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Recently, a novel technology was published, utilizing the strengths of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) and immunohistochemistry (IHC), achieving highly multiplexed, targeted imaging of biomolecules in tissue. This new technique, called MALDIIHC, opened up workflows to target molecules of interest using MALDI-MSI that are usually targeted by standard IHC. In this paper, the utility of targeted MALDIIHC and its complementarity with untargeted on-tissue bottom-up spatial proteomics is explored using breast cancer tissue. Furthermore, the MALDI-2 effect was investigated and demonstrated to improve MALDI-IHC. Formalin-fixed paraffin-embedded (FFPE) human breast cancer tissue sections were stained for multiplex MALDI-IHC with six photocleavable mass-tagged (PC-MT) antibodies constituting a breast cancer antibody panel (CD20, actin-alpha SM, HER2, CD68, vimentin, and panCK). K-means spatial clusters were created based on the MALDI-IHC images and cut out using laser-capture microdissection (LMD) for further untargeted LC-MS-based bottom-up proteomics analyses. Numerous peptides could be tentatively assigned to multiple proteins, of which three proteins were also part of the antibody panel (vimentin, keratins, and actin). Post-ionization with MALDI-2 showed an increased intensity of the PC-MTs and suggests options for the development of new mass-tags. Although the on-tissue digestion covered a wider range of proteins, the MALDI-IHC allowed for easy and straightforward identification of proteins that were not detected in untargeted approaches. The combination of the multiplexed MALDI-IHC with image-guided proteomics showed great potential to further investigate diseases by providing complementary information from the same tissue section and without the need for customized instrumentation.

Every year, thousands of suspicious deaths are accounted for by an overdose of opioids. Occasionally all traditional matrices are unavailable due to decomposition. Skeletal tissue may pose a valid alternative. However, reference data on postmortem concentrations in bone tissue and bone marrow (BM) is sparse. Therefore, a liquid chromatography--tandem mass spectrometry method was developed and fully validated for the analysis of four opioids and two metabolites (tramadol, O-desmethyltramadol, morphine, fentanyl, norfentanyl, codeine) in bone tissue and BM. Sample preparation was performed using solid phase extraction (BM), methanolic extraction (bone) and a protein precipitation (whole blood). All validation parameters were successfully fulfilled. This method was applied to analyze 22 forensic cases involving opioids. All six opioids were proven to be detectable and quantifiable in all specimens sampled. When tramadol blood concentrations were correlated with bone concentrations, a linear trend could be detected. The same was seen between tramadol blood and BM concentration. A similar linear trend was seen when correlating codeine blood concentration with bone and BM concentration. Although some variability was detected, the same linear trend was seen for morphine. For fentanyl and norfentanyl, the sample size was too small to draw conclusions, regarding correlation. As far as the authors know this is the first-time fentanyl and norfentanyl are quantified in skeletal tissue. In conclusion, due to the absence of reference data for drugs in skeletal tissue, these findings are a step forward toward a more thorough understanding of drug concentration found in postmortem skeletal tissue. ispartof: JOURNAL OF ANALYTICAL TOXICOLOGY vol:46 issue:7 pages:783-790 ispartof: location:England status: published

Detecting, identifying, and characterizing molecules and structures as the substance of life are essential steps toward unveiling their role as a sole entity or more commonly as a part of a larger molecular assembly for specific biological functionalities. Next, it is of pivotal importance to translate the acquired knowledge of molecular structures and their activity to a more comprehensive understanding of the biological process, such as the biochemical foundations of diseases or metabolic disorders. The aim and ultimate outcomes of this thesis were to address these two aspects by offering new analytical strategies such as focusing on the molecular structure assessment of small molecules, including drug metabolites and potential biomarkers, and drug targets. Addressing challenges encountered in protein characterization and protein interactions studies. And lastly, taking a step forward and analyzing a dynamic chemical reaction in an online fashion by mass, tandem mass, and ion mobility spectrometry.

Iron(II) and Ru(II) half-sandwich compounds encompass some promising pre-clinical anticancer agents whose efficacy may be tuned by structural modification of the coordinated ligands. Here, we combine two such bioactive metal centres in cationic bis(diphenylphosphino)alkane-bridged heterodinuclear [Fe2+, Ru2+] com-plexes to delineate how ligand structural variations modulate compound cytotoxicity. Specifically, Fe(II) complexes of the type [(eta(5)-C5H5)Fe(CO)(2)(kappa(1)-PPh2(CH2)(n)PPh2)]{PF6} (n = 1-5), compounds 1-5, and heterodinuclear [Fe2+, Ru2+] complexes, [(eta(5)-C5H5)Fe(CO)(2)(mu-PPh2(CH2)nPPh(2)))(eta(6)-p-cymene)RuCl2]{PF6} (n = 2-5) (compounds 7-10), were synthesized and characterised. The mononuclear complexes were moderately cytotoxic against two ovarian cancer cell lines (A2780 and cisplatin resistant A2780cis) with IC50 values ranging from 2.3 +/- 0.5 mu M to 9.0 +/- 1.4 mu M. For 7-10, the cytotoxicity increased with increasing Fe center dot center dot center dot Ru distance, consistent with their DNA affinity. UV-visible spectroscopy suggested the chloride ligands in heterodinuclear 8-10 undergo stepwise substitution by water on the timescale of the DNA interaction experiments, probably affording the species [RuCl(OH2)(eta(6)-p-cymene)(PRPh2)](2+) and [Ru(OH)(OH2)(eta(6)-p-cymene)(PRPh2)](2+) (where PRPh2 has R = [-(CH2)(5)PPh2-Fe(C5H5)(CO)(2)](+)). One interpretation of the combined DNA-interaction and kinetic data is that the mono(aqua) complex may interact with dsDNA through nucleobase coordination. Heterodinuclear 10 reacts with glutathione (GSH) to form stable mono-and bis(thiolate) adducts, 10-SG and 10-SG(2), with no evidence of metal ion reduction (k(1) = 1.07 +/- 0.17 x 10(-1) min(-1) and k(2) = 6.04 +/- 0.59 x 10-3 min(-1) at 37 degrees C). This work highlights the synergistic effect of the Fe2+/Ru2+ centres on both the cytotoxicity and biomolecular interactions of the present heterodinuclear complexes.

Mass spectrometry imaging (MSI) is a surface analysis technique that produces chemical images and is commonly used for biological and biomedical research. Multimodal imaging combines multiple imaging modes in order to get a more comprehensive view of a sample. Multimodal MSI images are often acquired using multiple MSI instruments, which leads to issues regarding image registration and increases the chance of sample damage or degradation during sample transfer. These problems can be solved by using a single instrument that can image in multiple modes. In order to improve the efficiency of multimodal imaging and investigate complementary modes of MSI, we have modified a prototype Bruker timsTOF fleX by adding secondary ion mass spectrometry (SIMS) and secondary electron (SE) imaging capabilities while preserving the ability to perform matrix-assisted laser desorption/ionization (MALDI). We show multimodal images collected on this instrument that required only trivial registration and were acquired without sample transfer between imaging trials. Furthermore, we characterize the performance of SIMS, SE, and MALDI imaging and compare the performance of the modified instrument to a commercial timsTOF fleX.

With the recent improvements in ion mobility resolution, it is now possible to separate small protomerictautomers, called protomers. In larger molecules above 1000 Dasuch as peptides, a few studies suggest that protomers do exist aswell and may contribute to their gas-phase conformationalheterogeneity. In this work, we observed a CCS distribution thatcan be explained by the presence of protomers of surfactin, a smalllipopeptide with no basic site. Following preliminary densityfunctional theoretical calculations, several protonation sites in thegas phase were energetically favorable in positive ionization mode.Experimentally, at least three near-resolved IM peaks wereobserved in positive ionization mode, while only one was detectedin negative ionization mode. These results were in good agreement with the DFT predictions. CID breakdown curve analysis afterIM separation showed different inflection points (CE50) suggesting that different intramolecular interactions were implied in thestabilization of the structures of surfactin. The fragment ratio observed after collision-induced fragmentation was also different,suggesting different ring-opening localizations. All these observations support the presence of protomers on the cyclic peptidemoieties of the surfactin. These data strongly suggest that protomeric tautomerism can still be observed on molecules above 1000 Daif the IM resolving power is sufficient. It also supports that the proton localization involves a change in the 3D structure that can affect the experimental CCS and the fragmentation channels of such peptides

Organoids are increasingly used as model systems in precision medicine, but they are delicate, making it difficult to get accurate spatial chemical information. This protocol describes how to prepare organoid samples for mass spectrometry imaging.Organoid culture systems are self-renewing, three-dimensional (3D) models derived from pluripotent stem cells, adult derived stem cells or cancer cells that recapitulate key molecular and structural characteristics of their tissue of origin. They generally form into hollow structures with apical-basolateral polarization. Mass spectrometry imaging (MSI) is a powerful analytical method for detecting a wide variety of molecules in a single experiment while retaining their spatiotemporal distribution. Here we describe a protocol for preparing organoids for MSI that (1) preserves the 3D morphological structure of hollow organoids, (2) retains the spatiotemporal distribution of a vast array of molecules (3) and enables accurate molecular identification based on tandem mass spectrometry. The protocol specifically focuses on the collection and embedding of the organoids in gelatin, and gives recommendations for MSI-specific sample preparation, data acquisition and molecular identification by tandem mass spectrometry. This method is applicable to a wide range of organoids from different origins, and takes 1 d from organoid collection to MSI data acquisition.

The third annual conference of the Imaging Mass Spectrometry Society (IMSS3) was held October 3-6, 2021 in a hybrid format that included virtual and in-person attendance (Colorado Springs, CO). Here, we highlight many of the methods and applications presented, the state of the field, and some insights into the emerging areas in the field of imaging mass spectrometry. We also reflect upon the processes behind planning a hybrid conference and discuss the successes and challenges of the event in retrospect.

Mass spectrometry imaging (MSI) is a powerful technique enabling the visualization of the spatial distribution of different molecules in tissue biopsies with different pathologies. Sample handling and preparing adipose tissue for MSI is challenging and prone to molecular delocalization due to tissue melting. In this work, we developed a method for matrix-assisted laser desorption/ionization (MALDI)-MSI to study lipids in human infrapatellar fat pad (IPFP), a biomarker source in musculoskeletal pathologies, while preserving molecular spatial distribution. Cryosectioning at 15 μm with a temperature below -30 °C, thaw-mounting, and sublimation, was demonstrated to preserve IPFP's heterogeneous appearance and spatial distribution of lipids.

Different developments necessary for the application of different mass spectrometry imaging techniques on skeletal tissue are presented in this thesis. Sample preparation protocols for undecalcified bone and fracture hematoma were optimized for analysis with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). Time-dependent changes in the lipid profiles were observed during fracture healing in human fracture hematoma. In addition, different lipid profiles were seen between the citrulline supplementation and control group during different fracture healing phases in a rat model. Protein and pathway analyses showed an enhancement of fracture healing in the citrulline supplementation group in comparison to the control group. An automated setup for laser-assisted rapid evaporative ionisation mass spectrometry (LA-REIMS) was developed for the analysis of sample surfaces with height variations. This setup allowed for the visualization of molecular distributions for a wide variety of samples, including human femoral heads. The different methodological developments and the applications shown in this thesis can be used for future research in skeletal tissue and its healing processes to improve molecular understanding and outcome prediction.

The analysis of samples with large height variations remains a challenge for mass spectrometry imaging (MSI), despite many technological advantages. Ambient sampling and ionization MS techniques allow for the molecular analysis of sample surfaces with height variations, but most techniques lack MSI capabilities. We developed a 3D MS scanner for the automated sampling and imaging of a 3D surface with laser-assisted rapid evaporative ionization mass spectrometry (LA-REIMS). The sample is moved automatically with a constant distance between the laser probe and sample surface in the 3D MS Scanner. The topography of the surface was scanned with a laser point distance sensor to define the MS measurement points. MS acquisition was performed with LA-REIMS using a surgical CO2 laser coupled to a qTOF instrument. The topographical scan and MS acquisition can be completed within 1 h using the 3D MS scanner for 300 measurement points on uneven samples with a spatial resolution of 2 mm in the top view, corresponding to 22.04 cm(2). Comparison between the automated acquisition with the 3D MS scanner and manual acquisition by hand showed that the automation resulted in increased reproducibility between the measurement points. 3D visualizations of molecular distributions related to structural differences were shown for an apple, a marrowbone, and a human femoral head to demonstrate the imaging feasibility of the system. The developed 3D MS scanner allows for the automated sampling of surfaces with uneven topographies with LA-REIMS, which can be used for the 3D visualization of molecular distributions of these surfaces.

RATIONALE: Isomeric separation of prostanoids is often a challenge and requires chromatography and time-consuming sample preparation. Multiple prostanoid isomers have distinct in vivo functions crucial to understand the inflammation process, including prostaglandins E2 (PGE2 ) and D2 (PGD2 ). High-resolution Ion Mobility Spectrometry (IMS) based on linear ion transport in low-to-moderate electric fields and nonlinear ion transport in strong electric fields emerges as a broad approach for rapid separations prior to mass spectrometry (MS).METHODS: Derivatization with Girard's reagent T (GT) was used to overcome inefficient ionization of prostanoids in negative ionization mode due to poor deprotonation of the carboxylic acid group. Three high-resolution IMS techniques including linear cyclic IMS (cIMS), linear Trapped IMS (TIMS), and nonlinear high-Field Asymmetric Waveform IMS (FAIMS) were compared for the isomeric separation and endogenous detection of prostanoids present in intestinal tissue.RESULTS: Direct infusion of GT derivatized prostanoids proved to increase the ionization efficiency in positive ionization mode by a factor of >10, which enabled detection of these molecules in endogenous concentration levels. The high-resolution IMS comparison revealed its potential for rapid isomeric analysis of biologically relevant prostanoids. Strengths and weaknesses of both linear and nonlinear IMS were discussed. Endogenous prostanoid detection in intestinal tissue extracts demonstrated the applicability of our approach in biomedical research.CONCLUSIONS: The applied derivatization strategy offers high sensitivity and improved stereoisomeric separation for screening of complex biological systems. The high-resolution IMS comparison indicated that the best sensitivity and resolution are achieved by linear and nonlinear IMS, respectively.

The Timepix (TPX) is a position- and time-sensitive pixelated charge detector that can be coupled with time-of-flight mass spectrometry (TOF MS) in combination with microchannel plates (MCPs) for the spatially and temporally resolved detection of biomolecules. Earlier generation TPX detectors used in previous studies were limited by a moderate time resolution (at best 10 ns) and single-stop detection for each pixel that hampered the detection of ions with high mass-to-charge (m/z) values at high pixel occupancies. In this study, we have coupled an MCP-phosphor screen-TPX3CAM detection assembly that contains a silicon-coated TPX3 chip to a matrix-assisted laser desorption/ionization (MALDI)-axial TOF MS. A time resolution of 1.5625 ns, per-pixel multihit functionality, simultaneous measurement of TOF and time-over-threshold (TOT) values, and kHz readout rates of the TPX3 extended the m/z detection range of the TPX detector family. The detection of singly charged intact Immunoglobulin M ions of m/z value approaching 1 × 106 Da has been demonstrated. We also discuss the utilization of additional information on impact coordinates and TOT provided by the TPX3 compared to conventional MS detectors for the enhancement of the quality of the mass spectrum in terms of signal-to-noise (S/N) ratio. We show how the reduced dead time and event-based readout in TPX3 compared to the TPX improves the sensitivity of high m/z detection in both low and high mass measurements (m/z range: 757-970,000 Da). We further exploit the imaging capabilities of the TPX3 detector for the spatial and temporal separation of neutral fragments generated by metastable decay at different locations along the field-free flight region by simultaneous application of deflection and retarding fields.

Continuous flow chemistry is an efficient, sustainable and green approach for chemical synthesis that surpasses some of the limitations of the traditional batch chemistry. Along with the multiple advantages of a flow reactor, it could be directly connected to the analytical techniques for on-line monitoring of a chemical reaction and ensure the quality by design. Here, we aim to use ion mobility, mass and tandem mass spectrometry (IMS-MS and MS/MS) for the on-line analysis of a pharmaceutically relevant chemical reaction. We carried out a model hetero-Diels Alder reaction in a microflow reactor directly connected to the IMS-MS and MS/MS using either electrospray or atmospheric pressure photo ionization methods. We were able to monitor the reaction mechanism of the Diels Alder reaction and structurally characterize the reaction product and synthesis side-products. The chosen approach enabled identification of two isomers of the main reaction product. A new strategy to annotate the ion mobility spectrum in the absence of standard molecules was introduced and tested for its validity. This was achieved by determining the survival yield of each isomer upon ion mobility separation and density functional theory calculations. This approach was verified by comparing the theoretically driven collision cross section values to the experimental data. In this paper, we demonstrated the potential of combined IMS-MS and MS/MS on-line analysis platform to investigate, monitor and characterize structural isomers in the millisecond time scale.

A novel and green route for the synthesis of cyclopentane-1,3-diamine (CPDA) from hemicellulosic feed-stock is established in this work. Through many explorations and optimizations, the single successful multi-step synthesis was found to comprise: (1) the Piancatelli rearrangement of furfuryl alcohol to 4-hydroxycyclopent-2-enone (4-HCP), (2) a highly improved isomerization of 4-HCP into cyclopentane-1,3-dione (CPDO) using the Ru Shvo catalyst, (3) conversion of CPDO into cyclopentane-1,3-dioxime (CPDX), and (4) a mild oxime hydrogenation of CPDX over Rh/C to afford the desired CPDA. In addition, diastereomerically pure cis- and trans-isomers of CPDA were reacted with (A) bio-based lactones, and (B) 5-(hydroxymethyl)furfural (HMF) to synthesize novel bifunctional diol monomers with internal amide and imine groups, respectively. Monomer 5, derived using gamma-valerolactone (GVL), was successfully applied in the synthesis of polyurethanes.

Synthetic biodegradable materials are commonly used to create constructs for medical devices and tissue engineered constructs. However, many of the homopolymers used in FDA approved devices such as poly(ε-caprolactone) (PCL), poly(lactic acid), or poly(carbonates) lack biogically relevant functional groups to steer biological responses in a controlled fashion. Commonly, an interconversion of the end groups is required to insert addressable moieties for the attachment of biologically active groups. In this study, the activation of the hydroxyl groups of a low molecular weight PCL-diol to the corresponding p-toluene sulfonate ester using p-toluenesulfonyl chloride was performed in both dichloromethane (DCM) and dimethylformamide (DMF). To our initial surprise, we only yielded the chlorinated product in DMF, while in DCM the tosylate ester was obtained. In a small series of reactions, we studied the solvent dependent switchability between tosylation and chlorination on PCL. We concluded that in polar aprotic solvents (DMF and dimethylsulfoxide), we rapidly and efficiently converted the hydroxyl into the chloride group, whereas in inert solvents (DCM and chloroform) we yielded the tosylated product. The data suggested that solvation effects of the polar aprotic solvents led to a Sn2 reaction of the tosyl group by the chloride. Furthermore, we utilized a polyethylene glycol (PEG) polymer to show translatability of the chlorination reaction to other (biomedical) polymers. This work highlights a new reaction pathway during the tosylation of a polymer end group, and presents a new useful strategy to insert clickable groups on synthetic polymers that are only soluble in polar aprotic solvents. We thank the CORE IMS lab, Division of Imaging Mass Spectrometry at The Maastricht Multimodal Molecular Imaging Institute (M4i), for the ToF-SIMS experiments. We are also grateful to the European Research Council starting grant “Cell Hybridge” for financial support under the Horizon2020 framework program (Grant #637308).

Over the last decades, mass spectrometry based diagnostic tests have become an important cornerstone in clinical decision making. This thesis evaluated the efficacy of existing reference techniques and developed new techniques that might improve the detection of drugs and the characterization and discrimination between normal and diseased tissue in human brain tumours, both in the laboratory and intra-operatively. The research presented in this thesis contributed to the development of molecular patterns-based classification to support personalised and precise intraoperative decision making. Thanks to the real-time information during electrosurgical dissection a more accurate tissue identification and significantly shorter surgery time becomes possible. Hence, the possibility of direct tissue and tumour identification would provide the surgeon with additional important information to be used for multimodal integration, allowing intraoperative decision making.

Mass spectrometry imaging (MSI) maps the spatial distributions of chemicals on surfaces. MSI requires improvements in throughput and spatial resolution, and often one is compromised for the other. In microprobe-mode MSI, improvements in spatial resolution increase the imaging time quadratically, thus limiting the use of high spatial resolution MSI for large areas or sample cohorts and time-sensitive measurements. Here, we bypass this quadratic relationship by combining a Timepix3 detector with a continuously sampling secondary ion mass spectrometry mass microscope. By reconstructing the data into large-field mass images, this new method, fast mass microscopy, enables orders of magnitude higher throughput than conventional MSI albeit yet at lower mass resolution. We acquired submicron, gigapixel images of fingerprints and rat tissue at acquisition speeds of 600,000 and 15,500 pixels s-1, respectively. For the first image, a comparable microprobe-mode measurement would take more than 2 months, whereas our approach took 33.3 min.

Myocardial infarction is the most common cause of death worldwide. An understanding of the alterations in protein pathways is needed in order to develop strategies that minimize myocardial damage. To identify the protein signature of cardiac ischemia/reperfusion (I/R) injury in rats, we combined, for the first time, protein matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) and label-free proteomics on the same tissue section placed on a conductive slide. Wistar rats were subjected to I/R surgery and sacrificed after 24 h. Protein MALDI-MSI data revealed ischemia specific regions, and distinct profiles for the infarct core and border. Firstly, the infarct core, compared to histologically unaffected tissue, showed a significant downregulation of cardiac biomarkers, while an upregulation was seen for coagulation and immune response proteins. Interestingly, within the infarct tissue, alterations in the cytoskeleton reorganization and inflammation were found. This work demonstrates that a single tissue section can be used for protein-based spatial-omics, combining MALDI-MSI and label-free proteomics. Our workflow offers a new methodology to investigate the mechanisms of cardiac I/R injury at the protein level for new strategies to minimize damage after MI.

Mass spectrometry imaging (MSI) of lipids within tissues has significant potential for both biomolecular discovery and histopathological applications. Conventional MSI technologies are, however, challenged by the prevalence of phospholipid regioisomers that differ only in the location(s) of carbon-carbon double bonds and/or the relative position of fatty acyl attachment to the glycerol backbone (i.e., sn position). The inability to resolve isomeric lipids within imaging experiments masks underlying complexity, resulting in a critical loss of metabolic information. Herein, ozone-induced dissociation (OzID) is implemented on a mobility-enabled quadrupole time-of-flight (Q-TOF) mass spectrometer capable of matrix-assisted laser desorption/ionization (MALDI). Exploiting the ion mobility region in the Q-TOF, high number densities of ozone were accessed, leading to similar to 1000-fold enhancement in the abundance of OzID product ions compared to earlier MALDI-OzID implementations. Translation of this uplift into imaging resulted in a 50-fold improvement in acquisition rate, facilitating large-area mapping with resolution of phospholipid isomers. Mapping isomer distributions across rat brain sections revealed distinct distributions of lipid isomer populations with region-specific associations of isomers differing in double bond and sn positions. Moreover, product ions arising from sequential ozone- and collision-induced dissociation enabled double bond assignments in unsaturated fatty acyl chains esterified at the noncanonical sn-1 position.

Objective The potential chondroprotective effect of celecoxib, a nonsteroidal anti-inflammatory drug and selective cyclooxygenase-2 inhibitor used to reduce pain and inflammation in knee osteoarthritis patients, is disputed. This study aimed at investigating the chondroprotective effects of celecoxib on (1) human articular cartilage explants and (2) in an in vivo osteoarthritis rat model. Design Articular cartilage explants from 16 osteoarthritis patients were cultured for 24 hours with celecoxib or vehicle. Secreted prostaglandins (prostaglandin E2, prostaglandin F2α, prostaglandin D2) and thromboxane B2 (TXB2) concentrations were determined in medium by ELISA, and protein regulation was measured with label-free proteomics. Cartilage samples from 7 of these patients were analyzed for gene expression using real-time quantitative polymerase chain reaction. To investigate the chondroprotective effect of celecoxib in vivo, 14 rats received an intra-articular injection of celecoxib or 0.9% NaCl after osteoarthritis induction by anterior cruciate ligament transection and partial medial meniscectomy (ACLT/pMMx model). Histopathological scoring was used to evaluate osteoarthritis severity 12 weeks after injection. Results Secretion of prostaglandins, target of Nesh-SH3 (ABI3BP), and osteonectin proteins decreased, whereas tissue inhibitor of metalloproteinase 2 (TIMP-2) increased significantly after celecoxib treatment in the human ( ex vivo) explant culture. Gene expression of a disintegrin and metalloproteinase with thrombospondin motifs 4 and 5 (ADAMTS4/5) and metalloproteinase 13 (MMP13) was significantly reduced after celecoxib treatment in human cartilage explants. Cartilage degeneration was reduced significantly in an in vivo osteoarthritis knee rat model. Conclusions Our data demonstrated that celecoxib acts chondroprotective on cartilage ex vivo and a single intra-articular bolus injection has a chondroprotective effect in vivo.

IntroductionSpatial analysis of lipids in inflammatory microenvironments is key to understand the pathogenesis of infectious disease. Granulomatous inflammation is a hallmark of leishmaniasis and changes in host and parasite lipid metabolism have been observed at the bulk tissue level in various infection models. Here, mass spectrometry imaging (MSI) is applied to spatially map hepatic lipid composition following infection with Leishmania donovani, an experimental mouse model of visceral leishmaniasis.MethodsLivers from naïve and L. donovani-infected C57BL/6 mice were harvested at 14- and 20-days post-infection (n=5 per time point). 12 µm transverse sections were cut and covered with norhamane, prior to lipid analysis using MALDI-MSI. MALDI-MSI was performed in negative mode on a Rapiflex (Bruker Daltonics) at 5 and 50 µm spatial resolution and data-dependent analysis (DDA) on an Orbitrap-Elite (Thermo-Scientific) at 50 µm spatial resolution for structural identification analysis of lipids.ResultsAberrant lipid abundances were observed in a heterogeneous distribution across infected mouse livers compared to naïve mouse liver. Distinctive localized correlated lipid masses were found in granulomas and surrounding parenchymal tissue. Structural identification revealed 40 different lipids common to naïve and d14/d20 infected mouse livers, whereas 15 identified lipids were only detected in infected mouse livers. For pathology-guided MSI imaging, we deduced lipids from manually annotated granulomatous and parenchyma regions of interests (ROIs), identifying 34 lipids that showed significantly different intensities between parenchyma and granulomas across all infected livers.DiscussionOur results identify specific lipids that spatially correlate to the major histopathological feature of Leishmania donovani infection in the liver, viz. hepatic granulomas. In addition, we identified a three-fold increase in the number of unique phosphatidylglycerols (PGs) in infected liver tissue and provide direct evidence that arachidonic acid-containing phospholipids are localized with hepatic granulomas. These phospholipids may serve as important precursors for downstream oxylipin generation with consequences for the regulation of the inflammatory cascade. This study provides the first description of the use of MSI to define spatial-temporal lipid changes at local sites of infection induced by Leishmania donovani in mice.

Osteoarthritis (OA) is a multifactorial pathology and comprises a wide range of distinct phenotypes. In this context, the characterization of the different molecular profiles associated with each phenotype can improve the classification of OA. In particular, OA can coexist with type 2 diabetes mellitus (T2DM). This study investigates lipidomic and proteomic differences between human OA/T2DM(-) and OA/T2DM(+) cartilage through a multimodal mass spectrometry approach. Human cartilage samples were obtained after total knee replacement from OA/T2DM(-) and OA/T2DM(+) patients. Label-free proteomics was employed to study differences in protein abundance and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) for spatially resolved-lipid analysis. Label-free proteomic analysis showed differences between OA/T2DM(-) and OA/T2DM(+) phenotypes in several metabolic pathways such as lipid regulation. Interestingly, phospholipase A2 protein was found increased within the OA/T2DM(+) cohort. In addition, MALDI-MSI experiments revealed that phosphatidylcholine and sphingomyelin species were characteristic of the OA/T2DM(-) group, whereas lysolipids were more characteristic of the OA/T2DM(+) phenotype. The data also pointed out differences in phospholipid content between superficial and deep layers of the cartilage. Our study shows distinctively different lipid and protein profiles between OA/T2DM(-) and OA/T2DM(+) human cartilage, demonstrating the importance of subclassification of the OA disease for better personalized treatments.

Mass spectrometry imaging (MSI) provides insight into the molecular distribution of a broad range of compounds and, therefore, is frequently applied in the pharmaceutical industry. Pharmacokinetic and toxicological studies deploy MSI to localize potential drugs and their metabolites in biological tissues but currently require other analytical tools to quantify these pharmaceutical compounds in the same tissues. Quantitative mass spectrometry imaging (Q-MSI) is a field with challenges due to the high biological variability in samples combined with the limited sample cleanup and separation strategies available prior to MSI. In consequence, more selectivity in MSI instruments is required. This can be provided by multiple reaction monitoring (MRM) which uses specific precursor ion-product ion transitions. This targeted approach is in particular suitable for pharmaceutical compounds because their molecular identity is known prior to analysis. In this work, we compared different analytical platforms to assess the performance of MRM detection compared to other MS instruments/MS modes used in a Q-MSI workflow for two drug candidates (A and B). Limit of detection (LOD), linearity, and precision and accuracy of high and low quality control (QC) samples were compared between MS instruments/modes. MRM mode on a triple quadrupole mass spectrometer (QqQ) provided the best overall performance with the following results for compounds A and B: LOD 35.5 and 2.5 μg/g tissue, R2 0.97 and 0.98 linearity, relative standard deviation QC R2 0.86–0.98 and 0.86–0.98 linearity, relative standard deviation QC Graphical abstract

Highlights • Lipids change overtime in normal fascial healing in the early post-surgery period. • Specific lipid species are correlated with the changes of inflammation cells and fibroblasts. • Lipid species in the present study are considered as predictive markers for the formation of incisional hernia., Background Failure of fascial healing in the abdominal wall can result in incisional hernia, which is one of the most common complications after laparotomy. Understanding the molecular healing process of abdominal fascia may provide lipid markers of incisional hernia or therapeutic targets that allow prevention or treatment of incisional hernias. Purpose This study aims to investigate temporal and in situ changes of lipids during the normal healing process of abdominal fascia in the first postoperative week. Methods Open hemicolectomy was performed in a total of 35 Wistar rats. The midline fascia was closed identically for all rats using a single continuous suturing technique. These animals were sacrificed with equal numbers (n = 5) at each of 7-time points (6, 12, 24, 48, 72, 120, and 168 h. The local and temporal changes of lipids were examined with mass spectrometry imaging and correlated to histologically scored changes during healing using hematoxylin and eosin staining. Results Two phosphatidylcholine lipid species (PC O-38:5 and PC 38:4) and one phosphatidylethanolamine lipid (PE O-16:1_20:4) were found to significantly correlate with temporal changes of inflammation. A phosphatidylcholine (PC 32:0) and a monosialodihexosylganglioside (GM3 34:1;2) were found to correlate with fibroblast cell growth. Conclusion Glycerophospholipids and gangliosides are strongly involved in the normal healing process of abdominal fascia and their locally fluctuating concentrations are considered as potential lipid markers and therapeutic targets of fascial healing.

Liver resident CD8 T cells have an essential role in immunopathology in a mouse model of nonalcoholic steatohepatitis, by becoming auto-aggressive following sequential transcriptional and metabolic activation steps . Nonalcoholic steatohepatitis (NASH) is a manifestation of systemic metabolic disease related to obesity, and causes liver disease and cancer(1,2). The accumulation of metabolites leads to cell stress and inflammation in the liver(3), but mechanistic understandings of liver damage in NASH are incomplete. Here, using a preclinical mouse model that displays key features of human NASH (hereafter, NASH mice), we found an indispensable role for T cells in liver immunopathology. We detected the hepatic accumulation of CD8 T cells with phenotypes that combined tissue residency (CXCR6) with effector (granzyme) and exhaustion (PD1) characteristics. Liver CXCR6(+) CD8 T cells were characterized by low activity of the FOXO1 transcription factor, and were abundant in NASH mice and in patients with NASH. Mechanistically, IL-15 induced FOXO1 downregulation and CXCR6 upregulation, which together rendered liver-resident CXCR6(+) CD8 T cells susceptible to metabolic stimuli (including acetate and extracellular ATP) and collectively triggered auto-aggression. CXCR6(+) CD8 T cells from the livers of NASH mice or of patients with NASH had similar transcriptional signatures, and showed auto-aggressive killing of cells in an MHC-class-I-independent fashion after signalling through P2X7 purinergic receptors. This killing by auto-aggressive CD8 T cells fundamentally differed from that by antigen-specific cells, which mechanistically distinguishes auto-aggressive and protective T cell immunity.

Real-time tissue classifiers based on molecular patterns are emerging tools for fast tumor diagnosis. Here, we used rapid evaporative ionization mass spectrometry (REIMS) and multivariate statistical analysis (principal component analysis–linear discriminant analysis) to classify tissues with subsequent comparison to gold standard histopathology. We explored whether REIMS lipid patterns can identify human liver tumors and improve the rapid characterization of their underlying metabolic features. REIMS-based classification of liver parenchyma (LP), hepatocellular carcinoma (HCC), and metastatic adenocarcinoma (MAC) reached an accuracy of 98.3%. Lipid patterns of LP were more similar to those of HCC than to those of MAC and allowed clear distinction between primary and metastatic liver tumors. HCC lipid patterns were more heterogeneous than those of MAC, which is consistent with the variation seen in the histopathological phenotype. A common ceramide pattern discriminated necrotic from viable tumor in MAC with 92.9% accuracy and in other human tumors. Targeted analysis of ceramide and related sphingolipid mass features in necrotic tissues may provide a new classification of tumor cell death based on metabolic shifts. Real-time lipid patterns may have a role in future clinical decision-making in cancer precision medicine. Real-time lipid patterns can identify liver tumors and their inter-tumor and intra-tumor heterogeneity. Ceramides and related sphingolipids are a common feature of necrotic tumors and can characterize the tumor phenotype based on metabolic shifts relevant for cell death pathways. Lipid patterns have the potential to improve clinical decision-making in the near future.

Mass spectrometry imaging (MSI) can analyze the spatial distribution of hundreds of different molecules directly from tissue sections usually placed on conductive glass slides to provide conductivity on the sample surface. Additional experiments are often required for molecular identification using consecutive sections on membrane slides compatible with laser capture microdissection (LMD). In this work, we demonstrate for the first time the use of a single conductive slide for both matrix-assisted laser desorption ionization (MALDI)-MSI and direct proteomics. In this workflow, regions of interest can be directly ablated with LMD while preserving protein integrity. These results offer an alternative for MSI-based multimodal spatial-omics.

Mass spectrometry imaging (MSI) has become an indispensible tool for spatially resolved molecular investigation of tissues. One of the key application areas is biomedical research, where matrix-assisted laser desorption/ionization (MALDI) MSI is predominantly used due to its high-throughput capability, flexibility in the molecular class to investigate, and ability to achieve single cell spatial resolution. While many of the initial technical challenges have now been resolved, so-called batch effects, a phenomenon already known from other omics fields, appear to significantly impede reliable comparison of data from particular midsized studies typically performed in translational clinical research. This critical insight will discuss at what levels (pixel, section, slide, time, and location) batch effects can manifest themselves in MALDI-MSI data and what consequences this might have for biomarker discovery or multivariate classification. Finally, measures are presented that could be taken to recognize and/or minimize these potentially detrimental effects, and an outlook is provided on what is still needed to ultimately overcome these effects.

The hydrogenative conversions of the biobased platform molecules 4-hydroxycyclopent-2-enone and cyclopentane-1,3-dione to their corresponding 1,3-diols are established using a pre-activated Knolker-type iron catalyst. The catalyst exhibits a high selectivity for ketone reduction, and does not induce dehydration. Moreover, by using different substituents of the ligand, the cis-trans ratio of the products can be affected substantially. A decent compatibility of this catalytic system with various structurally related substrates is demonstrated.

ESX-1 is a major virulence factor of Mycobacterium tuberculosis, a secretion machinery directly involved in the survival of the microorganism from the immune system defence. It disrupts the phagosome membrane of the host cell through a contact-dependent mechanism. Recently, the structure of the inner-membrane core complex of the homologous ESX-3 and ESX-5 was resolved; however, the elements involved in the secretion through the outer membrane or those acting on the host cell membrane are unknown. Protein substrates might form this missing element. Here, we describe the oligomerisation process of the ESX-1 substrate EspB, which occurs upon cleavage of its C-terminal region and is favoured by an acidic environment. Cryo-electron microscopy data shows that quaternary structure of EspB is conserved across slow growing species, but not in the fast growing M. smegmatis. EspB assembles into a channel with dimensions and characteristics suitable for the transit of ESX-1 substrates, as shown by the presence of another EspB trapped within. Our results provide insight into the structure and assembly of EspB, and suggests a possible function as a structural element of ESX-1., Graphical abstract Image 1, Highlights • Acidic environment and removal of C-terminal end primes EspB oligomerisation. • EspB from slow-growing species oligomerise while EspB from M. smegmatis does not. • New structure of EspB from M. marinum presented. • Residues within the C-terminal region interact with hydrophobic surfaces. • Monomer inside the channel supports the idea that EspB is part of ESX-1 machinery.

Cell death & disease 12(1), 95 (2021). doi:10.1038/s41419-020-03379-9, Published by Nature Publishing Group, London [u.a.]

Target-controlled infusion systems are increasingly used to administer intravenous anaesthetic drugs to achieve a user-specified plasma or effect-site target concentration. While several studies have investigated the ability of the underlying pharmacokinetic-dynamic models to predict plasma concentrations, there are no data on their performance in predicting drug concentrations in the human brain. We assessed the predictive performance of the Marsh propofol model and Minto remifentanil model for plasma and brain tissue concentrations. Plasma samples were obtained during neurosurgery from 38 patients, and brain tissue samples from nine patients. Propofol and remifentanil concentrations were measured using gas chromatography mass spectrometry and liquid chromatography tandem mass spectrometry. Data were analysed from the nine patients in whom both plasma and brain samples were simultaneously obtained. For the Minto model (five patients), the median performance error was 72% for plasma and -14% for brain tissue concentration predictions. The model tended to underestimate plasma remifentanil concentrations, and to overestimate brain tissue remifentanil concentrations. For the Marsh model (five patients), the median prediction errors for plasma and brain tissue concentrations were 12% and 81%, respectively. However, when the data from all blood propofol assays (36 patients) were analysed, the median prediction error was 11%, with overprediction in 15 (42%) patients and underprediction in 21 (58%). These findings confirm earlier reports demonstrating inaccuracy for commonly used pharmacokinetic-dynamic models for plasma concentrations and extend these findings to the prediction of effect-site concentrations.

We describe an innovative use for the recently reported fast lipid analysis technique (FLAT) that allows for the generation of MALDI tandem mass spectrometry data suitable for lipid A structure analysis directly from a single Gram-negative bacterial colony. We refer to this tandem MS version of FLAT as FLATn. Neither technique requires sophisticated sample preparation beyond the selection of a single bacterial colony, which significantly reduces overall analysis time (∼1 h), as compared to conventional methods. Moreover, the tandem mass spectra generated by FLATn provides comprehensive information on fragments of lipid A, for example, ester bonded acyl chain dissociations, cross-ring cleavages, and glycosidic bond dissociations, all of which allow the facile determination of novel lipid A structures or confirmation of expected structures. In addition to generating tandem mass spectra directly from single colonies, we also show that FLATn can be used to analyze lipid A structures taken directly from a complex biological clinical sample without the need for ex vivo growth. From a urine sample from a patient with an E. coli infection, FLATn identified the organism and demonstrated that this clinical isolate carried the mobile colistin resistance-1 gene (mcr-1) that results in the addition of a phosphoethanolamine moiety and subsequently resistance to the antimicrobial, colistin (polymyxin E). Moreover, FLATn allowed for the determination of the existence of a structural isomer in E. coli lipid A that had either a 1- or 4'-phosphate group modification by phosphoethanolamine generated by a change of bacterial culture conditions.

Skeletal stem cells (SSCs, or mesenchymal stromal cells typically referred to as mesenchymal stem cells from the bone marrow) are a dynamic progenitor population that can enter quiescence, self-renew or differentiate depending on regenerative demand and cues from their niche environment. However, ex vivo, in culture, they are grown typically on hard polystyrene surfaces, and this leads to rapid loss of the SSC phenotype. While materials are being developed that can control SSC growth and differentiation, very few examples of dynamic interfaces that reflect the plastic nature of the stem cells have, to date, been developed. Achieving such interfaces is challenging because of competing needs: growing SSCs require lower cell adhesion and intracellular tension while differentiation to, for example, bone-forming osteoblasts requires increased adhesion and intracellular tension. We previously reported a dynamic interface where the cell adhesion tripeptide arginine-glycine-aspartic acid (RGD) was presented to the cells upon activation by user-added elastase that cleaved a bulky blocking group hiding RGD from the cells. This allowed for a growth phase while the blocking group was in place and the cells could only form smaller adhesions, followed by an osteoblast differentiation phase that was induced after elastase was added which triggered exposure of RGD and subsequent cell adhesion and contraction. Here, we aimed to develop an autonomous system where the surface is activated according to the need of the cell by using matrix metalloprotease (MMP) cleavable peptide sequences to remove the blocking group with the hypothesis that the SSCs would produce higher levels of MMP as the cells reached confluence. The current studies demonstrate that SSCs produce active MMP-2 that can cleave functional groups on a surface. We also demonstrate that SSCs can grow on the uncleaved surface and, with time, produce osteogenic marker proteins on the MMP-responsive surface. These studies demonstrate the concept for cell-controlled surfaces that can modulate adhesion and phenotype with significant implications for stem cell phenotype modulation.

Radical resection for patients with oral cavity cancer remains challenging. Rapid evaporative ionization mass spectrometry (REIMS) of electrosurgical vapors has been reported for real-time classification of normal and tumor tissues for numerous surgical applications. However, the infiltrative pattern of invasion of oral squamous cell carcinomas (OSCC) challenges the ability of REIMS to detect low amounts of tumor cells. We evaluate REIMS sensitivity to determine the minimal amount of detected tumors cells during oral cavity cancer surgery. A total of 11 OSCC patients were included in this study. The tissue classification based on 185 REIMS ex vivo metabolic profiles from five patients was compared to histopathology classification using multivariate analysis and leave-one-patient-out cross-validation. Vapors were analyzed in vivo by REIMS during four glossectomies. Complementary desorption electrospray ionization-mass spectrometry imaging (DESI-MSI) was employed to map tissue heterogeneity on six oral cavity sections to support REIMS findings. REIMS sensitivity was assessed with a new cell-based assay consisting of mixtures of cell lines (tumor, myoblasts, keratinocytes). Our results depict REIMS classified tumor and soft tissues with 96.8% accuracy. In vivo REIMS generated intense mass spectrometric signals. REIMS detected 10% of tumor cells mixed with 90% myoblasts with 83% sensitivity and 82% specificity. DESI-MSI underlined distinct metabolic profiles of nerve features and a metabolic shift phosphatidylethanolamine PE(O-16:1/18:2))/cholesterol sulfate common to both mucosal maturation and OSCC differentiation. In conclusion, the assessment of tissue heterogeneity with DESI-MSI and REIMS sensitivity with cell mixtures characterized sensitive metabolic profiles toward in vivo tissue recognition during oral cavity cancer surgeries.

Human cells produce thousands of lipids that change during cell differentiation and can vary across individual cells of the same type. However, we are only starting to characterize the function of these cell-to-cell differences in lipid composition. Here, we measured the lipidomes and transcriptomes of individual human dermal fibroblasts by coupling high-resolution mass spectrometry imaging with single-cell transcriptomics. We found that the cell-to-cell variations of specific lipid metabolic pathways contribute to the establishment of cell states involved in the organization of skin architecture. Sphingolipid composition is shown to define fibroblast subpopulations, with sphingolipid metabolic rewiring driving cell-state transitions. Therefore, cell-to-cell lipid heterogeneity affects the determination of cell states, adding a new regulatory component to the self-organization of multicellular systems.

The molecular pathology of breast cancer is challenging due to the complex heterogeneity of cellular subtypes. The ability to directly identify and visualize cell subtype distribution at the single-cell level within a tissue section enables precise and rapid diagnosis and prognosis. Here, we applied mass spectrometry imaging (MSI) to acquire and visualize the molecular profiles at the single-cell and subcellular levels of 14 different breast cancer cell lines. We built a molecular library of genetically well-characterized cell lines. Multistep processing, including deep learning, resulted in a breast cancer subtype, the cancer's hormone status, and a genotypic recognition model based on metabolic phenotypes with cross-validation rates of up to 97%. Moreover, we applied our single-cell-based recognition models to complex tissue samples, identifying cell subtypes in tissue context within seconds during measurement. These data demonstrate "on the spot" digital pathology at the single-cell level using MSI, and they provide a framework for fast and accurate high spatial resolution diagnostics and prognostics.

Time-of-flight (TOF) systems are one of the most widely used mass analyzers in native mass spectrometry (nMS) for the analysis of non-covalent multiply charged bio-macromolecular assemblies (MMAs). Typically, microchannel plates (MCPs) are employed for high mass native ion detection in TOF MS. MCPs are well known for their reduced detection efficiency when impinged by large slow moving ions. Here, a position- and time-sensitive Timepix (TPX) detector has been added to the back of a dual MCP stack to study the key factors that affect MCP performance for MMA ions generated by nMS. The footprint size of the secondary electron cloud generated by the MCP on the TPX for each individual ion event is analyzed as a measure of MCP performance at each mass-to-charge (m/z) value and resulted in a Poisson distribution. This allowed us to investigate the dependency of ion mass, ion charge, ion velocity, acceleration voltage, and MCP bias voltage on MCP response in the high mass low velocity regime. The study of measurement ranges; ion mass = 195 to 802,000 Da, ion velocity = 8.4 to 67.4 km/s, and ion charge = 1+ to 72+, extended the previously examined mass range and characterized MCP performance for multiply charged species. We derived a MCP performance equation based on two independent ion properties, ion mass and charge, from these results, which enables rapid MCP tuning for single MMA ion detection.

INTRODUCTION: The main goal of brain tumour surgery is to maximize tumour resection while avoiding neurological deficits. Accurate characterization of tissue and delineation of resection margins are, therefore, essential to achieve optimal surgical results. OBJECTIVES: The primary objective of this study was to develop and validate a mass spectrometry- based technique for the molecular characterization of high- and low-grade glioma tissue during surgery. METHODS: An electrosurgical knife is connected to a mass spectrometer (iKnife). Using this system, an aerosol created during electrosurgical resection is aspirated to a mass spectrometer to determine the molecular profile of the tissue within seconds. This rapid evaporative ionization mass spectrometry (REIMS) technique is used to create a chemical profile database and develop a real-time tissue recognition system based on machine learning. RESULTS: Classification models were built by analysing biopsies from 36 patients who underwent brain tumour resection. Our multivariate statistical model could differentiate between astrocytoma grade II and III, glioblastoma, oligodendroglioma grade II and III, and normal brain tissue with an 88% overall accuracy. Astrocytoma and oligodendroglioma grade II were separated from normal brain with a 96% correct classification rate. REIMS could differentiate between different percentages of GBM with 99.2% sensitivity and different percentages of astrocytoma grade II with 97.5% sensitivity. CONCLUSION: Real-time information during electrosurgical dissection can improve intra-operative decision-making, leading to a more accurate tumour removal for different glioma subtypes. ispartof: JOURNAL OF MASS SPECTROMETRY AND ADVANCES IN THE CLINICAL LAB vol:24 pages:80-89 ispartof: location:Netherlands status: published

Bile acids are steroid compounds involved in biological mechanisms of neurodegenerative diseases making them potential biomarkers for diagnosis or treatment. These compounds exist as structural and conformational isomers, which hinder distinguishing them in physiological processes. We aimed to develop tandem mass spectrometry-ion mobility spectrometry (MS/MS-IMS) methodologies to explore and understand the behaviour of isomeric steroids in the gas-phase and rapidly separate them. Unlike previously published ion mobility data, various isomers were investigated in mixtures to better mimic complex (pre-) clinical samples. The experimental collision cross sections (CCS)s were compared to the theoretical CCS values for an in-depth analysis of isomeric ions' behaviour in the gas-phase. Based on density-functional theory, we identified the impact of adduct positioning on the 3D conformation of enantiomers, diastereomers and structural isomers. The curling of the large side chains hedged the small differences among the isomers and lowered the CCS values. On the other hand, fragmenting off the identical side branches as well as imposing the bending of the steroid ring resulted in ion mobility differentiation. Careful data evaluation revealed the tendency of isomers to form homo-cluster in the mixture solutions and assist the separation. Our fundamental and experimental findings enable the ion mobility separation of isomeric steroids to be predicted. The introduced rapid and optimal MS/MS-IMS analytical methodology can be applied to distinguish isomeric bile acids both in a solution and potentially in patients' tissue samples, and consequently, reveal their molecular pathways.

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) imaging provides molecular speciation at the micrometer scale, while the penetration depth of the primary ion beam is limited to the top-layers of a sample. These combined properties make TOF-SIMS potentially an ideal technique to study oil-gas interfaces. TOF-SIMS spectra of three crude oils were evaluated, and only low-mass fragment ions could be assigned to molecular structures unambiguously. Films of crude oils were incubated under air, oil vapor, or water vapor for various times. TOF-SIMS images of a polar crude oil revealed feeble structures of similar to 10 mu m large round patches that grew to similar to 30 mu m large crystals when incubated under air and oil vapor, respectively. Principal component analysis of the images showed that the continuous phase had typical aromatic signatures, while the patches and crystals had alkane-like characteristics. No features showed up when the oil film was incubated under water vapor, which indicated that saturated water vapor prevented the accumulation of nonpolar alkane-like compounds at the oil-gas interface. These examples showed that crude oils do not behave as dead fluids but that their constituents accumulate at the oil-gas interfaces in a dynamic way.

We developed a method to directly detect and map the Gram-negative bacterial virulence factor lipid A derived from lipopolysaccharide (LPS) by coupling acid hydrolysis with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). As the structure of lipid A (endotoxin) determines the innate immune outcome during infection, the ability to map its location within an infected organ or animal is needed to understand localized inflammatory responses that results during host-pathogen interactions. We previously demonstrated detec- tion of free lipid A from infected tissue; however detection of lipid A derived from intact (smooth) LPS from host-pathogen MSI studies, proved elusive. Here, we detected LPS-derived lipid A from the Gram-negative pathogens, Escherichia coli (Ec, m/z 1797) and Pseudomonas aeruginosa (Pa, m/z 1446) using on-tissue acid hydrolysis to cleave the glycosidic linkage between the polysaccharide (core and 0-antigen) and lipid A moieties of LPS. Using accurate mass methods, the ion corresponding to the major Ec and Pa lipid A species (m/z 1797 and 1446, respectively) were unambiguously discriminated from complex tissue substrates. Further, we evaluated potential delocalization and signal loss of other tissue lipids and found no evidence for either, making this LPS-to-Lipid A-MSI (LLA-MSI) method, compatible with simultaneous host-pathogen lipid imaging following acid hydrolysis. This spatially sensitive technique is the first step in mapping host-influenced de novo lipid A modifications, such as those associated with antimicrobial resistance phenotypes, during Gram-negative bacterial infection and will advance our understanding of the host-pathogen interface.

The ability to spatially resolve the chemical distribution of compounds on a surface is important in many applications ranging from biological to material science. To this extent, we have recently introduced a hybrid atomic force microscopy (AFM)-mass spectrometry (MS) system for direct thermal desorption and pyrolysis of material with nanoscale chemical resolution. However, spatially resolved direct surface heating using local thermal desorption becomes challenging on material surfaces with low melting points, because the material will undergo a melting phase transition due to heat dissipation prior to onset of thermal desorption. Therefore, we developed an approach using mechanical sampling and collection of surface materials on an AFM cantilever probe tip for real-time analysis directly from the AFM tip. This approach allows for material to be concentrated directly onto the probe for subsequent MS analysis. We evaluate the performance metrics of the technique and demonstrate localized MS sampling from a candelilla wax matrix containing UV stabilizers avobenzone and oxinoxate from areas down to 250 nm × 250 nm. Overall, this approach removes heat dissipation into the bulk material allowing for a faster desorption and concentration of the gas phase analyte from a single heating pulse enabling higher signal levels from a given amount of material in a single sampling spot. Graphical abstract

In the past decades, matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has been applied to a broad range of biological samples, e.g., forensics and preclinical samples. The use of MALDI-MSI for the analysis of bone tissue has been limited due to the insulating properties of the material but more importantly the absence of a proper sample preparation protocol for undecalcified bone tissue. Undecalcified sections are preferred to retain sample integrity as much as possible or to study the tissue-bone bio interface in particular. Here, we optimized the sample preparation protocol of undecalcified bone samples, aimed at both targeted and untargeted applications for forensic and preclinical applications, respectively. Different concentrations of gelatin and carboxymethyl cellulose (CMC) were tested as embedding materials. The composition of 20% gelatin and 7.5% CMC showed to support the tissue best while sectioning. Bone tissue has to be sectioned with a tungsten carbide knife in a longitudinal fashion, while the sections need to be supported with double-sided tapes to maintain the morphology of the tissue. The developed sectioning method was shown to be applicable on rat and mouse as well as human bone samples. Targeted (methadone and EDDP) as well as untargeted (unknown lipids) detection was demonstrated. DHB proved to be the most suitable matrix for the detection of methadone and EDDP in positive ion mode. The limit of detection (LOD) is estimated to approximately 50 pg/spot on bone tissue. The protocol was successfully applied to detect the presence of methadone and EDDP in a dosed rat femur and a dosed human clavicle. The best matrices for the untargeted detection of unknown lipids in mouse hind legs in positive ion mode were CHCA and DHB based on the number of tissue-specific peaks and signal-to-noise ratios. The developed and optimized sample preparation method, applicable on animal and human bones, opens the door for future forensic and (pre)clinical investigations. Electronic supplementary material The online version of this article (10.1007/s00216-020-02920-1) contains supplementary material, which is available to authorized users.

Cardiolipins (CLs) are an important, regulated lipid class both in prokaryotic and eukaryotic cells, yet they remain largely unexplored by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) in tissues. To date, no in-depth optimization studies of label-free visualization of CLs in complex biological samples have been reported. Here we report a streamlined modification to our previously reported MALDI-MSI method for detection of endogenous CLs in prokaryotic and eukaryotic cells based on preparation with norharmane (NRM) matrix. Notably, the use of NRM matrix permitted sensitive detection (4.7 pg/mm(2)) of spotted CL synthetic standards. By contrast, four other MALDI matrices commonly used for lipid analysis failed to generate CL ions. Using this NRM-based method, endogenous CLs were detected from two types of complex biological samples: dried bacterial arrays and mouse tissue sections. In both cases, using NRM resulted in a better signal/noise for CL ions than the other matrices. Furthermore, inclusion of a washing step improved CL detection from tissue and this combined tissue preparation method (washing and NRM matrix) was used to profile normal mouse lung. Mouse lung yielded 26 unique CLs that were mapped and identified. Consistent with previous findings, CLs containing polyunsaturated fatty acids (PUFAs) were found in abundance in the airway and vascular features of the lung. This work represents a comprehensive investigation of detection conditions for CL using MALDI-MSI in complex biological samples that resulted in a streamlined method that enables future studies of the biological role(s) of CL in tissue.

The large‐scale and label‐free molecular characterization of single cells in their natural tissue habitat remains a major challenge in molecular biology. We present a method that integrates morphometric image analysis to delineate and classify individual cells with their single‐cell‐specific molecular profiles. This approach provides a new means to study spatial biological processes such as cancer field effects and the relationship between morphometric and molecular features., Understanding how cells regulate their bioenergetic demands, adjust to the microenvironment, or respond to stimuli is the aim of molecular biology. Yet, methods able to describe the molecular content, histological identity, and spatial context of a single cell do not exist. The presented method integrates morphometric image analysis to delineate and classify individual cells with their cell‐specific molecular profiles obtained by MALDI‐MSI.

Low-back pain affects 80 % of the world population at some point in their lives and 40 % of the cases are attributed to intervertebral disc (IVD) degeneration. Over the years, many animal models have been developed for the evaluation of prevention and treatment strategies for IVD degeneration. Ex vivo organ culture systems have also been developed to better control mechanical loading and biochemical conditions, but a reproducible ex vivo model that mimics moderate human disc degeneration is lacking. The present study described an ex vivo caprine IVD degeneration model that simulated the changes seen in the nucleus pulposus during moderate human disc degeneration.Following pre-load under diurnal, simulated physiological loading (SPL) conditions, lumbar caprine IVDs were degenerated enzymatically by injecting collagenase and chondroitinase ABC (cABC). After digestion, IVDs were subjected to SPL for 7 d. No intervention and phosphate-buffered saline injection were used as controls. Disc deformation was continuously monitored to assess disc height recovery. Histology and immunohistochemistry were performed to determine the histological grade of degeneration, matrix expression, degrading enzyme and catabolic cytokine expression.Injection of collagenase and cABC irreversibly affected the disc mechanical properties. A decrease in extracellular matrix components was found, along with a consistent increase in degradative enzymes and catabolic proteins [interleukin (IL)-1 beta, -8 and vascular endothelial growth factor (VEGF)]. The changes observed were commensurate with those seen in moderate human-IVD degeneration. This model should allow for controlled ex vivo testing of potential biological, cellular and biomaterial treatments of moderate human-IVD degeneration.

Vitamin D plays a key role in the maintenance of calcium/phosphate homeostasis and elicits biological effects that are relevant to immune function and metabolism. It is predominantly formed through UV exposure in the skin by conversion of 7-dehydrocholsterol (vitamin D3). The clinical biomarker, 25-hydroxyvitamin D (25-(OH)-D), is enzymatically generated in the liver with the active hormone 1,25-dihydroxyvitamin D then formed under classical endocrine control in the kidney. Vitamin D metabolites are measured in biomatrices by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In LC-MS/MS, chemical derivatization (CD) approaches have been employed to achieve the desired limit of quantitation. Recently, matrix-assisted laser desorption/ionization (MALDI) has also been reported as an alternative method. However, these quantitative approaches do not offer any spatial information. Mass spectrometry imaging (MSI) has been proven to be a powerful tool to image the spatial distribution of molecules from the surface of biological tissue sections. On-tissue chemical derivatization (OTCD) enables MSI to image molecules with poor ionization efficiently. In this technical report, several derivatization reagents and OTCD methods were evaluated using different MSI ionization techniques. Here, a method for detection and spatial distribution of vitamin D metabolites in murine kidney tissue sections using an OTCD-MALDI-MSI platform is presented. Moreover, the suitability of using the Bruker ImagePrep for OTCD-based platforms has been demonstrated. Importantly, this method opens the door for expanding the range of other poor ionizable molecules that can be studied by OTCD-MSI by adapting existing CD methods.