to formaldehyde using a methanol dehydrogenase by aerobes. Methanol is an important substrate in deep-subsurface environments, where thermophilic sulfate-reducing bacteria of the genus Desulfotomaculum have key roles. Here, we study the methanol metabolism of Desulfotomaculum kuznetsovii strain 17^T, isolated from a 3000-m deep geothermal water reservoir. We use proteomics to analyze cells grown with methanol and sulfate in the presence and absence of cobalt and vitamin B12. The results indicate the presence of two methanol-degrading pathways in D. kuznetsovii, a cobalt-dependent methanol methyltransferase and a cobalt-independent methanol dehydrogenase, which is further confirmed by stable isotope fractionation. This is the first report of a microorganism utilizing two distinct methanol conversion pathways. We hypothesize that this gives D. kuznetsovii a competitive advantage in its natural environment.