Advancing rare cancer research by MALDI mass spectrometry imaging: Applications, challenges, and future perspectives in sarcoma.

MALDI mass spectrometry imaging (MALDI imaging) uniquely advances cancer research, by measuring spatial distribution of endogenous and exogenous molecules directly from tissue sections. These molecular maps provide valuable insights into basic and translational cancer research, including tumor biology, tumor microenvironment, biomarker identification, drug treatment, and patient stratification. Despite its advantages, MALDI imaging is underutilized in studying rare cancers. Sarcomas, a group of malignant mesenchymal tumors, pose unique challenges in medical research due to their complex heterogeneity and low incidence, resulting in understudied subtypes with suboptimal management and outcomes. In this review, we explore the applicability of MALDI imaging in sarcoma research, showcasing its value in understanding this highly heterogeneous and challenging rare cancer. We summarize all MALDI imaging studies in sarcoma to date, highlight their impact on key research fields, including molecular signatures, cancer heterogeneity, and drug studies. We address specific challenges encountered when employing MALDI imaging for sarcomas, and propose solutions, such as using formalin-fixed paraffin-embedded tissues, and multiplexed experiments, and considerations for multi-site studies and digital data sharing practices. Through this review, we aim to spark collaboration between MALDI imaging researchers and clinical colleagues, to deploy the unique capabilities of MALDI imaging in the context of sarcoma.

MALDI Imaging Assisted Discovery of a Di-O-glycosyltransferase from Platycodon grandiflorum Root.

A matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) assisted genome mining strategy was developed for the discovery of glycosyltransferase (GT) from the root of Platycodon grandiflorum. A di-O-glycosyltransferase PgGT1 was discovered and characterized that is capable of catalyzing platycoside E (PE) synthesis through the attachment of two ß-1,6-linked glucosyl residues sequentially to the glucosyl residue at the C3 position of platycodin D (PD). Although UDP-glucose is the preferred sugar donor for PgGT1, it could also utilize UDP-xylose and UDP-N-acetylglucosamine as weak donors. Residues S273, E274, and H350 played important roles in stabilizing the glucose donor and positioning the glucose in the optimal orientation for the glycosylation reaction. This study clarified two key steps involved in the biosynthetic pathway of PE and could greatly contribute to improving its industrial biotransformation.

An optimized MALDI MSI protocol for spatial detection of tryptic peptides in fresh frozen prostate tissue.

MALDI MS imaging (MSI) is a powerful analytical tool for spatial peptide detection in heterogeneous tissues. Proper sample preparation is crucial to achieve high quality, reproducible measurements. Here we developed an optimized protocol for spatially resolved proteolytic peptide detection with MALDI time-of-flight MSI of fresh frozen prostate tissue sections. The parameters tested included four different tissue washes, four methods of protein denaturation, four methods of trypsin digestion (different trypsin densities, sprayers, and incubation times), and five matrix deposition methods (different sprayers, settings, and matrix concentrations). Evaluation criteria were the number of detected and excluded peaks, percentage of high mass peaks, signal-to-noise ratio, spatial localization, and average intensities of identified peptides, all of which were integrated into a weighted quality evaluation scoring system. Based on these scores, the optimized protocol included an ice-cold EtOH+H2 O wash, a 5 min heating step at 95°C, tryptic digestion incubated for 17h at 37°C and CHCA matrix deposited at a final amount of 1.8 µg/mm2 . Including a heat-induced protein denaturation step after tissue wash is a new methodological approach that could be useful also for other tissue types. This optimized protocol for spatial peptide detection using MALDI MSI facilitates future biomarker discovery in prostate cancer and may be useful in studies of other tissue types.

Spatial quantitation of antibiotics in bone tissue compartments by laser-capture microdissection coupled with UHPLC-tandem mass spectrometry.

Bones are the site of multiple diseases requiring chemotherapy, including cancer, arthritis, osteoporosis and infections. Yet limited methodologies are available to investigate the spatial distribution and quantitation of small molecule drugs in bone compartments, due to the difficulty of sectioning undecalcified bones and the interference of decalcification methods with spatially resolved drug quantitation. To measure drug concentrations in distinct anatomical bone regions, we have developed a workflow that enables spatial quantitation of thin undecalcified bone sections by laser-capture microdissection coupled to HPLC/tandem mass spectrometry, and spatial mapping on adjacent sections by mass spectrometry imaging. The adhesive film and staining methods were optimized to facilitate histology staining on the same sections used for mass spectrometry image acquisition, revealing drug accumulation in the underlying bone tissue architecture, for the first time. Absolute spatial concentrations of rifampicin, bedaquiline, doxycycline, vancomycin and several of their active metabolites are shown for both small rodent bones and larger rabbit bones that more closely resemble human bone density. Overlaid MALDI mass spectrometry images of drugs and histology staining enabled the generation of semi-quantitative data from regions of interest within anatomical bone compartments. These data correlated with absolute drug concentrations determined by HPLC-MS/MS in laser-capture microdissection samples. Collectively, these techniques enable semi- and fully quantitative drug distribution investigations within bone tissue compartments for the first time. Our workflow can be translated to image and quantify not only drugs but also biomarkers of disease to investigate drug penetration as well as mechanisms underlying bone disorders.

The peptide secreted at the water to land transition in a model amphibian has antioxidant effects.

In addition to the morphophysiological changes experienced by amphibians during metamorphosis, they must also deal with a different set of environmental constraints when they shift from the water to the land. We found that Pithecopus azureus secretes a single peptide ([M + H]+ = 658.38 Da) at the developmental stage that precedes the onset of terrestrial behaviour. De novo peptide and cDNA sequencing revealed that the peptide, named PaT-2, is expressed in tandem and is a member of the tryptophyllins family. In silico studies allowed us to identify the position of reactive sites and infer possible antioxidant mechanisms of the compounds. Cell-based assays confirmed the predicted antioxidant activity in mammalian microglia and neuroblast cells. The potential neuroprotective effect of PaT-2 was further corroborated in FRET-based live cell imaging assays, where the peptide prevented lipopolysaccharide-induced ROS production and glutamate release in human microglia. In summary, PaT-2 is the first peptide expressed during the ontogeny of P. azureus, right before the metamorphosing froglet leaves the aquatic environment to occupy terrestrial habitats. The antioxidant activity of PaT-2, predicted by in silico analyses and confirmed by cell-based assays, might be relevant for the protection of the skin of P. azureus adults against increased O2 levels and UV exposure on land compared with aquatic environments.

MALDI-TOF mass spectrometry imaging for N-glycans on FFPE tissue sections of mouse NASH liver through Sialic acid Benzylamidation.

Glycans play an important physiological role and are drawing attention as biomarkers that capture pathophysiological changes. Glycans can be detected by mass spectrometry, but recently matrix-assisted laser desorption/ionization- mass spectrometry imaging (MALDI-MSI) has enabled the visualization of glycans distribution on tissues. In this study, focusing on sialylated glycan (sialoglycans), we investigated the amidation reaction used to visualize glycans distribution, and developed a method of sialic acid derivatization by benzylamidation which is more sensitive than conventional amidation. Furthermore, we adapted this method for visualizing glycans in formalin-fixed paraffin-embedded (FFPE) liver tissue from normal mice and non-alcoholic steatohepatitis (NASH) model mice using MALDI-MSI. As a result, an increase in the distribution of glycan N-Acetylneuraminic acid-(NeuAc) ions was observed in the NASH mouse liver, and the change in glycan structure in the NASH model was clarified.

High-resolution AP-SMALDI MSI as a tool for drug imaging in Schistosoma mansoni.

Schistosoma mansoni is a parasitic flatworm causing schistosomiasis, an infectious disease affecting several hundred million people worldwide. Schistosomes live dioeciously, and upon pairing with the male, the female starts massive egg production, which causes pathology. Praziquantel (PZQ) is the only drug used, but it has an inherent risk of resistance development. Therefore, alternatives are needed. In the context of drug repurposing, the cancer drug imatinib was tested, showing high efficacy against S. mansoni in vitro. Besides the gonads, imatinib mainly affected the integrity of the intestine in males and females. In this study, we investigated the potential uptake and distribution of imatinib in adult schistosomes including its distribution kinetics. To this end, we applied for the first time atmospheric-pressure scanning microprobe matrix-assisted laser desorption/ionization mass spectrometry imaging (AP-SMALDI MSI) for drug imaging in paired S. mansoni. Our results indicate that imatinib was present in the esophagus and intestine of the male as early as 20 min after in vitro exposure, suggesting an oral uptake route. After one hour, the drug was also found inside the paired female. The detection of the main metabolite, N-desmethyl imatinib, indicated metabolization of the drug. Additionally, a marker signal for the female ovary was successfully applied to facilitate further conclusions regarding organ tropism of imatinib. Our results demonstrate that AP-SMALDI MSI is a useful method to study the uptake, tissue distribution, and metabolization of imatinib in S. mansoni. The results suggest using AP-SMALDI MSI also for investigating other antiparasitic compounds and their metabolites in schistosomes and other parasites.

Mapping Spatiotemporal Microproteomics Landscape in Experimental Model of Traumatic Brain Injury Unveils a link to Parkinson's Disease.

Traumatic brain injury (TBI) represents a major health concerns with no clinically-approved FDA drug available for therapeutic intervention. Several genomics and neuroproteomics studies have been employed to decipher the underlying pathological mechanisms involved that can serve as potential neurotherapeutic targets and unveil a possible underlying relation of TBI to other secondary neurological disorders. In this work, we present a novel high throughput systems biology approach using a spatially resolved microproteomics platform conducted on different brain regions in an experimental rat model of moderate of controlled cortical injury (CCI) at a temporal pattern postinjury (1 day, 3 days, 7 days, and 10 days). Mapping the spatiotemporal landscape of signature markers in TBI revealed an overexpression of major protein families known to be implicated in Parkinson's disease (PD) such as GPR158, HGMB1, synaptotagmin and glutamate decarboxylase in the ipsilateral substantia nigra. In silico bioinformatics docking experiments indicated the potential correlation between TBI and PD through alpha-synuclein. In an in vitro model, stimulation with palmitoylcarnitine triggered an inflammatory response in macrophages and a regeneration processes in astrocytes which also further confirmed the in vivo TBI proteomics data. Taken together, this is the first study to assess the microproteomics landscape in TBI, mainly in the substantia nigra, thus revealing a potential predisposition for PD or Parkinsonism post-TBI.

Electrospun α-Lactalbumin Nanofibers for Site-Specific and Fast-Onset Delivery of Nicotine in the Oral Cavity: An In Vitro, Ex Vivo, and Tissue Spatial Distribution Study.

Nicotine replacement therapy (NRT) formulations for oromucosal administration induce a delayed rise in nicotine blood levels as opposed to the immediate nicotine increase obtained from cigarette smoking, this being a shortcoming of the therapy. Here, we demonstrate that α-lactalbumin/polyethylene oxide (ALA/PEO) electrospun nanofibers constitute an efficient oromucosal delivery system for fast-onset nicotine delivery of high relevance for acute dosing NRT applications. In vitro, nicotine-loaded nanofibers showed fast disintegration in water, with a weight loss up to 40% within minutes, and a faster nicotine release (26.1 ± 4.6% after 1 min of incubation) of the loaded nicotine compared to two relevant marketed NRT formulations with a comparable nicotine dose (i.e., 7.9 ± 5.1 and 2.2 ± 0.3% nicotine was released from a lozenge and a sublingual tablet, respectively). Model-fitting of the release data indicated that the release mechanism of nicotine from the hydrophilic nanofibers was possibly governed by more than one type of release phenomena. Remarkably, ex vivo studies using porcine buccal mucosa demonstrated a more efficient permeation of the nicotine released from the nanofibers [flux of 1.06 ± 0.22 nmol/(cm2·min)] compared to when dosing even a ten-fold concentrated nicotine solution [flux of 0.17 ± 0.14 nmol/(cm2·min)]. Moreover, matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MS) imaging of ex vivo porcine buccal mucosa exposed to nicotine-loaded nanofibers clearly revealed higher amounts of nicotine throughout the epithelium, as well as in the lamina propria and submucosa of the tissue. Our findings suggest that nicotine-loaded ALA/PEO nanofibers have potential as a mucosal, fast-releasing, and biocompatible delivery system for nicotine, which can overcome the limitations of the currently marketed NRTs.

Glioblastoma multiforme: Metabolic differences to peritumoral tissue and IDH-mutated gliomas revealed by mass spectrometry imaging.

Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor. High infiltration rates and poor therapy responses make it the deadliest glioma. The tumor metabolism is known to differ from normal one and is influenced through various factors which can lead to longer survival. Metabolites are small molecules (< 1500 Da) that display the metabolic pathways in the tissue. To determine the metabolic alterations between tumor and peritumoral tissue in human GBMs, mass spectrometry imaging (MSI) was performed on thin sections from 25 resected tumors. In addition, the GBMs were compared with six gliomas harboring a mutation in the isocitrate dehydrogenase (IDH1) gene (IDH1). With this technique, a manifold of analytes can be easily visualized on a single tissue section. Metabolites were annotated based on their accurate mass using high resolution MSI. Differences in their mean intensities in the tumor and peritumoral areas were statistically evaluated and abundances were visualized on the tissue. Enhanced levels of the antioxidants ascorbic acid, taurine, and glutathione in tumor areas suggest protective effects on the tumor. Increased levels of purine and pyrimidine metabolism compounds in GBM areas indicate the high energy demand. In accordance with these results, enhanced abundances of lactate and glutamine were detected. Moreover, decreased abundance of N-acetylaspartate, a marker for neuronal health, was measured in tumor areas. Obtained metabolic information could potentially support and personalize therapeutic approaches, hence emphasizing the suitability of MSI for GBM research.

Label-free distribution of anti-amyloid D-AIP in Drosophila melanogaster: prevention of Aß42-induced toxicity without side effects in transgenic flies.

Soluble oligomers of the 42-amino acid amyloid beta (Aß42) peptide are highly toxic and suspected as the causative agent of synaptic dysfunction and neuronal loss in Alzheimer's disease (AD). Previously, we have shown that a small, D-amino acid Aß42-oligomer interacting peptide (D-AIP) can neutralize human Aß42-mediated toxicity using in vitro and cell-based assays. In the present longitudinal study using a transgenic Drosophila melanogaster model, advanced live confocal imaging and mass spectrometry imaging (MALDI-MSI) showed that the eight amino acid D-AIP can attenuate Aß42-induced toxicity in vivo. By separating male and female flies into distinct groups, the resultant distribution of ingested D-AIP was different between the sexes. The Aß42-induced 'rough eye' phenotype could be rescued in the female transgenics, likely because of the co-localization of D-AIP with human Aß42 in the female fly heads. Interestingly, the phenotype could not be rescued in the male transgenics, likely because of the co-localization of D-AIP with a confounding male-specific sex peptide (Acp70A candidate in MSI spectra) in the gut of the male flies. As a novel, more cost-effective strategy to prevent toxic amyloid formation during the early stages of AD (i.e. neutralization of toxic low-order Aß42 oligomers without creating larger aggregates in the process), our longitudinal study establishes that D-AIP is a stable and highly effective neutralizer of toxic Aß42 peptides in vivo. Cover Image for this issue: doi: 10.1111/jnc.14512.

Mucoadhesive Electrospun Patch Delivery of Lidocaine to the Oral Mucosa and Investigation of Spatial Distribution in a Tissue Using MALDI-Mass Spectrometry Imaging.

Many oral mucosal conditions cause considerable and prolonged pain that to date has been difficult to alleviate via topical delivery, and the use of injection causes many patients dental anxiety and needle-prick pain. Therefore, developing a noninjectable drug delivery system as an alternative administration procedure may vastly improve the health and wellbeing of these patients. Recent advances in the development of mucoadhesive electrospun patches for the direct delivery of therapeutics to the oral mucosa offer a potential solution, but as yet, the release of local anesthetics from this system and their uptake by oral tissue have not been demonstrated. Here, we demonstrate the fabrication of lidocaine-loaded electrospun fiber patches, drug release, and subsequent uptake and permeation through the porcine buccal mucosa. Lidocaine HCl and lidocaine base were incorporated into the electrospun patches to evaluate the difference in drug permeation for the two drug compositions. Lidocaine released from the lidocaine HCl-containing electrospun patches was significantly quicker than from the lidocaine base patches, with double the amount of drug released from the lidocaine HCl patches in the first 15 min (0.16 ± 0.04 mg) compared to that from the lidocaine base patches (0.07 ± 0.01 mg). The permeation of lidocaine from the lidocaine HCl electrospun patches through ex vivo porcine buccal mucosa was also detected in 15 min, whereas permeation of lidocaine from the lidocaine base patch was not detected. Matrix-assisted laser desorption ionization-mass spectrometry imaging was used to investigate localization of lidocaine within the oral tissue. Lidocaine in the solution as well as from the mucoadhesive patch penetrated into the buccal mucosal tissue in a time-dependent manner and was detectable in the lamina propria after only 15 min. Moreover, the lidocaine released from lidocaine HCl electrospun patches retained biological activity, inhibiting veratridine-mediated opening of voltage-gated sodium channels in SH-SY5Y neuroblastoma cells. These data suggest that a mucoadhesive electrospun patch may be used as a vehicle for rapid uptake and sustained anesthetic drug delivery to treat or prevent oral pain.

Mass spectrometry imaging reveals ganglioside and ceramide localization patterns during cerebellar degeneration in the Npc1-/- mouse model.

Mass spectrometry imaging (MSI) is a powerful tool to perform untargeted mapping of biomolecules in situ. In the current study, we performed matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) to evaluate lipid changes during disease progression (asymptomatic to symptomatic time points) in Niemann-Pick disease, type C1 (NPC1), a cerebellar neurodegenerative, lipid storage disorder. Our data show that gangliosides GM2 and GM3 are elevated in NPC1 disease and localize in the posterior lobules of the cerebellum, which is enhanced over a time-course analysis of the disease. Further analysis of sphingolipids in negative ion mode indicated reduction of sulfatides in white matter of the cerebellum and patterned distribution and co-localization of ceramide species Cer(d36:1), HexCer(d36:1), and the ganglioside GM1(d36:1) during disease progression. Finally, a putative lipid of unknown structure demonstrated similar patterning during NPC1 cerebellar degeneration. These studies provide insight into lipid markers of neurodegeneration in NPC1 and link lipid alterations to altered pathways that lead to cell death.

The management of haemoglobin interference for the MALDI-MSI proteomics analysis of thyroid fine needle aspiration biopsies.

MALDI-MSI represents an ideal tool to explore the spatial distribution of proteins directly in situ, integrating molecular and cytomorphological information, enabling the discovery of potential diagnostic markers in thyroid cytopathology. However, red cells present in the fine needle aspiration biopsy (FNAB) specimens caused ion suppression of other proteins during the MALDI-MSI analysis due to large amount of haemoglobin. Aim of this study was to set up a sample preparation workflow able to manage this haemoglobin interference. Three protocols were compared using ex vivo cytological samples collected from fresh thyroid nodules of 9 patients who underwent thyroidectomy: (A) conventional air-dried smears, (B) cytological smears immediately fixed in ethanol, and (C) ThinPrep liquid-based preparation. Protocols C and A were also evaluated using real FNABs. Results show that protocol C markedly decreased the amount of haemoglobin, with respect to protocols A and B. Protein profiles obtained with protocols A and B were characterised by high inter-patient variability, probably related to the abundance of the haemoglobin, whereas similar spectra were observed for protocol C, where haemoglobin contents were lower. Our findings suggest protocol C as the sample preparation method for MALDI-MSI analysis. Graphical abstract.

PAXgene fixation enables comprehensive metabolomic and proteomic analyses of tissue specimens by MALDI MSI.

An alcohol-based non-crosslinking tissue fixative, PAXgene Tissue System, has been proposed as alternative fixation method to formalin, providing superior and morphological preservation. To date, metabolites have not been assessed in PAXgene-fixed tissues. The study focuses on a comparison between PAXgene and standard formalin fixation for metabolomic analysis by MALDI mass spectrometry imaging. Therefore, fifty-six samples from seven mice organs were fixed with PAXgene (PFPE) or formalin (FFPE), embedded in paraffin, and processed to a tissue microarray. PAXgene was able to spatially preserve metabolites in organs achieving an overlap of common metabolites ranging from 34 to 78% with FFPE. Highly similar signal intensities and visualization of molecules demonstrated negligible differences for metabolite imaging on PFPE compared to FFPE tissues. In addition, we performed proteomic analysis of intact proteins and peptides derived from enzymatic digestion. An overlap of 33 to 58% was found between FFPE and PFPE tissue samples in peptide analysis with a higher number of PFPE-specific peaks. Analysis of intact proteins achieved an overlap in the range of 0 to 28% owing to the poor detectability of cross-linked proteins in formalin-fixed tissues. Furthermore, metabolite and peptide profiles obtained from PFPE tissues were able to correctly classify organs independent of the fixation method, whereas a distinction of organs by protein profiles was only achieved by PAXgene fixation. Finally, we applied MALDI MSI to human biopsies by sequentially analyzing metabolites and peptides within the same tissue section. Concerning prospective studies, PAXgene can be used as an alternative fixative for multi-omic tissue analysis.

In Situ Characterization of Tissue-Resident Immune Cells by MALDI Mass Spectrometry Imaging.

Tissue-resident immune cells differ from their corresponding blood cells in many functional aspects. Although the proteome of blood immune cells has been well-investigated, there are almost no data on tissue-resident immune cells. Here, we explored the potential of using MALDI-TOF-MS imaging (MSI) to investigate these cells in colon tissue, which exhibits a strong infiltration of immune cells. MSI identified several proteinaceous markers that colocalized with specific structures of the colon, such as mucosa or muscularis mucosae, in six patients. In addition, we showed that certain m/z values have the same spatial distribution as CD3+ T lymphocytes in the lymphoid follicular structures or as CD206+ macrophages in the lamina propria. For further corroboration, blood lymphocytes and monocytes from 10 healthy volunteers were analyzed by intact cell mass spectrometry (ICMS). Furthermore, we analyzed monocyte-derived macrophages that had been polarized in vitro into proinflammatory M1 and anti-inflammatory M2 phenotypes. The mass spectra differed clearly among all immune cell types. Additionally, it was found that distinct signals from ICMS analysis were identical to the m/z values found in the MSI experiment in lymphoid follicular structures. These data show for the first time that MSI is well-suited to visualize the spatial distribution of immune cells in human colon tissue. We consider MALDI mass spectrometry imaging to be a technique with high potential for use in rapid investigations of tissue-specific features of cells.

Unraveling Drug Penetration of Echinocandin Antifungals at the Site of Infection in an Intra-abdominal Abscess Model.

Intra-abdominal candidiasis (IAC) is a prominent invasive fungal infection associated with high mortality. Prompt antifungal therapy and source control are crucial for successful treatment. Echinocandin antifungal drugs are first-line agents; however, their clinical effectiveness is highly variable, with known potential for breakthrough resistance, and little is known about drug exposure at the site of infection. Using matrix-assisted desorption ionization mass spectrometry imaging technology, we investigated the spatial and quantitative distribution in tissue lesions for two echinocandin drugs, micafungin and CD101, in a clinically relevant IAC mouse model. Drug accumulation within lesions was observed with both drugs at their humanized therapeutic doses. CD101, but not micafungin, accumulated in lesions at levels above the mutant prevention concentration of the infecting strain. These findings indicate that current echinocandin drugs are limited by penetration at the site of infection and have implications for clinical outcomes and emergence of resistance in patients with IAC.

Brain Region-Specific Dynamics of On-Tissue Protein Digestion Using MALDI Mass Spectrometry Imaging.

In mass spectrometry imaging (MSI), on-tissue proteolytic digestion is performed to access larger protein species and to assign protein identities through matching the detected peaks with those obtained by LC-MS/MS analyses of tissue extracts. The on-tissue proteolytic digestion also allows the analysis of proteins from formalin-fixed, paraffin-embedded tissues. For these reasons, on-tissue digestion-based MSI is frequently used in clinical investigations, for example, to determine changes in protein content and distribution associated with a disease. In this work, we sought to investigate the completeness and uniformity of the digestion in on-tissue digestion MSI. On the basis of an extensive experiment investigating three groups with varying incubation times: (i) 1.5 h, (ii) 3 h, and (iii) 18 h, we have found that longer incubation times improve the repeatability of the analyses. Furthermore, we discovered morphology-associated differences in the completeness of the proteolysis for short incubation times. These results support the notion that a more complete proteolysis allows better quantitation.

2D and 3D MALDI-imaging: conceptual strategies for visualization and data mining.

3D imaging has a significant impact on many challenges in life sciences, because biology is a 3-dimensional phenomenon. Current 3D imaging-technologies (various types MRI, PET, SPECT) are labeled, i.e. they trace the localization of a specific compound in the body. In contrast, 3D MALDI mass spectrometry-imaging (MALDI-MSI) is a label-free method imaging the spatial distribution of molecular compounds. It complements 3D imaging labeled methods, immunohistochemistry, and genetics-based methods. However, 3D MALDI-MSI cannot tap its full potential due to the lack of statistical methods for analysis and interpretation of large and complex 3D datasets. To overcome this, we established a complete and robust 3D MALDI-MSI pipeline combined with efficient computational data analysis methods for 3D edge preserving image denoising, 3D spatial segmentation as well as finding colocalized m/z values, which will be reviewed here in detail. Furthermore, we explain, why the integration and correlation of the MALDI imaging data with other imaging modalities allows to enhance the interpretation of the molecular data and provides visualization of molecular patterns that may otherwise not be apparent. Therefore, a 3D data acquisition workflow is described generating a set of 3 different dimensional images representing the same anatomies. First, an in-vitro MRI measurement is performed which results in a three-dimensional image modality representing the 3D structure of the measured object. After sectioning the 3D object into N consecutive slices, all N slices are scanned using an optical digital scanner, enabling for performing the MS measurements. Scanning the individual sections results into low-resolution images, which define the base coordinate system for the whole pipeline. The scanned images conclude the information from the spatial (MRI) and the mass spectrometric (MALDI-MSI) dimension and are used for the spatial three-dimensional reconstruction of the object performed by image registration techniques. Different strategies for automatic serial image registration applied to MS datasets are outlined in detail. The third image modality is histology driven, i.e. a digital scan of the histological stained slices in high-resolution. After fusion of reconstructed scan images and MRI the slice-related coordinates of the mass spectra can be propagated into 3D-space. After image registration of scan images and histological stained images, the anatomical information from histology is fused with the mass spectra from MALDI-MSI. As a result of the described pipeline we have a set of 3 dimensional images representing the same anatomies, i.e. the reconstructed slice scans, the spectral images as well as corresponding clustering results, and the acquired MRI. Great emphasis is put on the fact that the co-registered MRI providing anatomical details improves the interpretation of 3D MALDI images. The ability to relate mass spectrometry derived molecular information with in vivo and in vitro imaging has potentially important implications. This article is part of a Special Issue entitled: Computational Proteomics in the Post-Identification Era. Guest Editors: Martin Eisenacher and Christian Stephan.

Inter- and intra-organ spatial distributions of sea star saponins by MALDI imaging.

Saponins are secondary metabolites that are abundant and diversified in echinoderms. Mass spectrometry is increasingly used not only to identify saponin congeners within animal extracts but also to decipher the structure/biological activity relationships of these molecules by determining their inter-organ and inter-individual variability. The usual method requires extensive purification procedures to prepare saponin extracts compatible with mass spectrometry analysis. Here, we selected the sea star Asterias rubens as a model animal to prove that direct analysis of saponins can be performed on tissue sections. We also demonstrated that carboxymethyl cellulose can be used as an embedding medium to facilitate the cryosectioning procedure. Matrix-assisted laser desorption/ionization (MALDI) imaging was also revealed to afford interesting data on the distribution of saponin molecules within the tissues. We indeed highlight that saponins are located not only inside the body wall of the animals but also within the mucus layer that probably protects the animal against external aggressions. Graphical Abstract Saponins are the most abundant secondary metabolites in sea stars. They should therefore participate in important biological activities. Here, MALDI imaging is presented as a powerful method to determine the spatial distribution of saponins within the animal tissues. The inhomogeneity of the intra-organ saponin distribution is highlighted, paving the way for future elegant structure/activity relationship investigations.