We explore mass-resolved imaging of fragments generated from single macromolecular assembly (MMA) ions on a custom-built Orbitrap/time-of-flight (TOF) mass spectrometer with integrated UV photodissociation (UVPD) and a position- and time-sensitive Timepix3 imaging detector assembly. We postulated that the 2D detector images provide information about the 3D geometry of the MMAs in the gas phase as the TOF analyzer has the ability to retain the relative positions of the product ions following the fragmentation process and until they reach the imaging detector, when the fragmentation occurs at the level of single-precursor MMA ion. We demonstrate that the Orbitrap/TOF mass spectrometer enables fragmentation at the single-precursor MMA ion level using dimeric and tetrameric noncovalently bound assemblies. Timepix3-derived relative position data from single-precursor fragmentation events of two distinct tetrameric MMAs reveal different higher-order structural signatures that enable their differentiation. Furthermore, mapping these single-precursor fragmentation events to possible dissociation pathways provides insight into the underlying dissociation mechanisms. Overall, this study demonstrates the potential of single-ion mass-resolved imaging to understand UVPD dissociation mechanisms, fragmentation pathways of MMA ions, and their higher-order structure.

Understanding tissue complexity requires spatially resolved multi-omics data at single-cell resolution. Here, we present a workflow integrating high-resolution matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) with Xenium spatial transcriptomics (SPT) on a single tissue section. This strategy ensures pixel-scale spatial correspondence between metabolic and transcriptomic features, avoiding misalignment issues of serial sections, where even minor offsets result in sampling different cells. We investigated MALDI-MSI compatibility with downstream SPT revealing that the number of transcripts per cell decreased by ~ 30% after MSI, whilst cell recovery and cell-type assignments are preserved. Validated using mouse brain and demonstrated using human glioblastoma tissues, we achieved pixel-scale modality co-registration, enabling per-cell MALDI spectra extraction aligned with gene expression. Integrated clustering revealed enhanced cell-type resolution and identified metabolic heterogeneity within transcriptionally defined populations. This facilitates precise correlations of a cell's function and its biochemical state, providing a holistic view of cellular function, heterogeneity, and interaction in health and disease. Our workflow provides a scalable path to multi-omic atlases, advancing both data integration and translational research.

The prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) progressing to metabolic dysfunction-associated steatohepatitis (MASH), characterized by hepatic inflammation, has significantly increased in recent years due to unhealthy dietary practices and sedentary lifestyles. Cathepsin D (CTSD), a lysosomal protease involved in lipid homeostasis, is linked to abnormal lipid metabolism and inflammation in MASH. Although primarily intracellular, CTSD can be secreted extracellularly. Our previous proteomics research has shown that inhibition of extracellular CTSD results in more anti-inflammatory effects and fewer potential side effects compared to intracellular CTSD inhibition. However, the correlation between reduced side effects and alterations in the hepatic lipid composition remains unknown. This study aims to investigate the correlation between intra- and extracellular CTSD inhibition and potential alterations in the hepatic lipid composition in MASH. Low-density lipoprotein receptor knockout (Ldlr-/-) mice were fed a high-fat diet for 10 weeks and received subcutaneous injections every 2 days of vehicle, intracellular CTSD inhibitor (GA-12), or extracellular CTSD inhibitor (CTD-002). Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was used to visualize and compare the lipid composition in liver tissues. Hepatic phosphatidylcholine remodeling was observed with both inhibitors, suggesting their therapeutic potential in treating MASH. Treatment with an intracellular CTSD inhibitor resulted in elevated levels of cardiolipin, reactive oxygen species, phosphatidylinositol, phosphatidylethanolamine, and lipids that are linked to mitochondrial dysfunction and inflammation, and induced more oxidative stress. The observed modifications in lipid composition demonstrate the clinical advantages of extracellular CTSD inhibition as a potentially beneficial therapeutic approach for MASH.

The incidence of osteoarthritis (OA) has been expected to increase due to an aging population, as well as an increased incidence of intra-articular (osteo-) chondral damage. Lipids have already been shown to be involved in the inflammatory process of OA. This study aims at revealing region-specific lipid profiles of the infrapatellar fat pad (IPFP) of OA or cartilage defect patients by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), which could be used as biomarkers for early OA detection. A higher presence of phospholipids was found in OA patients compared with cartilage defect patients. In addition, a higher abundance of ether-linked phosphatidylethanolamines (PE O-s) containing arachidonic acid was specifically found in OA patients compared with cartilage defect patients. These lipids were mainly found in the connective tissue of the IPFP. Specific lipid species were associated to OA patients compared with cartilage defect patients. PE O-s have been suggested as possible biomarkers for OA. As these were found more abundantly in the connective tissue, the IPFP's intra-tissue heterogeneity might play an important role in biomarker discovery, implying that the amount of fibrous tissue is associated with OA.

We discuss the design, development, and evaluation of an Orbitrap/time-of-flight (TOF) mass spectrometry (MS)-based instrument with integrated UV photodissociation (UVPD) and time/mass-to-charge ratio (m/z)-resolved imaging for the comprehensive study of the higher-order molecular structure of macromolecular assemblies (MMAs). A bespoke TOF analyzer has been coupled to the higher-energy collisional dissociation cell of an ultrahigh mass range hybrid quadrupole-Orbitrap MS. A 193 nm excimer laser was employed to photofragment MMA ions. A combination of microchannel plates (MCPs)-Timepix (TPX) quad and MCPs-phosphor screen-TPX3CAM assemblies have been used as axial and orthogonal imaging detectors, respectively. The instrument can operate in four different modes, where the UVPD-generated fragment ions from the native MMA ions can be measured with high-mass resolution or imaged in a mass-resolved manner to reveal the relative positions of the UVPD fragments postdissociation. This information is intended to be utilized for retrieving higher-order molecular structural details that include the conformation, subunit stoichiometry, and molecular interactions as well as to understand the dissociation dynamics of the MMAs in the gas phase.

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.

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 cm2. 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.

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.

Background Metabolic reprogramming is a common phenomenon in tumorigenesis and tumor progression. Amino acids are important mediators in cancer metabolism, and their kinetics in tumor tissue are far from being understood completely. Mass spectrometry imaging is capable to spatiotemporally trace important endogenous metabolites in biological tissue specimens. In this research, we studied L-[ring- 13 C 6 ]-labeled phenylalanine and tyrosine kinetics in a human non-small cell lung carcinoma (NSCLC) xenografted mouse model using matrix-assisted laser desorption/ionization Fourier-transform ion cyclotron resonance mass spectrometry imaging (MALDI-FTICR-MSI). Methods We investigated the L-[ring- 13 C 6 ]-Phenylalanine ( 13 C 6 -Phe) and L-[ring- 13 C 6 ]-Tyrosine ( 13 C 6 -Tyr) kinetics at 10 min ( n = 4), 30 min ( n = 3), and 60 min ( n = 4) after tracer injection and sham-treated group ( n = 3) at 10 min in mouse-xenograft lung tumor tissues by MALDI-FTICR-MSI. Results The dynamic changes in the spatial distributions of 19 out of 20 standard amino acids are observed in the tumor tissue. The highest abundance of 13 C 6 -Phe was detected in tumor tissue at 10 min after tracer injection and decreased progressively over time. The overall enrichment of 13 C 6 -Tyr showed a delayed temporal trend compared to 13 C 6 -Phe in tumor caused by the Phe-to-Tyr conversion process. Specifically, 13 C 6 -Phe and 13 C 6 -Tyr showed higher abundances in viable tumor regions compared to non-viable regions. Conclusions We demonstrated the spatiotemporal intra-tumoral distribution of the essential aromatic amino acid 13 C 6 -Phe and its de-novo synthesized metabolite 13 C 6 -Tyr by MALDI-FTICR-MSI. Our results explore for the first time local phenylalanine metabolism in the context of cancer tissue morphology. This opens a new way to understand amino acid metabolism within the tumor and its microenvironment.

Native mass spectrometry (native MS) has emerged as a powerful technique to study the structure and stoichiometry of large protein complexes. Traditionally, native MS has been performed on modified time-of-flight (TOF) systems combined with detectors that do not provide information on the arrival coordinates of each ion at the detector. In this study, we describe the implementation of a Timepix (TPX) pixelated detector on a modified orthogonal TOF (O-TOF) mass spectrometer for the analysis and imaging of native protein complexes. In this unique experimental setup, we have used the impact positions of the ions at the detector to visualize the effects of various ion optical parameters on the flight path of ions. We also demonstrate the ability to unambiguously detect and image individual ion events, providing the first report of single-ion imaging of protein complexes in native MS. Furthermore, the simultaneous space- and time-sensitive nature of the TPX detector was critical in the identification of the origin of an unexpected TOF signal. A signal that could easily be mistaken as a fragment of the protein complex was explicitly identified as a secondary electron signal arising from ion-surface collisions inside the TOF housing. This work significantly extends the mass range previously detected with the TPX and exemplifies the value of simultaneous space- and time-resolved detection in the study of ion optical processes and ion trajectories in TOF mass spectrometers.

The incidence of osteoarthritis (OA) is increasing in our younger population. OA development early in life is often related to cartilage damage, caused by (sport) injury or trauma. Detection of ear...

In osteoarthritis (OA), impairment of cartilage regeneration can be related to a defective chondrogenic differentiation of mesenchymal stromal cells (MSCs). Therefore, understanding the proteomic- and metabolomic-associated molecular events during the chondrogenesis of MSCs could provide alternative targets for therapeutic intervention. Here, a SILAC-based proteomic analysis identified 43 proteins related with metabolic pathways whose abundance was significantly altered during the chondrogenesis of OA human bone marrow MSCs (hBMSCs). Then, the level and distribution of metabolites was analyzed in these cells and healthy controls by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), leading to the recognition of characteristic metabolomic profiles at the early stages of differentiation. Finally, integrative pathway analysis showed that UDP-glucuronic acid synthesis and amino sugar metabolism were downregulated in OA hBMSCs during chondrogenesis compared with healthy cells. Alterations in these metabolic pathways may disturb the production of hyaluronic acid (HA) and other relevant cartilage extracellular matrix (ECM) components. This work provides a novel integrative insight into the molecular alterations of osteoarthritic MSCs and potential therapeutic targets for OA drug development through the enhancement of chondrogenesis.

Purpose: Drugs able to stimulate chondrogenic differentiation in mesenchymal stem cells (MSCs) located in the joint represent an attractive approach for developing chondroprotective treatments or disease-modifying osteoarthritis drugs (DMOADs). Therefore, elucidation of the mechanisms governing chondrogenic differentiation of MSCs can lead to the identification of new molecular markers that could be used as targets for new DMOADs therapies. The aim of this study is to investigate proteomic and metabolic-associated molecular events during the chondrogenesis of OA human bone MSCs (hBMSCs) in a 3D chondrogenic model, in order to characterize molecular pathways that can be potential targets for therapeutic intervention in OA. Methods: SILAC-labelled OA and control hBMSCs populations were cultured in 3D high-density pellet mass cultures or micromasses with a home-made chondrogenic differentiation medium containing 10 ng/ml TGF-β3 for 14 days. Total protein extracts from micromasses collected at day 2 and 14 of differentiation were quantitatively compared using gel-based liquid chromatography coupled to mass spectrometry. Gene expression levels of differentially abundant proteins were determined by quantitative real-time PCR assays. Additionally, sections of OA and control micromasses collected at day 2 and day 14 were compared by using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to identify potential metabolic alterations during the chondrogenic differentiation of OA hBMSCs. Multivariate statistical analysis was used to determine the significant metabolic differences between the two chondrogenic time points. Metabolite identification was based on tandem MS analysis in comparison with Human Metabolome Database and Metlin databases. MALDI images of differentially abundant metabolites were visualized with FlexImaging 4.1 software. Differentially expressed metabolites and proteins were further subjected to an integrated metabolic pathway analysis using MetaboAnalyst software 4.0. Results: SILAC-based quantitative proteomic analysis of OA hBMSCs revealed that 41 proteins showed statistically significant alterations in their abundance during chondrogenesis. According to Gene Ontology database, more than 40% of these proteins were involved in metabolic pathways, mainly glycolysis, UDP-glucuronic acid synthesis and pentose phosphate pathway (Table 1). Metabolomics analysis performed by MALDI-MSI revealed that hBMSCs from OA patients and control donors displayed a differential metabolic profile at day 2 and 14 of chondrogenesis. For instance, several fatty acids and sugar phosphates were found more abundant in OA hBMSCs after 14 days of chondrogenesis compared to control cells. To further elucidate the metabolic pathways perturbed in OA hBMSCs undergoing chondrogenic differentiation, we also performed an integrated pathway analysis combining proteomics and metabolomics data. Remarkably, we found three metabolic pathways significantly altered in OA compared to control. These pathways included pentose and glucuronic acid interconversion, amino sugar and nucleotide sugar metabolism and synthesis of unsaturated fatty acids (Figure 1A). Since glucuronic acid is a precursor of cartilage hyaluronic acid (HA) synthesis, we evaluated the changes in the abundance and spatial distribution of metabolites involved in the glucuronic acid interconversion in OA hBMSCs micromasses (Figure 1B). We demonstrated a clear correlation between the upregulation of UTP-glucose-1-phosphate uridylyltransferase (UGP2) (see Table 1) with the synthesis of UDP-glucose and the reduction in the abundance of glucose monophosphate after 14 days of chondrogenesis. Additionally, a significantly reduced gene expression of UDP-glucose 6-dehydrogenase (UGDH) was found at day 14 in OA by qRT-PCR (Figure 1C), leading to an accumulation of UDP-glucose in the micromasses collected at day 14. The downregulation of UGDH negatively affects UDP-glucuronic acid synthesis, which might alter HA production and therefore, other relevant cartilage extracellular matrix components. Conclusions: Our work demonstrated that the downregulation of UDP-glucuronic acid synthesis is a key metabolic regulator of chondrogenesis in OA hBMSCs. Alterations in UDP-glucuronic acid synthesis pathway provides molecular targets that could be deeply explored for future therapeutic development for treating cartilage degenerative diseases such as OA.Tabled 1List of chondrogenic-regulated proteins involved in metabolism after nanoLC-MS/MS analysis of hBMSCsAccession numberProtein namePeptides (95%)Ratio Heavy (14d)/Light (2d)p-valueMetabolic pathwayP04406Glyceraldehyde-3-phosphate dehydrogenase491,4220,010GlycolysisP00338L-lactate dehydrogenase A chain261,5750,000GlycolysisP00558Phosphoglycerate kinase 1241,4110,016GlycolysisP60174Triosephosphate isomerase171,8310,000GlycolysisP18669Phosphoglycerate mutase 1141,4020,000GlycolysisO60701UDP-glucose 6-dehydrogenase80,6830,041UDP-glucuronic acid synthesisQ044461,4-alpha-glucan-branching enzyme62,4110,005Glycogen synthesisP07195L-lactate dehydrogenase B chain41,9750,031GlycolysisP29401Transketolase41,6810,032Pentose phosphate pathwayP15121Aldose reductase32,7790,046Carbohydrate metabolic processP30041Peroxiredoxin-631,3840,004Lipid metabolismP37837Transaldolase33,2750,024Pentose phosphate pathwayQ16851UTP-glucose-1-phosphate uridylyltransferase21,4490,049UDP-glucuronic acid synthesisP98160Basement membrane-specific heparan sulfate proteoglycan core protein21,9400,030Glycosaminoglycan synthetic Open table in a new tab

We report a method that enables automated data-dependent acquisition of lipid tandem mass spectrometry data in parallel with a high-resolution mass spectrometry imaging experiment. The method does not increase the total image acquisition time and is combined with automatic structural assignments. This lipidome-per-pixel approach automatically identified and validated 104 unique molecular lipids and their spatial locations from rat cerebellar tissue.

Purpose: Inflammatory processes that occur during cartilage degenerative diseases, such as osteoarthritis (OA), result in more reactive oxygen species (ROS) as well as alterations in oxygen levels within the joint. Chondrocytes maintain the extracellular matrix (ECM) by keeping a fine balance between anabolic and catabolic activities. Chondrocytes are generally exposed to low oxygen levels (1-6%). When exposed to supraphysiological oxygen levels chondrocytes produce less ECM, shifting the balance to ECM breakdown. Using mass spectrometry imaging (MSI), we have previously shown that cholesterol levels were lower in physiological oxygen levels and that phosphatidylglycerol and cardiolipins were elevated in supraphysiological oxygen levels. To understand the biological implications of these changes in the lipidome, we studied the proteomic and metabolomics changes in chondrocyte pellet cultures under various oxygen tensions. Methods: Human OA chondrocytes were isolated from articular cartilage of three patients undergoing total knee replacement and were cultured for 7 days in 3D pellets in high or physiological oxygen levels (20% and 2.5%, respectively). Proteins were extracted followed by trypsin digestion. Nano LC-MS was performed on a Thermo Scientific (Dionex) Ultimate 3000 RSLCnano system coupled to a Q Exactive HF mass spectrometer (Thermo Scientific). Proteins were identified using data dependent acquisition (DDA) and protein fold changes between culture conditions analyzed with Proteome Discoverer. Pathway analysis of proteins with a fold change >2 and P-value <0.05 was performed using the database STRING 10.5. Additionally, succinate dehydrogenase (SDH) activity was assessed in chondrocyte monolayer cultures. To detect metabolic changes, MALDI imaging was employed. Chondrocyte cell pellets were embedded in 10% gelatin, cryosectioned and sprayed with 9-aminoacridine using the TM-sprayer (HTX Imaging). Mass spectra of each pixel (40 μm raster size) were acquired in the negative ion mode between 100 and 1000 Da with a Bruker 9.4T SolariX Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. Principal component analysis (PCA) and linear discriminant analysis (LDA) were used to search for spectral similarities and differences between the conditions. The human metabolome database was employed for metabolite identification. Results: Bottom-up proteomics identified 1144 common proteins in the chondrocyte pellet cultures. Label-free quantification revealed differences in the protein abundance between physiological and high oxygen levels. Pathway analysis showed a higher level of ECM remodeling in high oxygen levels and metabolic processes were elevated in physiological oxygen levels. In our previous study, we found changes in the lipidome that signified mitochondrial changes. Several mitochondrial proteins were elevated (fold change >2 ) in high oxygen conditions, including proteins that are part of the electron transport chain complex III, IV and V, superoxide dismutase and proteins involved in cell metabolism. SDH is present in complex II of the electron transport chain and its protein levels were not altered by oxygen. However, its activity showed a negative correlation with oxygen levels in monolayer cultures of chondrocytes. MALDI-MSI followed by LDA was used to identify oxygen induced changes in metabolites. Amongst others, we could identify adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP). Conclusions: Using multimodal mass spectrometry approaches, we show that human chondrocytes have a distinct, oxygen-dependent molecular profile. These changes in the lipidome, metabolome and proteome signify changes in mitochondria and may be a sign of elevated levels of ROS in high oxygen conditions. These mitochondrial changes may explain why chondrocytes perform poorly and lose their phenotype in supraphysiological oxygen levels and cartilage degenerative disease Targeting these mitochondrial changes may restore the balance between anabolic and catabolic activities and thereby halt cartilage degeneration.

Hepatocellular lipid accumulation characterizes nonalcoholic fatty liver disease (NAFLD). However, the types of lipids associated with disease progression are debated, as is the impact of their localization. Traditional lipidomics analysis using liver homogenates or plasma dilutes and averages lipid concentrations, and does not provide spatial information about lipid distribution. We aimed to characterize the distribution of specific lipid species related to NAFLD severity by performing label-free molecular analysis by mass spectrometry imaging (MSI). Fresh frozen liver biopsies from obese subjects undergoing bariatric surgery ( n = 23) with various degrees of NAFLD were cryosectioned and analyzed by matrix-assisted laser desorption/ionization (MALDI)-MSI. Molecular identification was verified by tandem MS. Tissue sections were histopathologically stained, annotated according to the Kleiner classification, and coregistered with the MSI data set. Lipid pathway analysis was performed and linked to local proteome networks. Spatially resolved lipid profiles showed pronounced differences between nonsteatotic and steatotic tissues. Lipid identification and network analyses revealed phosphatidylinositols and arachidonic acid metabolism in nonsteatotic regions, whereas low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) metabolism was associated with steatotic tissue. Supervised and unsupervised discriminant analysis using lipid based classifiers outperformed simulated analysis of liver tissue homogenates in predicting steatosis severity. We conclude that lipid composition of steatotic and nonsteatotic tissue is highly distinct, implying that spatial context is important for understanding the mechanisms of lipid accumulation in NAFLD. MSI combined with principal component-linear discriminant analysis linking lipid and protein pathways represents a novel tool enabling detailed, comprehensive studies of the heterogeneity of NAFLD.

Mass spectrometry imaging (MSI) has proven to be a valuable tool for drug and metabolite imaging in pharmaceutical toxicology studies and can reveal, for example, accumulation of drug candidates in early drug development. However, the lack of sample cleanup and chromatographic separation can hamper the analysis due to isobaric interferences. Multiple reaction monitoring (MRM) uses unique precursor ion-product ion transitions to add specificity which leads to higher selectivity. Here, we present a targeted imaging platform where desorption electrospray ionization is combined with a triple quadrupole (QqQ) system to perform MRM imaging. The platform was applied to visualize (i) lipids in mouse brain tissue sections and (ii) a drug candidate and metabolite in canine liver tissue. All QqQ modes were investigated to show the increased detection time provided by MRM as well as the possibility to perform dual polarity imaging. This is very beneficial for lipid imaging because some phospholipid classes ionize in opposite polarity (e.g., phosphatidylcholine/sphingomyelin in positive ion mode and phosphatidylserine/phosphatidylethanolamine in negative ion mode). Drug and metabolite images were obtained to show its strength in drug distribution studies. Multiple MRM transitions were used to confirm the local presence and selective detection of pharmaceutical compounds.

Abstract Mass spectrometry imaging (MSI) enables the spatial distributions of molecules possessing different mass‐to‐charge ratios to be mapped within complex environments revealing regional changes at the molecular level. Even at high mass resolving power, however, these images often reflect the summed distribution of multiple isomeric molecules, each potentially possessing a unique distribution coinciding with distinct biological function(s) and metabolic origin. Herein, this chemical ambiguity is addressed through an innovative combination of ozone‐induced dissociation reactions with MSI, enabling the differential imaging of isomeric lipid molecules directly from biological tissues. For the first time, we demonstrate both double bond‐ and sn‐positional isomeric lipids exhibit distinct spatial locations within tissue. This MSI approach enables researchers to unravel local lipid molecular complexity based on both exact elemental composition and isomeric structure directly from tissues.

Purpose: Articular cartilage is generally exposed to a finely regulated gradient of oxygen levels ranging from 8% at the superficial surface to 1% in the deepest layers. While most cartilage research is performed in supraphysiological oxygen levels (19-21%), culturing chondrocytes under hypoxic oxygen levels (≤ 8%) promotes the chondrogenic phenotype and cartilage-specific matrix formation. Exposure of cells to various oxygen levels has been shown to alter their lipid metabolism. To better understand the chondrocyte's behavior in response to oxygen, we studied the effect of various oxygen levels on their lipid composition using Matrix Assisted Laser Desorption Ionization mass spectrometry imaging (MALDI-IMS). Methods: Human primary chondrocytes were isolated from cartilage knee biopsies of patients (n=5) undergoing total knee replacement, expanded in monolayer in normoxia (20% oxygen) or hypoxia (2.5% oxygen) and subsequently cultured in 3D pellets in normoxia or hypoxia for 7 days. To assess which lipids are altered by oxygen and to visualize their spatial distribution, we performed MALDI-IMS on cryosections of chondrocyte pellets in the negative and positive ion mode. Briefly, cell pellets were cryo-sectioned (10 μm section) and sprayed with Norharmane 7 mg/mL in chloroform/methanol (2:1, (v/v)) or α-Cyano-4-hydroxycinnamic acid (CHCA) 5 mg/ml in methanol/water/trifluoroacetic acid (70:30:0.01) using a SunCollect (SunChrom). The Synapt HDMS MALDI-Q-TOF (Waters) was used for the MSI experiments with a spatial raster size of 100 μm. Principal component analysis (PCA) and linear discriminant analysis (LDA) were used to search for spectral similarities and differences between the conditions. Biomap software was used to visualize molecular distributions. Tandem MS and the Lipid Maps database were used for molecular identification. Results: MALDI-IMS followed by LDA reveal that normoxic and hypoxic cultures of primary chondrocytes can be easily separated based on their molecular lipid profile. In the negative ion mode spectra phosphatidylglycerols (PG) were the species that had a high contribution in normoxic pellets, whereas in hypoxic pellets phosphatidylinositol (PI) species were the most prominent lipids (Table 1). Spatial mapping of individual molecules revealed that several PI species have the tendency to be more prominent in the center of hypoxic and normoxic pellets where there is generally less oxygen present. PG is a low abundant phospholipid and mostly present in mitochondria where it is a precursor for cardiolipin (CL), a mitochondrial membrane stabilizing lipid.Table 1Positive and negative m/z values with the highest discriminant function loadings. PG, phosphatidylglycerol; PI, phosphatidylinositol; SM, sphingomyelin; PC, phosphocholineNegatively charged ionsm/z valuelipid assignmentDesignationCondition773.5PG 36:2[M-H]–795.5PG 38:5[M-H]–819.5PG 40:7[M-H]–841.5PG 42:10[M-H]–865.5PG 44:12[M-H]–885.5PI 38:4[M-H]–722.5PE p36:4[M-H]–788.5PS 36:1[M-H]–835.5PI 34:1[M-H]–863.5PI 36:1[M-H]–887.5PI 38:2[M-H]–913.5PI 40:4[M-H]–Positively charged ionsm/z valueLipid assignmentDesignationCondition788.5PC 36:1[M+H]+804.5PC 36:4[M+Na]+832.5PC 38:4[M+Na]+848.5PC 38:4[M+K]+725.5SM d18:1/16:0[M+Na]+782.5PC 34:1[M+Na]+808.5PC 36:2[M+Na]+ Open table in a new tab Additional experiments revealed a higher abundance of the CL species at m/z 1454.0, 1478.0, 1510.1, 1512.1, 1538.1 in normoxia than hypoxia. The positive ion mode spectra showed that the sphingomyelin (SM) at m/z 725.5 is more prominent in hypoxia. Furthermore, spatial mapping of this molecule showed co-localization with Alcian blue stained areas. Conclusions: It is increasingly recognized that culturing chondrocytes in physiological oxygen levels, often referred to as hypoxia, better retains the chondrogenic phenotype and promotes chondrogenesis of mesenchymal stromal cells. This has been attributed to the activation of the chondrogenic transcription factor SOX9 by Hypoxia Inducible Factors (HIFs) in low oxygen levels. Here we provide an additional mechanism for improved chondrogenesis under low oxygen levels. We show that culturing chondrocytes in normoxia induces PG and CL species which may signify mitochondrial stress and may be a result of more ROS generation in normoxic cultures. Diminished activation of SOX9 in combination with cellular stress and production of ROS in normoxia contribute to impaired chondrocyte function in normoxic conditions. Our study further signifies the importance of studying chondrocyte function and dysfunction at physiological oxygen levels.