Probing Interkingdom Signaling Molecules via Liquid Extraction Surface Analysis-Mass Spectrometry.

Previously, metabolites diffused or secreted from microbial samples have been analyzed via liquid chromatography-mass spectrometry (LC-MS) approaches following lengthy extraction protocols. Here, we present a model system for growing biofilms on discs before utilizing rapid and direct surface sampling MS, namely, liquid extraction surface analysis, to study the microbial exometabolome. One of the benefits of this approach is its surface-specific nature, enabling mimicking biofilm formation in a way that the study of planktonic liquid cultures cannot imitate. Even though Pseudomonas aeruginosa (P. aeruginosa), Staphylococcus aureus (S. aureus), and Candida albicans (C. albicans) have been studied previously in isolation, very few studies consider the complexity of the interplay between these pathogens, which are commonly combined causative agents of infection. Our model system provides a route to investigate changes in the exometabolome, such as metabolites that become circulatory in the presence of multiple pathogens. Our results agree with previous reports showing that 2-alkyl-4(1H)-quinolone signal molecules produced by P. aeruginosa are important markers of infection and suggest that methods for monitoring levels of 2-heptyl-4-hydroxyquinoline and 2,4-dihydroxyquinoline, as well as pyocyanin, could be beneficial in the determination of causative agents in interkingdom infection including P. aeruginosa. Furthermore, studying changes in exometabolome metabolites between pqs quorum sensing antagonists in treated and nontreated samples suggests suppression of phenazine production by P. aeruginosa. Hence, our model provides a rapid analytical approach to gaining a mechanistic understanding of bacterial signaling.

Untargeted Metabolomic Characterization of Glioblastoma Intra-Tumor Heterogeneity Using OrbiSIMS.

Glioblastoma (GBM) is an incurable brain cancer with a median survival of less than two years from diagnosis. The standard treatment of GBM is multimodality therapy comprising surgical resection, radiation, and chemotherapy. However, prognosis remains poor, and there is an urgent need for effective anticancer drugs. Since different regions of a single GBM contain multiple cancer subpopulations ("intra-tumor heterogeneity"), this likely accounts for therapy failure as certain cancer cells can escape from immune surveillance and therapeutic threats. Here, we present metabolomic data generated using the Orbitrap secondary ion mass spectrometry (OrbiSIMS) technique to investigate brain tumor metabolism within its highly heterogeneous tumor microenvironment. Our results demonstrate that an OrbiSIMS-based untargeted metabolomics method was able to discriminate morphologically distinct regions (viable, necrotic, and non-cancerous) within single tumors from formalin-fixed paraffin-embedded tissue archives. Specifically, cancer cells from necrotic regions were separated from viable GBM cells based on a set of metabolites including cytosine, phosphate, purine, xanthine, and 8-hydroxy-7-methylguanine. Moreover, we mapped ubiquitous metabolites across necrotic and viable regions into metabolic pathways, which allowed for the discovery of tryptophan metabolism that was likely essential for GBM cellular survival. In summary, this study first demonstrated the capability of OrbiSIMS for in situ investigation of GBM intra-tumor heterogeneity, and the acquired information can potentially help improve our understanding of cancer metabolism and develop new therapies that can effectively target multiple subpopulations within a tumor.

Metabolic alterations in dairy cattle with lameness revealed by untargeted metabolomics of dried milk spots using direct infusion-tandem mass spectrometry and the triangulation of multiple machine learning models.

Lameness is a major challenge in the dairy cattle industry in terms of animal welfare and economic implications. Better understanding of metabolic alteration associated with lameness could lead to early diagnosis and effective treatment, there-fore reducing its prevalence. To determine whether metabolic signatures associated with lameness could be discovered with untargeted metabolomics, we developed a novel workflow using direct infusion-tandem mass spectrometry to rapidly analyse (2 min per sample) dried milk spots (DMS) that were stored on commercially available Whatman® FTA® DMPK cards for a prolonged period (8 and 16 days). An orthogonal partial least squares-discriminant analysis (OPLS-DA) method validated by triangulation of multiple machine learning (ML) models and stability selection was employed to reliably identify important discriminative metabolites. With this approach, we were able to differentiate between lame and healthy cows based on a set of lipid molecules and several small metabolites. Among the discriminative molecules, we identified phosphatidylglycerol (PG 35:4) as the strongest and most sensitive lameness indicator based on stability selection. Overall, this untargeted metabolomics workflow is found to be a fast, robust, and discriminating method for determining lameness in DMS samples. The DMS cards can be potentially used as a convenient and cost-effective sample matrix for larger scale research and future routine screening for lameness.

Time resolved growth of (N)-polycyclic aromatic hydrocarbons in engine deposits uncovered with OrbiSIMS depth profiling.

Carbonaceous deposits are ubiquitous, being formed on surfaces in engines, fuel systems and on catalysts operating at high temperatures for hydrocarbon transformations. In internal combustion engines, their formation negatively affects worldwide vehicle emissions and fuel economy, leading to premature deaths and environmental damage. Deposit composition and formation pathways are poorly understood due to their insolubility and the intrinsic complexity of their layered carbonaceous matrix. Here, we apply the in situ high mass resolving power capabilities of 3D Orbitrap secondary ion mass spectrometry (3D OrbiSIMS) argon cluster depth profiling on 16 lab grown deposits and evidence common molecular distributions in deposit depth and in positions relative to the combustion chamber. We observe the products of the growth of both planar and curved polycyclic aromatic hydrocarbons to form small fullerenes over time in the engine and propose possible formation pathways which explain the molecular distributions observed. These include alkyl scission, cyclisation of aliphatic side chains and hydrogen abstraction C2H2 addition to form larger aromatic structures. We apply this pathway to previously unidentified nitrogen containing structures in deposits including quinolines and carbazoles. For the first time, 3D OrbiSIMS results were compared and validated with data from atmospheric pressure matrix assisted laser desorption ionization MS. The comprehensive characterization provided will help the development of a new generation of chemical additives to reduce deposits, and thus improve vehicle emissions and global air quality.

Comprehensive LESA Mass Spectrometry Imaging of Intact Proteins by Integration of Cylindrical FAIMS.

The benefits of high field asymmetric waveform ion mobility spectrometry (FAIMS) for mass spectrometry imaging of intact proteins in thin tissue sections have been demonstrated previously. In those works, a planar FAIMS device coupled with a Thermo Elite mass spectrometer was employed. Here, we have evaluated a newly introduced cylindrical FAIMS device (the FAIMS Pro) coupled with a Thermo Fusion Lumos mass spectrometer for liquid extraction surface analysis mass spectrometry imaging of intact proteins in thin tissue sections from rat testes, kidney, and brain. The method makes use of multiple FAIMS compensation values at each location (pixel) of the imaging array. A total of 975 nonredundant protein species were detected in the testes imaging dataset, 981 in the kidney dataset, and 249 in the brain dataset. These numbers represent a 7-fold (brain) and over 10-fold (testes, kidney) improvement on the numbers of proteins previously detected in LESA FAIMS imaging, and a 10-fold to over 20-fold improvement on the numbers detected without FAIMS on this higher performance mass spectrometer, approaching the same order of magnitude as those obtained in top-down proteomics of cell lines. Nevertheless, high throughput identification within the LESA FAIMS imaging workflow remains a challenge.

Direct Mass Spectrometry Analysis of Protein Complexes and Intact Proteins up to >70 kDa from Tissue.

Native liquid extraction surface analysis (LESA) mass spectrometry allows direct analysis of folded proteins and protein complexes from biological substrates, such as dried blood spots and thin tissue sections, by use of native-like extraction/ionization solvents. Previously, we have demonstrated native LESA mass spectrometry of folded proteins up to 16 kDa as well as the 64 kDa hemoglobin tetramer, from mouse tissues. With denaturing LESA solvents, the highest mass protein detected in tissue to date is ∼37 kDa. Here, we demonstrate native LESA mass spectrometry by use of a Q Exactive UHMR Hybrid Quadrupole-Orbitrap (QE-UHMR) mass spectrometer, pushing the upper mass limit of proteins detected in tissue to >70 kDa. Moreover, a protein trimer of 42 kDa was detected and its stoichiometry confirmed by higher energy collision dissociation (HCD). The benefits of inclusion of detergents in the LESA sampling solvent are also demonstrated.

Quantitative Imaging of Proteins in Tissue by Stable Isotope Labeled Mimetic Liquid Extraction Surface Analysis Mass Spectrometry.

Absolute quantification of proteins in tissue is important for numerous fields of study. Liquid chromatography-mass spectrometry (LC-MS) methods are the norm but typically involve lengthy sample preparation including tissue homogenization, which results in the loss of information relating to spatial distribution. Here, we propose liquid extraction surface analysis (LESA) mass spectrometry (MS) of stable isotope labeled mimetic tissue models for the spatially resolved quantification of intact ubiquitin in rat and mouse brain tissue. Measured ubiquitin concentrations are in agreement with values found in the literature. Images of rat and mouse brain tissue demonstrate spatial variation in the concentration of ubiquitin and demonstrate the utility of spatially resolved quantitative measurement of proteins in tissue. Although we have focused on ubiquitin, the method has the potential for broader application to the absolute quantitation of any endogenous protein or protein-based drug in tissue.

Direct liquid extraction surface analysis mass spectrometry of cell wall lipids from mycobacteria: Salt additives for decreased spectral complexity.

RATIONALE: Lipids are important mycobacterium cell wall constituents; changes are linked with drug resistance. Liquid extraction surface analysis (LESA) enables direct sampling in a highly sensitive manner. Here we describe protocols for the analysis of lipids from bacterial colonies. Lipids form various adducts, complicating spectra. Salt additives were investigated to circumvent this problem. METHODS: Chloroform:methanol mixtures were studied for lipid extraction and analysis by LESA-MS. The inclusion of (ESI-compatible) acetate salts of sodium, potassium or lithium in the extraction solvent was investigated. RESULTS: We report the detection of bacterial cell wall lipids from mycobacterial species using LESA for the first time. Sampling protocols were optimised for the use of volatile extraction solvents. The inclusion of acetate salt additives in the sampling solvent significantly reduces spectral complexity in comparison with no additives being used. CONCLUSIONS: LESA offers a sensitive technique for bacterial lipid phenotyping. The inclusion of an acetate salt in the sampling solvent drives adduct formation towards a specific adduct type and thus significantly reduces spectral complexity.

LESA MS Imaging of Heat-Preserved and Frozen Tissue: Benefits of Multistep Static FAIMS.

We have previously demonstrated liquid extraction surface analysis (LESA) high field asymmetric waveform ion mobility spectrometry (FAIMS) mass spectrometry imaging of proteins in thin tissue sections of brain and liver. Here, we present an improved approach that makes use of multiple static FAIMS parameters at each sampled location and allows a significant improvement in the number of proteins, lipids, and drugs that can be imaged simultaneously. The approach is applied to the mass spectrometry imaging of control and cassette-dosed rat kidneys. Mass spectrometry imaging of kidneys typically requires washing to remove excess hemoglobin; however, that is not necessary with this approach. Multistep static FAIMS mass spectrometry resulted in a 6- to 16-fold increase in the number of proteins detected in the absence of FAIMS, in addition to smaller increases over single step static FAIMS (chosen for optimum transmission of total protein ions). The benefits of multistep static FAIMS mass spectrometry for protein detection are also shown for sections of testes. The numbers of proteins detected following multistep FAIMS increased between 2- and 3-fold over single step FAIMS and between 2- and 14-fold over LESA alone. Finally, to date, LESA mass spectrometry of proteins in tissue has been undertaken solely on fresh frozen samples. In this work, we demonstrate that heat-preserved tissues are also suitable for these analyses. Heat preservation of tissue improved the number of proteins detected by LESA MS for both kidney and testes tissue (by between 2- and 4-fold). For both tissue types, the majority of the proteins additionally detected in the heat-treated samples were subsequently detected in the frozen samples when FAIMS was incorporated. Improvements in the numbers of proteins detected were observed for LESA FAIMS MS for the kidney tissue; for testes tissue, fewer total proteins were detected following heat preservation; however, approximately one-third were unique to the heat-preserved samples.

Raster-Mode Continuous-Flow Liquid Microjunction Mass Spectrometry Imaging of Proteins in Thin Tissue Sections.

Mass spectrometry imaging by use of continuous-flow liquid microjunction sampling at discrete locations (array mode) has previously been demonstrated. In this Letter, we demonstrate continuous-flow liquid microjunction mass spectrometry imaging of proteins from thin tissue sections in raster mode and discuss advantages (a 10-fold reduction in analysis time) and challenges (suitable solvent systems, data interpretation) of the approach. Visualization of data is nontrivial, requiring correlation of solvent-flow, mass spectral data acquisition rate, data quality, and liquid microjunction sampling area. The latter is particularly important for determining optimum pixel size. The minimum achievable pixel size is related to the scan time of the instrument used. Here we show a minimum achievable pixel size of 50 µm (x-dimension) when using an Orbitrap Elite; however a pixel size of 600 µm is recommended in order to minimize the effects of oversampling on image accuracy.

Direct Tissue Profiling of Protein Complexes: Toward Native Mass Spectrometry Imaging.

Native mass spectrometry seeks to probe noncovalent protein interactions in terms of protein quaternary structure, protein-protein and protein-ligand complexes. The ultimate goal is to link the understanding of protein interactions to the protein environment by visualizing the spatial distribution of noncovalent protein interactions within tissue. Previously, we have shown that noncovalently bound protein complexes can be directly probed via liquid extraction surface analysis from dried blood spot samples, where hemoglobin is highly abundant. Here, we show that the intact hemoglobin complex can be sampled directly from thin tissue sections of mouse liver and correlated to a visible vascular feature, paving the way for native mass spectrometry imaging.

LESA FAIMS Mass Spectrometry for the Spatial Profiling of Proteins from Tissue.

We have shown previously that coupling of high field asymmetric waveform ion mobility spectrometry (FAIMS), also known as differential ion mobility, with liquid extraction surface analysis (LESA) mass spectrometry of tissue results in significant improvements in the resulting protein mass spectra. Here, we demonstrate LESA FAIMS mass spectrometry imaging of proteins in sections of mouse brain and liver tissue. The results are compared with LESA mass spectrometry images obtained in the absence of FAIMS. The results show that the number of different protein species detected can be significantly increased by incorporating FAIMS into the workflow. A total of 34 proteins were detected by LESA FAIMS mass spectrometry imaging of mouse brain, of which 26 were unique to FAIMS, compared with 15 proteins (7 unique) detected by LESA mass spectrometry imaging. A number of proteins were identified including α-globin, 6.8 kDa mitochondrial proteolipid, macrophage migration inhibitory factor, ubiquitin, ß-thymosin 4, and calmodulin. A total of 40 species were detected by LESA FAIMS mass spectrometry imaging of mouse liver, of which 29 were unique to FAIMS, compared with 24 proteins (13 unique) detected by LESA mass spectrometry imaging. The spatial distributions of proteins identified in both LESA mass spectrometry imaging and LESA FAIMS mass spectrometry imaging were in good agreement indicating that FAIMS is a suitable tool for inclusion in mass spectrometry imaging workflows.

Liquid Extraction Surface Analysis Mass Spectrometry Coupled with Field Asymmetric Waveform Ion Mobility Spectrometry for Analysis of Intact Proteins from Biological Substrates.

Previously we have shown that liquid extraction surface analysis (LESA) mass spectrometry is suitable for the analysis of intact proteins from a range of biological substrates. Here we show that LESA mass spectrometry may be coupled with high field asymmetric waveform ion mobility spectrometry (FAIMS) for top-down protein analysis directly from thin tissue sections (mouse liver, mouse brain) and from bacterial colonies (Escherichia coli) growing on agar. Incorporation of FAIMS results in significant improvements in signal-to-noise and reduced analysis time. Abundant protein signals are observed in single scan mass spectra. In addition, FAIMS enables gas-phase separation of molecular classes, for example, lipids and proteins, enabling improved analysis of both sets of species from a single LESA extraction.

Liquid extraction surface analysis field asymmetric waveform ion mobility spectrometry mass spectrometry for the analysis of dried blood spots.

Liquid extraction surface analysis (LESA) is a surface sampling technique that allows electrospray mass spectrometry analysis of a wide range of analytes directly from biological substrates. Here, we present LESA mass spectrometry coupled with high field asymmetric waveform ion mobility spectrometry (FAIMS) for the analysis of dried blood spots on filter paper. Incorporation of FAIMS in the workflow enables gas-phase separation of lipid and protein molecular classes, enabling analysis of both haemoglobin and a range of lipids (phosphatidylcholine or phosphatidylethanolamine, and sphingomyelin species) from a single extraction sample. The work has implications for multiplexed clinical assays of multiple analytes.

Formal lithium fixation improves direct analysis of lipids in tissue by mass spectrometry.

Mass spectrometry imaging is a powerful method for imaging and in situ characterization of lipids in thin tissue sections. Structural elucidation of lipids is often achieved via collision induced dissociation, and lithium-lipid adducts have been widely reported as providing the most structurally informative fragment ions. We present a method for the incorporation of lithium salts into tissue imaging experiments via fixation of samples in formal lithium solutions. The method is suitable for preparation of single tissue sections, or as an immersion fixation method for whole tissue blocks or organs prior to sectioning. We compare lithium adduct detection and MALDI-MSI of murine brain from analysis of tissues prepared in different ways. Tissues prepared in formal solutions containing lithium or sodium salts before coating in matrix via air-spray deposition are compared with fresh samples coated in lithium-doped matrix preparations by either dry-coating or air-spray deposition. Sample preparation via fixation in formal lithium is shown to yield the highest quality images of lithium adducts, resulting in acquisition of more informative product ion spectra in MALDI MS/MS profiling and imaging experiments. Finally, the compatibility of formal lithium solutions with standard histological staining protocols (hemotoxylin and eosin, Van Giessen and Oil Red O) is demonstrated in a study of human liver tissue.

Hyperspectral visualization of mass spectrometry imaging data.

The acquisition of localized molecular spectra with mass spectrometry imaging (MSI) has a great, but as yet not fully realized, potential for biomedical diagnostics and research. The methodology generates a series of mass spectra from discrete sample locations, which is often analyzed by visually interpreting specifically selected images of individual masses. We developed an intuitive color-coding scheme based on hyperspectral imaging methods to generate a single overview image of this complex data set. The image color-coding is based on spectral characteristics, such that pixels with similar molecular profiles are displayed with similar colors. This visualization strategy was applied to results of principal component analysis, self-organizing maps and t-distributed stochastic neighbor embedding. Our approach for MSI data analysis, combining automated data processing, modeling and display, is user-friendly and allows both the spatial and molecular information to be visualized intuitively and effectively.

Surface-sampling mass spectrometry to study proteins and protein complexes.

This review aims to summarise the current capabilities of surface mass spectrometry (MS) approaches that offer intact protein analysis, and that of non-covalent complexes. Protein analysis is largely achieved via matrix-assisted laser desorption/ionisation (MALDI), which is in itself a surface analysis approach or solvent-based electrospray ionisation (ESI). Several surface sampling approaches have been developed based on ESI, and those that have been used for intact protein analysis will be discussed below. The extent of protein coverage, top-down elucidation, and probing of protein structure for native proteins and non-covalent complexes will be discussed for each approach. Strategies for improving protein analysis, ranging from sample preparation, and sampling methods to instrument modifications and the inclusion of ion mobility separation in the workflow will also be discussed. The relative benefits and drawbacks of each approach will be summarised, providing an overview of current capabilities.

Identifying new biomarkers of aggressive Group 3 and SHH medulloblastoma using 3D hydrogel models, single cell RNA sequencing and 3D OrbiSIMS imaging.

The most common malignant brain tumour in children, medulloblastoma (MB), is subdivided into four clinically relevant molecular subgroups, although targeted therapy options informed by understanding of different cellular features are lacking. Here, by comparing the most aggressive subgroup (Group 3) with the intermediate (SHH) subgroup, we identify crucial differences in tumour heterogeneity, including unique metabolism-driven subpopulations in Group 3 and matrix-producing subpopulations in SHH. To analyse tumour heterogeneity, we profiled individual tumour nodules at the cellular level in 3D MB hydrogel models, which recapitulate subgroup specific phenotypes, by single cell RNA sequencing (scRNAseq) and 3D OrbiTrap Secondary Ion Mass Spectrometry (3D OrbiSIMS) imaging. In addition to identifying known metabolites characteristic of MB, we observed intra- and internodular heterogeneity and identified subgroup-specific tumour subpopulations. We showed that extracellular matrix factors and adhesion pathways defined unique SHH subpopulations, and made up a distinct shell-like structure of sulphur-containing species, comprising a combination of small leucine-rich proteoglycans (SLRPs) including the collagen organiser lumican. In contrast, the Group 3 tumour model was characterized by multiple subpopulations with greatly enhanced oxidative phosphorylation and tricarboxylic acid (TCA) cycle activity. Extensive TCA cycle metabolite measurements revealed very high levels of succinate and fumarate with malate levels almost undetectable particularly in Group 3 tumour models. In patients, high fumarate levels (NMR spectroscopy) alongside activated stress response pathways and high Nuclear Factor Erythroid 2-Related Factor 2 (NRF2; gene expression analyses) were associated with poorer survival. Based on these findings we predicted and confirmed that NRF2 inhibition increased sensitivity to vincristine in a long-term 3D drug treatment assay of Group 3 MB. Thus, by combining scRNAseq and 3D OrbiSIMS in a relevant model system we were able to define MB subgroup heterogeneity at the single cell level and elucidate new druggable biomarkers for aggressive Group 3 and low-risk SHH MB.

A survey of useful salt additives in matrix-assisted laser desorption/ionization mass spectrometry and tandem mass spectrometry of lipids: introducing nitrates for improved analysis.

RATIONALE: Matrix-assisted laser desorption/ionization (MALDI) is a powerful technique for the direct analysis of lipids in complex mixtures and thin tissue sections, making it an extremely attractive method for profiling lipids in health and disease. Lipids are readily detected as [M+H](+), [M+Na](+) and [M+K](+) ions in positive ion MALDI mass spectrometry (MS) experiments. This not only decreases sensitivity, but can also lead to overlapping m/z values of the various adducts of different lipids. Additives can be used to promote formation of a particular adduct, improving sensitivity, reducing spectral complexity and enhancing structural characterization in collision-induced dissociation (CID) experiments. METHODS: Li(+), Na(+), K(+), Cs(+) and NH(4)(+) cations were considered as a range of salt types (acetates, chlorides and nitrates) incorporated into DHB matrix solutions at concentrations between 5 and 80 mM. The study was extended to evaluate the effect of these additives on CID experiments of a lipid standard, after optimization of collision energy parameters. Experiments were performed on a hybrid quadrupole time-of-flight (QqTOF) instrument. RESULTS: The systematic evaluation of new and existing additives in MALDI-MS and MS/MS of lipids demonstrated the importance of additive cation and anion choice and concentration for tailoring spectral results. CONCLUSIONS: The recommended choice of additive depends on the desired outcomes of the experiment to be performed (MS or MS/MS). Nitrates are found to be particularly useful additives for lipid analysis.