Update DESI Mass Spectrometry Imaging (MSI).

Desorption electrospray ionization (DESI) is an ambient technique that allows chemical information to be obtained directly from a wide range of surfaces, without pretreatment. Here we describe the improvements that have been developed to be able to achieve low tens of microns pixel size MSI experiments with high sensitivity for metabolites and lipids from biological tissue sections.In the last decade, DESI mass spectrometry has undergone developmental improvements, with regard to the method of desorption and ionization as well as the mass spectrometer to which the DESI source has been coupled to. DESI is becoming a mass spectrometry imaging technique, which can match and complement the currently most widely adopted ionization technique, the matrix-assisted laser desorption/ionization (MALDI).

Novel Hybrid Quadrupole-Multireflecting Time-of-Flight Mass Spectrometry System.

A novel mass spectrometry system is described here comprising a quadrupole-multireflecting time-of-flight design. The new multireflecting time-of-flight analyzer has an effective path length of 48 m and employs planar, gridless ion mirrors providing fourth-order energy focusing resulting in resolving power over 200 000 fwhm and sub-ppm mass accuracy. We show how these attributes are maintained with relatively fast acquisition speeds, setting the system apart from other high resolution mass spectrometers. We have integrated this new system into both liquid chromatography-mass spectrometry and mass spectrometry imaging workflows to demonstrate how the instrument characteristics are of benefit to these applications.

High Resolution Ambient MS Imaging of Biological Samples by Desorption Electro-Flow Focussing Ionization.

In this study, we examine the suitability of desorption electro-flow focusing ionization (DEFFI) for mass spectrometry imaging (MSI) of biological tissue. We also compare the performance of desorption electrospray ionization (DESI) with and without the flow focusing setup. The main potential advantages of applying the flow focusing mechanism in DESI is its rotationally symmetric electrospray jet, higher intensity, more controllable parameters, and better portability due to the robustness of the sprayer. The parameters for DEFFI have therefore been thoroughly optimized, primarily for spatial resolution but also for intensity. Once the parameters have been optimized, DEFFI produces similar images to the existing DESI. MS images for mouse brain samples, acquired at a nominal pixel size of 50 µm, are comparable for both DESI setups, albeit the new sprayer design yields better sensitivity. Furthermore, the two methods are compared with regard to spectral intensity as well as the area of the desorbed crater on rhodamine-coated slides. Overall, the implementation of a flow focusing mechanism in DESI is shown to be highly suitable for imaging biological tissue and has potential to overcome some of the shortcomings experienced with the current geometrical design of DESI.

3D DESI-MS lipid imaging in a xenograft model of glioblastoma: a proof of principle.

Desorption electrospray ionisation mass spectrometry (DESI-MS) can image hundreds of molecules in a 2D tissue section, making it an ideal tool for mapping tumour heterogeneity. Tumour lipid metabolism has gained increasing attention over the past decade; and here, lipid heterogeneity has been visualised in a glioblastoma xenograft tumour using 3D DESI-MS imaging. The use of an automatic slide loader automates 3D imaging for high sample-throughput. Glioblastomas are highly aggressive primary brain tumours, which display heterogeneous characteristics and are resistant to chemotherapy and radiotherapy. It is therefore important to understand biochemical contributions to their heterogeneity, which may be contributing to treatment resistance. Adjacent sections to those used for DESI-MS imaging were used for H&E staining and immunofluorescence to identify different histological regions, and areas of hypoxia. Comparing DESI-MS imaging with biological staining allowed association of different lipid species with hypoxic and viable tissue within the tumour, and hence mapping of molecularly different tumour regions in 3D space. This work highlights that lipids are playing an important role in the heterogeneity of this xenograft tumour model, and DESI-MS imaging can be used for lipid 3D imaging in an automated fashion to reveal heterogeneity, which is not apparent in H&E stains alone.

De Novo Lipogenesis Alters the Phospholipidome of Esophageal Adenocarcinoma.

The incidence of esophageal adenocarcinoma is rising, survival remains poor, and new tools to improve early diagnosis and precise treatment are needed. Cancer phospholipidomes quantified with mass spectrometry imaging (MSI) can support objective diagnosis in minutes using a routine frozen tissue section. However, whether MSI can objectively identify primary esophageal adenocarcinoma is currently unknown and represents a significant challenge, as this microenvironment is complex with phenotypically similar tissue-types. Here, we used desorption electrospray ionization-MSI (DESI-MSI) and bespoke chemometrics to assess the phospholipidomes of esophageal adenocarcinoma and relevant control tissues. Multivariate models derived from phospholipid profiles of 117 patients were highly discriminant for esophageal adenocarcinoma both in discovery (AUC = 0.97) and validation cohorts (AUC = 1). Among many other changes, esophageal adenocarcinoma samples were markedly enriched for polyunsaturated phosphatidylglycerols with longer acyl chains, with stepwise enrichment in premalignant tissues. Expression of fatty acid and glycerophospholipid synthesis genes was significantly upregulated, and characteristics of fatty acid acyls matched glycerophospholipid acyls. Mechanistically, silencing the carbon switch ACLY in esophageal adenocarcinoma cells shortened glycerophospholipid chains, linking de novo lipogenesis to the phospholipidome. Thus, DESI-MSI can objectively identify invasive esophageal adenocarcinoma from a number of premalignant tissues and unveils mechanisms of phospholipidomic reprogramming. SIGNIFICANCE: These results call for accelerated diagnosis studies using DESI-MSI in the upper gastrointestinal endoscopy suite, as well as functional studies to determine how polyunsaturated phosphatidylglycerols contribute to esophageal carcinogenesis.

Rapid identification of species, sex and maturity by mass spectrometric analysis of animal faeces.

BACKGROUND: We describe a new approach to the recovery of information from faecal samples, based on the analysis of the molecular signature generated by rapid evaporative ionisation mass spectrometry (REIMS). RESULTS: Faecal pellets from five different rodent species were analysed by REIMS, and complex mass spectra were acquired rapidly (typically a few seconds per sample). The uninterpreted mass spectra (signatures) were then used to seed linear discriminant analysis and classification models based on random forests. It was possible to classify each species of origin with a high rate of accuracy, whether faeces were from animals maintained under standard laboratory conditions or wild-caught. REIMS signatures were stable to prior storage of the faecal material under a range of different conditions and were not altered rapidly or radically by changes in diet. Further, within species, REIMS signatures could be used to discriminate faeces from adult versus juvenile mice, male versus female mice and those from three different laboratory strains. CONCLUSIONS: REIMS offers a completely novel method for the rapid analysis of faecal samples, extending faecal analysis (previously focused on DNA) to an assessment of phenotype, and has considerable potential as a new tool in the armamentarium of the field biologist.

Matrix Assisted Rapid Evaporative Ionization Mass Spectrometry.

Rapid evaporative ionization mass spectrometry (REIMS) is a highly versatile technique allowing the sampling of a range of biological solid or liquid samples with no sample preparation. The cost of such a direct approach is that certain sample types provide only moderate amounts of chemical information. Here, we introduce a matrix assisted version of the technique (MA-REIMS), where an aerosol of a pure solvent, such as isopropanol, is mixed with the sample aerosol generated by rapid evaporation of the sample, and it is shown to enhance the signal intensity obtained from a REIMS sampling event by over 2 orders of magnitude. Such an increase greatly expands the scope of the technique, while providing additional benefits such as reducing the fouling of the REIMS source and allowing for a simple method of constant introduction of a calibration correction compound for accurate mass measurements. A range of experiments are presented in order to investigate the processes that occur within this modified approach, and applications where such enhancements are critical, such as intrasurgical tissue identification, are discussed.

Enhanced Fatty Acid Scavenging and Glycerophospholipid Metabolism Accompany Melanocyte Neoplasia Progression in Zebrafish.

Alterations in lipid metabolism in cancer cells impact cell structure, signaling, and energy metabolism, making lipid metabolism a potential diagnostic marker and therapeutic target. In this study, we combined PET, desorption electrospray ionization-mass spectrometry (DESI-MS), nonimaging MS, and transcriptomic analyses to interrogate changes in lipid metabolism in a transgenic zebrafish model of oncogenic RAS-driven melanocyte neoplasia progression. Exogenous fatty acid uptake was detected in melanoma tumor nodules by PET using the palmitic acid surrogate tracer 14(R,S)-18F-fluoro-6-thia-heptadecanoic acid ([18F]-FTHA), consistent with upregulation of genes associated with fatty acid uptake found through microarray analysis. DESI-MS imaging revealed that FTHA uptake in tumors was heterogeneous. Transcriptome and lipidome analyses further highlighted dysregulation of glycerophospholipid pathways in melanoma tumor nodules, including increased abundance of phosphatidyl ethanolamine and phosphatidyl choline species, corroborated by DESI-MS, which again revealed heterogeneous phospholipid composition in tumors. Overexpression of the gene encoding lipoprotein lipase (LPL), which was upregulated in zebrafish melanocyte tumor nodules and expressed in the majority of human melanomas, accelerated progression of oncogenic RAS-driven melanocyte neoplasia in zebrafish. Depletion or antagonism of LPL suppressed human melanoma cell growth; this required simultaneous fatty acid synthase (FASN) inhibition when FASN expression was also elevated. Collectively, our findings implicate fatty acid acquisition as a possible therapeutic target in melanoma, and the methods we developed for monitoring fatty acid uptake have potential for diagnosis, patient stratification, and monitoring pharmacologic response. SIGNIFICANCE: These findings demonstrate the translational potential of monitoring fatty acid uptake and identify lipoprotein lipase as a potential therapeutic target in melanoma.

Targeted Drug and Metabolite Imaging: Desorption Electrospray Ionization Combined with Triple Quadrupole Mass Spectrometry.

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.

Optimised Desorption Electrospray Ionisation Mass Spectrometry Imaging (DESI-MSI) for the Analysis of Proteins/Peptides Directly from Tissue Sections on a Travelling Wave Ion Mobility Q-ToF.

Desorption electrospray ionisation mass spectrometry imaging (DESI-MSI) is typically known for the ionisation of small molecules such as lipids and metabolites, in singly charged form. Here we present a method that allows the direct detection of proteins and peptides in multiply charged forms directly from tissue sections by DESI. Utilising a heated mass spectrometer inlet capillary, combined with ion mobility separation (IMS), the conditions with regard to solvent composition, nebulising gas flow, and solvent flow rate have been explored and optimised. Without the use of ion mobility separation prior to mass spectrometry analysis, only the most abundant charge series were observed. In addition to the dominant haemoglobin subunit(s) related trend line in the m/z vs drift time (DT) 2D plot, trend lines were found relating to background solvent peaks, residual lipids and, more importantly, small proteins/large peptides of lower abundance. These small proteins/peptides were observed with charge states from 1+ to 12+, the majority of which could only be resolved from the background when using IMS. By extracting charge series from the 2D m/z vs DT plot, a number of proteins could be tentatively assigned by accurate mass. Tissue images were acquired with a pixel size of 150 µm showing a marked improvement in protein image resolution compared to other liquid-based ambient imaging techniques such as liquid extraction surface analysis (LESA) and continuous-flow liquid microjunction surface sampling probe (LMJ-SSP) imaging. Graphical Abstract ᅟ.

Lipidomics for translational skin research: A primer for the uninitiated.

Healthy skin depends on a unique lipid profile to form a barrier that confers protection and prevents excessive water loss, aids cell-cell communication and regulates cutaneous homoeostasis and inflammation. Alterations in the cutaneous lipid profile can have severe consequences for skin health and have been implicated in numerous inflammatory skin conditions. Thus, skin lipidomics is increasingly of interest, and recent developments in mass spectrometry-based analytical technologies can deliver in-depth investigation of cutaneous lipids, providing insight into their role and mechanism of action. The choice of tissue sampling technique and analytical approach depends on the location and chemistry of the lipid of interest. Lipidomics can be conducted by various mass spectrometry approaches, including different chromatography and ionisation techniques. Targeted mass spectrometry is a sensitive approach for measuring low-abundance signalling lipids, such as eicosanoids, endocannabinoids and ceramides. This approach requires specific extraction, chromatography and mass spectrometry protocols to quantitate the lipid targets. Untargeted mass spectrometry reveals global changes and allows analysis of hundreds of complex lipids across a range of lipid classes, including phospholipids, glycerophospholipids, cholesteryl esters and sphingolipids. Mass spectrometry lipid imaging, including matrix-assisted laser desorption ionisation mass spectrometry and desorption electrospray ionisation mass spectrometry, can reveal information about abundance and anatomical distribution of lipids within a single skin sample. Skin lipidomics can provide qualitative and quantitative data on hundreds of biologically relevant lipid species with different properties and activities, all found within a single skin sample, and support translational studies exploring the involvement of lipids in skin health and disease.

Faster, More Reproducible DESI-MS for Biological Tissue Imaging.

A new, more robust sprayer for desorption electrospray ionization (DESI) mass spectrometry imaging is presented. The main source of variability in DESI is thought to be the uncontrolled variability of various geometric parameters of the sprayer, primarily the position of the solvent capillary, or more specifically, its positioning within the gas capillary or nozzle. If the solvent capillary is off-center, the sprayer becomes asymmetrical, making the geometry difficult to control and compromising reproducibility. If the stiffness, tip quality, and positioning of the capillary are improved, sprayer reproducibility can be improved by an order of magnitude. The quality of the improved sprayer and its potential for high spatial resolution imaging are demonstrated on human colorectal tissue samples by acquisition of images at pixel sizes of 100, 50, and 20 µm, which corresponds to a lateral resolution of 40-60 µm, similar to the best values published in the literature. The high sensitivity of the sprayer also allows combination with a fast scanning quadrupole time-of-flight mass spectrometer. This provides up to 30 times faster DESI acquisition, reducing the overall acquisition time for a 10 mm × 10 mm rat brain sample to approximately 1 h. Although some spectral information is lost with increasing analysis speed, the resulting data can still be used to classify tissue types on the basis of a previously constructed model. This is particularly interesting for clinical applications, where fast, reliable diagnosis is required. Graphical Abstract ᅟ.

DESI Mass Spectrometry Imaging (MSI).

Desorption Electrospray Ionization (DESI) mass spectrometry is a technique that allows chemical information to be obtained directly from a wide range of surfaces. Using a 2D stage, DESI can be implemented in an imaging mode whereby MS spectra are collected by rastering the spray across the whole surface. Here, we describe the implementation and optimization of DESI imaging for metabolites and lipids from tissue sections using oa-TOF mass spectrometers.

Mass spectrometry imaging shows major derangements in neurogranin and in purine metabolism in the triple-knockout 3×Tg Alzheimer mouse model.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) can simultaneously measure hundreds of biomolecules directly from tissue. Using different sample preparation strategies, proteins and metabolites have been profiled to study the molecular changes in a 3×Tg mouse model of Alzheimer's disease. In comparison with wild-type (WT) control mice MALDI-MSI revealed Alzheimer's disease-specific protein profiles, highlighting dramatic reductions of a protein with m/z 7560, which was assigned to neurogranin and validated by immunohistochemistry. The analysis also revealed substantial metabolite changes, especially in metabolites related to the purine metabolic pathway, with a shift towards an increase in hypoxanthine/xanthine/uric acid in the 3×Tg AD mice accompanied by a decrease in AMP and adenine. Interestingly these changes were also associated with a decrease in ascorbic acid, consistent with oxidative stress. Furthermore, the metabolite N-arachidonyl taurine was increased in the diseased mouse brain sections, being highly abundant in the hippocampus. Overall, we describe an interesting shift towards pro-inflammatory molecules (uric acid) in the purinergic pathway associated with a decrease in anti-oxidant level (ascorbic acid). Together, these observations fit well with the increased oxidative stress and neuroinflammation commonly observed in AD. This article is part of a Special Issue entitled: MALDI Imaging, edited by Dr. Corinna Henkel and Prof. Peter Hoffmann.

Real-Time Screening of Biocatalysts in Live Bacterial Colonies.

Screening of bacterial colonies to identify new biocatalytic activities is a widely adopted tool in biotechnology, but is constrained by the requirements for colorimetric or tag-based detection methods. Herein we report a label-free screening platform for biotransformations in live colonies using desorption electrospray ionization coupled with ion mobility mass spectrometry imaging (DiBT-IMMS). The screening method is demonstrated for both ammonia lyases and P450 monooxygenases expressed within live bacterial colonies and is shown to enable multiplexing of enzyme variants and substrate libraries simultaneously.

Imaging of Esophageal Lymph Node Metastases by Desorption Electrospray Ionization Mass Spectrometry.

Histopathological assessment of lymph node metastases (LNM) depends on subjective analysis of cellular morphology with inter-/intraobserver variability. In this study, LNM from esophageal adenocarcinoma was objectively detected using desorption electrospray ionization-mass spectrometry imaging (DESI-MSI). Ninety lymph nodes (LN) and their primary tumor biopsies from 11 esophago-gastrectomy specimens were examined and analyzed by DESI-MSI. Images from mass spectrometry and corresponding histology were coregistered and analyzed using multivariate statistical tools. The MSIs revealed consistent lipidomic profiles of individual tissue types found within LNs. Spatial mapping of the profiles showed identical distribution patterns as per the tissue types in matched IHC images. Lipidomic profile comparisons of LNM versus the primary tumor revealed a close association in contrast to benign LN tissue types. This similarity was used for the objective prediction of LNM in mass spectrometry images utilizing the average lipidomic profile of esophageal adenocarcinoma. The multivariate statistical algorithm developed for LNM identification demonstrated a sensitivity, specificity, positive predictive value, and negative predictive value of 89.5%, 100%, 100%, and 97.2%, respectively, when compared with gold-standard IHC. DESI-MSI has the potential to be a diagnostic tool for perioperative identification of LNM and compares favorably with techniques currently used by histopathology experts. Cancer Res; 76(19); 5647-56. ©2016 AACR.

Progress in mass spectrometry for the analysis of set-off phenomena in plastic food packaging materials.

In most cases, food packaging materials contain inks whose components can migrate to food by diffusion through the material as well as by set-off phenomena. In this work, different mass spectrometry approaches had been used in order to identify and confirm the presence of ink components in ethanol (95%) and Tenax(®) as food simulants. Three different sets of materials, manufactured with different printing technologies and with different structures, were analyzed. Sample analysis by ultra performance liquid chromatography mass spectrometry (UPLC-MS), using a quadrupole-time of flight (Q-TOF) as a mass analyser proved to be an excellent tool for identification purposes while ion mobility mass spectrometry (IM-MS) shown to be very useful for the confirmation of the candidates proposed. The results showed the presence of different non-volatile ink components in migration such as colorants (Solvent Red 49), plasticizers (dimethyl sebacate, tributyl o-acetyl citrate) or surfactants (SchercodineM, triethylene glycol caprilate). An oxidation product of an ink additive (triphenyl phosphine oxide) was also detected. In addition, a surface analysis technique, desorption electrospray mass spectrometry (DESI-MS), was used for analyzing the distribution of some ink components (tributyl o-acetyl citrate Schercodine L, phthalates) in the material. The detection of some of these compounds in the back-printed side confirmed the transference of this compound from the non-food to the food contact side. The results also showed that concentration of ink migrants decreased when an aluminum or polypropylene layer covered the ink. When aluminum was used, concentration of most of ink migrants decreased, and for 5 out of the 9 even disappeared.

Investigation of the Impact of Desorption Electrospray Ionization Sprayer Geometry on Its Performance in Imaging of Biological Tissue.

In this study, the impact of sprayer design and geometry on performance in desorption electrospray ionization mass spectrometry (DESI-MS) is assessed, as the sprayer is thought to be a major source of variability. Absolute intensity repeatability, spectral composition, and classification accuracy for biological tissues are considered. Marked differences in tissue analysis performance are seen between the commercially available and a lab-built sprayer. These are thought to be associated with the geometry of the solvent capillary and the resulting shape of the primary electrospray. Experiments with a sprayer with a fixed solvent capillary position show that capillary orientation has a crucial impact on tissue complex lipid signal and can lead to an almost complete loss of signal. Absolute intensity repeatability is compared for five lab-built sprayers using pork liver sections. Repeatability ranges from 1 to 224% for individual sprayers and peaks of different spectral abundance. Between sprayers, repeatability is 16%, 9%, 23%, and 34% for high, medium, low, and very low abundance peaks, respectively. To assess the impact of sprayer variability on tissue classification using multivariate statistical tools, nine human colorectal adenocarcinoma sections are analyzed with three lab-built sprayers, and classification accuracy for adenocarcinoma versus the surrounding stroma is assessed. It ranges from 80.7 to 94.5% between the three sprayers and is 86.5% overall. The presented results confirm that the sprayer setup needs to be closely controlled to obtain reliable data, and a new sprayer setup with a fixed solvent capillary geometry should be developed.

Enhanced capabilities for imaging gangliosides in murine brain with matrix-assisted laser desorption/ionization and desorption electrospray ionization mass spectrometry coupled to ion mobility separation.

The increased interest in lipidomics calls for improved yet simplified methods of lipid analysis. Over the past two decades, mass spectrometry imaging (MSI) has been established as a powerful technique for the analysis of molecular distribution of a variety of compounds across tissue surfaces. Matrix-assisted laser desorption/ionization (MALDI) MSI is widely used to study the spatial distribution of common lipids. However, a thorough sample preparation and necessity of vacuum for efficient ionization might hamper its use for high-throughput lipid analysis. Desorption electrospray ionization (DESI) is a relatively young MS technique. In DESI, ionization of molecules occurs under ambient conditions, which alleviates sample preparation. Moreover, DESI does not require the application of an external matrix, making the detection of low mass species more feasible due to the lack of chemical matrix background. However, irrespective of the ionization method, the final information obtained during an MSI experiment is very complex and its analysis becomes challenging. It was shown that coupling MSI to ion mobility separation (IMS) simplifies imaging data interpretation. Here we employed DESI and MALDI MSI for a lipidomic analysis of the murine brain using the same IMS-enabled instrument. We report for the first time on the DESI IMS-MSI of multiply sialylated ganglioside species, as well as their acetylated versions, which we detected directly from the murine brain tissue. We show that poly-sialylated gangliosides can be imaged as multiply charged ions using DESI, while they are clearly separated from the rest of the lipid classes based on their charge state using ion mobility. This represents a major improvement in MSI of intact fragile lipid species. We additionally show that complementary lipid information is reached under particular conditions when DESI is compared to MALDI MSI.

A Comparison of DESI-MS and LC-MS for the Lipidomic Profiling of Human Cancer Tissue.

In this study, we make a direct comparison between desorption electrospray ionization-mass spectrometry (DESI-MS) and ultraperformance liquid chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS) platforms for the profiling of glycerophospholipid (GPL) species in esophageal cancer tissue. In particular, we studied the similarities and differences in the range of GPLs detected and the congruency of their relative abundances as detected by each analytical platform. The main differences between mass spectra of the two modalities were found to be associated with the variance in adduct formation of common GPLs, rather than the presence of different GPL species. Phosphatidylcholines as formate adducts in UPLC-ESI-MS accounted for the majority of differences in negative ion mode and alkali metal adducts of phosphatidylcholines in DESI-MS for positive ion mode. Comparison of the relative abundance of GPLs, normalized to a common peak, revealed a correlation coefficient of 0.70 (P < 0.001). The GPL profile detected by DESI-MS is congruent to UPLC-ESI-MS, which reaffirms the role of DESI-MS for lipidomic profiling and a potential premise for quantification.