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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.
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Amônia-Liases/metabolismo , Anabaena variabilis/enzimologia , Escherichia coli/metabolismo , Oxigenases de Função Mista/metabolismo , Amônia-Liases/química , Biocatálise , Escherichia coli/citologia , Oxigenases de Função Mista/química , Estrutura Molecular , Espectrometria de Massas por Ionização por Electrospray , Fatores de TempoRESUMO
MALDI-mass spectrometry imaging (MALDI-MSI) has been shown to allow the study of protein distribution and identification directly within formalin-fixed paraffin-embedded (FFPE) tissue sections. However, direct protein identification from tissue sections remains challenging due to signal interferences and/or existing post-translational or other chemical modifications. The use of antigen retrieval (AR) has been demonstrated for unlocking proteins prior to in situ enzymatic digestion and MALDI-MSI analysis of FFPE tissue sections. In the work reported here, the identification of proline oxidation, which may occur when performing the AR protocol, is described. This facilitated and considerably increased the number of identified peptides when adding proline oxidation as a variable modification to the MASCOT search criteria. This article is part of a Special Issue entitled: MALDI Imaging, edited by Dr. Corinna Henkel and Prof. Peter Hoffmann.
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Antígenos/metabolismo , Formaldeído/química , Neoplasias/diagnóstico , Neoplasias/patologia , Parafina/química , Prolina/metabolismo , Humanos , Oxirredução , Peptídeos/metabolismo , Prolina/química , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
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.
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Química Encefálica , Gangliosídeos/isolamento & purificação , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Gangliosídeos/química , Íons/química , CamundongosRESUMO
The high chemical complexity of the lipidome is one of the major challenges in lipidomics research. Ion-mobility spectrometry (IMS), a gas-phase electrophoretic technique, makes possible the separation of ions in the gas phase according to their charge, shape, and size. IMS can be combined with mass spectrometry (MS), adding three major benefits to traditional lipidomic approaches. First, IMS-MS allows the determination of the collision cross section (CCS), a physicochemical measure related to the conformational structure of lipid ions. The CCS is used to improve the confidence of lipid identification. Second, IMS-MS provides a new set of hybrid fragmentation experiments. These experiments, which combine collision-induced dissociation with ion-mobility separation, improve the specificity of MS/MS-based approaches. Third, IMS-MS improves the peak capacity and signal-to-noise ratio of traditional analytical approaches. In doing so, it allows the separation of complex lipid extracts from interfering isobaric species. Developing in parallel with advances in instrumentation, informatics solutions enable analysts to process and exploit IMS-MS data for qualitative and quantitative applications. Here we review the current approaches for lipidomics research based on IMS-MS, including liquid chromatography-MS and direct-MS analyses of "shotgun" lipidomics and MS imaging.
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Cromatografia Líquida/métodos , Lipídeos/análise , Espectrometria de Massas/métodos , Animais , Humanos , Íons/análise , Metabolômica/métodos , Modelos MolecularesRESUMO
The combination of enhanced resolving power and improved duty cycle on a multireflecting time-of-flight mass spectrometer is described. Resolving power increases are achieved by extending the effective ion path length from 47 m to greater than 200 m. Path length increases are achieved through containment of ions within the analyzer for up to N = 5 passes using a pulsed deflection electrode. Resolving power was shown to increase from 220,000 to 402,000 (fwhm) at m/z 785 for N = 1 and N = 4 analyzer passes, respectively. Due to the timing of the pulsed deflection electrode, the approach is particularly suited to high resolution analysis over a targeted m/z range. Duty cycle enhancements are achieved for ions of the targeted m/z range via accumulation prior to orthogonal acceleration, providing signal improvements of 2 orders of magnitude. Achieving such high resolving powers at fast scan rates (30 Hz) can yield additional information such as fine isotope structure; when combined with ppb mass measurement accuracy, high confidence in analyte identification can be achieved. The technique is applied for N = 2 analyzer passes, demonstrating fine isotope structure for a typical UHPLC metabolite identification experiment at a 10 Hz acquisition rate. Additionally, mass spectrometry imaging data is acquired using DESI, demonstrating the improved image clarity achieved at >300,000 (fwhm).
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A multimodal mass spectrometry imaging (MSI) approach was used to investigate the chemotherapy drug-induced response of a Multicellular Tumour Spheroid (MCTS) 3D cell culture model of osteosarcoma (OS). The work addresses the critical demand for enhanced translatable early drug discovery approaches by demonstrating a robust spatially resolved molecular distribution analysis in tumour models following chemotherapeutic intervention. Advanced high-resolution techniques were employed, including desorption electrospray ionisation (DESI) mass spectrometry imaging (MSI), to assess the interplay between metabolic and cellular pathways in response to chemotherapeutic intervention. Endogenous metabolite distributions of the human OS tumour models were complemented with subcellularly resolved protein localisation by the detection of metal-tagged antibodies using Imaging Mass Cytometry (IMC). The first application of matrix-assisted laser desorption ionization-immunohistochemistry (MALDI-IHC) of 3D cell culture models is reported here. Protein localisation and expression following an acute dosage of the chemotherapy drug doxorubicin demonstrated novel indications for mechanisms of region-specific tumour survival and cell-cycle-specific drug-induced responses. Previously unknown doxorubicin-induced metabolite upregulation was revealed by DESI-MSI of MCTSs, which may be used to inform mechanisms of chemotherapeutic resistance. The demonstration of specific tumour survival mechanisms that are characteristic of those reported for in vivo tumours has underscored the increasing value of this approach as a tool to investigate drug resistance.
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BACKGROUND: Selection of the most promising radiotracer candidates for radiolabeling is a difficult step in the development of radiotracer pharmaceuticals, especially for the brain. Mass spectrometry (MS) is an alternative to study ex vivo the characteristics of candidates, but most MS studies are complicated by the pharmacologic doses injected and the dissection of regions to study candidate biodistribution. In this study, we tested the ability of a triple quadrupole analyzer (TQ LC-MS/MS) to quantify low concentrations of a validated precursor of a radiotracer targeting the DAT (LBT-999) in dissected regions. We also investigated its biodistribution on brain slices using MS imaging with desorption electrospray ionization (DESI) coupled to time-of-flight (TOF) vs. TQ mass analyzers. RESULTS: TQ LC-MS/MS enabled quantification of LBT-999 injected at sub-tracer doses in dissected striata. DESI-MS imaging (DESI-MSI) with both analyzers provided images of LBT-999 biodistribution on sagittal slices that were consistent with positron emission tomography (PET). However, the TOF analyzer only obtained biodistribution images at a high injected dose of LBT-999, while the TQ analyzer provided biodistribution images at lower injected doses of LBT-999 with a better signal-to-noise ratio. It also allowed simultaneous visualization of endogenous metabolites such as dopamine. CONCLUSIONS: Our results show that LC-TQ MS/MS in combination with DESI-MSI can provide important information (biodistribution, specific and selective binding) that can facilitate the selection of the most promising candidates for radiolabeling and support the development of radiotracers.
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Sexual offenders are increasingly reported to use condoms while committing the crime, mainly to prevent the transfer of DNA evidence. Although condoms are often removed from the crime scene, vaginal swabs can be taken from the victim to prove the presence of condom lubricants and therefore evidence of corpus delicti. However, late reporting to the police and the tendency of the victim to wash immediately after the crime, may compromise the detection of condom lubricants. Recently we showed that Matrix-Assisted Laser Desorption/Ionisation MS Imaging (MALDI MSI) of condom contaminated fingermarks enables images of the fingermark ridge pattern to be obtained simultaneously with the detection of the condom lubricant for two condom brands, thus becoming a potential alternative way to link the assailant to the crime. Building on the value of this information, it would be advantageous to identify the condom brand used during the sexual assault. Here we show the development of a multidisciplinary spectroscopic approach, including MALDI MSI, MS/MS, Raman microscopy and ATR-FTIR spectroscopy, applied to a range of condom brands/types. The techniques have complementary features and provide complementary information to retrieve a "condom brand spectroscopic fingerprint". Unique spectroscopic profiles would greatly aid in the screening and identification of the condom, thus adding intelligence to the case under investigation.
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Preservativos , Dermatoglifia , Medicina Legal/métodos , Lubrificantes/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Análise Espectral Raman/métodos , Feminino , Humanos , Masculino , Delitos SexuaisRESUMO
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).
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Diagnóstico por Imagem , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Background & Aims: Lipid metabolism plays an important role in liver pathophysiology. The liver lobule asymmetrically distributes oxygen and nutrition, resulting in heterogeneous metabolic functions. Periportal and pericentral hepatocytes have different metabolic functions, which lead to generating liver zonation. We developed spatial metabolic imaging using desorption electrospray ionisation mass spectrometry to investigate lipid distribution across liver zonation with high reproducibility and accuracy. Methods: Fresh frozen livers from healthy mice with control diet were analysed using desorption electrospray ionisation mass spectrometry imaging. Imaging was performed at 50 µm × 50 µm pixel size. Regions of interest (ROIs) were manually created by co-registering with histological data to determine the spatial hepatic lipids across liver zonation. The ROIs were confirmed by double immunofluorescence. The mass list of specific ROIs was automatically created, and univariate and multivariate statistical analysis were performed to identify statistically significant lipids across liver zonation. Results: A wide range of lipid species was identified, including fatty acids, phospholipids, triacylglycerols, diacylglycerols, ceramides, and sphingolipids. We characterised hepatic lipid signatures in three different liver zones (periportal zone, midzone, and pericentral zone) and validated the reproducibility of our method for measuring a wide range of lipids. Fatty acids were predominantly detected in the periportal region, whereas phospholipids were distributed in both the periportal and pericentral zones. Interestingly, phosphatidylinositols, PI(36:2), PI(36:3), PI(36:4), PI(38:5), and PI(40:6) were located predominantly in the midzone (zone 2). Triacylglycerols and diacylglycerols were detected mainly in the pericentral region. De novo triacylglycerol biosynthesis appeared to be the most influenced pathway across the three zones. Conclusions: The ability to accurately assess zone-specific hepatic lipid distribution in the liver could lead to a better understanding of lipid metabolism during the progression of liver disease. Impact and Implications: Zone-specific hepatic lipid metabolism could play an important role in lipid homoeostasis during disease progression. Herein, we defined the zone-specific references of hepatic lipid species in the three liver zones using molecular imaging. The de novo triacylglycerol biosynthesis was highlighted as the most influenced pathway across the three zones.
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BACKGROUND AND AIMS: Lipids play an important role in atherosclerotic plaque development and are interesting candidate predictive biomarkers. However, the link between circulating lipids, accumulating lipids in the vessel wall, and plaque destabilization processes in humans remains largely unknown. This study aims to provide new insights into the role of lipids in atherosclerosis using lipidomics and mass spectrometry imaging to investigate lipid signatures in advanced human carotid plaque and plasma samples. METHODS: We used lipidomics and desorption electrospray ionization mass spectrometry imaging (DESI-MSI) to investigate lipid signatures of advanced human carotid plaque and plasma obtained from patients who underwent carotid endarterectomy (n = 14 out of 17 whose plaque samples were analyzed by DESI-MSI). Multivariate data analysis and unsupervised clustering were applied to identify lipids that were the most discriminative species between different patterns in plaque and plasma. These patterns were interpreted by quantitative comparison with conventional histology. RESULTS: Lipidomics detected more than 300 lipid species in plasma and plaque, with markedly different relative abundances. DESI-MSI visualized the spatial distribution of 611 lipid-related m/z features in plaques, of which 330 m/z features could be assigned based on exact mass, comparison to the lipidomic data, and high mass resolution MSI. Matching spatial lipid patterns to histological areas of interest revealed several molecular species that were colocalized with pertinent disease processes in plaque including specific sphingomyelin and ceramide species with calcification, phospholipids and free fatty acids with inflammation, and triacylglycerols and phosphatidylinositols with fibrin-rich areas. CONCLUSIONS: By comparing lipid species in plaque and plasma, we identified those circulating species that were also prominently present in plaque. Quantitative comparison of lipid spectral patterns with histology revealed the presence of specific lipid species in destabilized plaque areas, corroborating previous in vitro and animal studies.
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Aterosclerose , Placa Aterosclerótica , Animais , Humanos , Espectrometria de Massas , Placa Aterosclerótica/química , Artérias Carótidas , Fosfolipídeos , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
Reversed-phase high performance thin-layer chromatography (RP-HPTLC) on C18 bonded silica gel was combined with desorption electrospray ionization (DESI) and high resolution time of flight mass spectrometry (HRToFMS) to detect, characterize and image (MSI) phytoecdysteroids (plant-derived insect moulting hormones) in ethanolic extracts of members of the Silene plant family. As seen previously for silica gel, DESI provided a simple and convenient method for recovering polar polyhydoxysteroids from RP-HPTLC plates for the purposes of both the MS and MSI of extracts obtained from three species of the Silene family (Silene otites, S. nutans and S. viridiflora). Using RP-HPTLC/DESI/MSI/HRToFMS a number of ecdysteroids, including 20-hydroxyecdysone, polypodine-B, 2-deoxy-20-hydroxyecdysone and 2-deoxyecdysone were identified in these extracts. Differences were noted in the mass spectra obtained depending upon both the stationary phase on which they were separated, and the temperatures used in the heated transfer line used for introduction into the ion source. Ecdysteroids detected after chromatography on C18 bonded silica showed increased fragmentation due to water loss compared to those imaged from silica. In addition, the benefits of the additional resolution provided by 2-dimensional TLC for increasing spectral quality compared to a 1-dimensional separation are demonstrated.
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Ecdisteroides , Espectrometria de Massas por Ionização por Electrospray , Cromatografia em Camada Fina/métodos , Ecdisterona , Extratos Vegetais/química , Sílica Gel , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
Desorption/ionization (DI)-mass spectrometric (MS) methods offer considerable advantages of rapidity and low-sample input for the analysis of solid biological matrices such as tissue sections. The concept of desorption electrospray ionization (DESI) offers the possibility to ionize compounds from solid surfaces at atmospheric pressure, without the addition of organic compounds to initiate desorption. However, severe drawbacks from former DESI hardware stability made the development of assays for drug quantification difficult. In the present study, the potential of new prototype source setups (High Performance DESI Sprayer and Heated Transfer Line) for the development of drug quantification assays in tissue sections was evaluated. It was demonstrated that following dedicated optimization, new DESI XS enhancements present promising options regarding targeted quantitative analyses. As a model compound for these developments, ulixertinib, an inhibitor of extracellular signal-regulated kinase (ERK) 1 and 2 was used.
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Lipidomics is a rapidly expanding area of scientific research and there are a number of analytical techniques that are employed to facilitate investigations. One such technique is matrix-assisted laser desorption ionisation (MALDI) mass spectrometry (MS). Previous MALDI-MS studies involving lipidomic investigation have included the analysis of a number of different ex vivo tissues, most of which were obtained from animal models, with only a few being of human origin. In this study, we describe the use of MALDI-MS, MS/MS and MS imaging methods for analysing lipids within cross-sections of ex vivo human skin. It has been possible to tentatively identify lipid species via accurate mass measurement MALDI-MS and also to confirm the identity of a number of these species via MALDI-MS/MS, in experiments carried out directly on tissue. The main lipid species detected include glycerophospholipids and sphingolipids. MALDI images have been generated at a spatial resolution of 150 and 30 µm, using a MALDI quadrupole time-of-flight Q-Star Pulsar-i (TM) (Applied Biosystems/MDS Sciex, Concord, ON, Canada) and a MALDI high-definition MS (HDMS) SYNAPT G2-HDMS(TM) system (Waters, Manchester, UK), respectively. These images show the normal distribution of lipids within human skin, which will provide the basis for assessing alterations in lipid profiles linked to specific skin conditions e.g. sensitisation, in future investigations.
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Lipídeos/análise , Pele/química , Pele/ultraestrutura , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espectrometria de Massas em Tandem/métodos , Diagnóstico por Imagem/métodos , Humanos , Bancos de TecidosRESUMO
Matrix assisted laser desorption ionisation mass spectrometry imaging (MALDI-MSI), was used to obtain images of lipids and metabolite distribution in formalin fixed and embedded in paraffin (FFPE) whole eye sections containing primary uveal melanomas (UM). Using this technique, it was possible to obtain images of lysophosphatidylcholine (LPC) type lipid distribution that highlighted the tumour regions. Laser ablation inductively coupled plasma mass spectrometry images (LA-ICP-MS) performed on UM sections showed increases in copper within the tumour periphery and intratumoural zinc in tissue from patients with poor prognosis. These preliminary data indicate that multi-modal MSI has the potential to provide insights into the role of trace metals and cancer metastasis.
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The ability to analyze complex (macro) molecules is of fundamental importance for understanding chemical, physical, and biological processes. Complexity may arise from small differences in structure, large dynamic range, as well as a vast range in solubility or ionization, imposing daunting tasks in areas as different as lipidomics and proteomics. Here, we describe a rapid matrix application that permits the deposition of matrix-assisted laser desorption/ionization (MALDI) matrix solvent-free. This solvent-free one-step automatic matrix deposition is achieved through vigorous movements of beads pressing the matrix material through a metal mesh. The mesh (20 mum) produces homogeneous coverage of <12 microm crystals (DHB, CHCA matrixes) in 1 min, as determined by optical microscopy, permitting fast uniform coverage of analyte and possible high-spatial resolution surface analysis. Homogenous tissue coverage of <5 microm sized crystals is achieved using a 3 microm mesh. Solvent-free MALDI analysis on a time-of-flight (TOF) mass analyzer of mouse brain tissue homogenously covered with CHCA matrix subsequently provides a homogeneous response in ion signal intensity. Total solvent-free analysis (TSA) by mass spectrometry (MS) of tissue sections is carried out by applying the MALDI matrix solvent-free for subsequent ionization and gas phase separation for decongestion of complexity in the absence of any solvent using ion mobility spectrometry (IMS) followed by MS detection. Isobaric compositions were well-delineated using TSA by MS.
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Encéfalo/anatomia & histologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Camundongos , Camundongos Endogâmicos C57BL , SolventesRESUMO
The development of tissue micro-array (TMA) technologies provides insights into high-throughput analysis of proteomics patterns from a large number of archived tumour samples. In the work reported here, matrix-assisted laser desorption/ionisation-ion mobility separation-mass spectrometry (MALDI-IMS-MS) profiling and imaging methodology has been used to visualise the distribution of several peptides and identify them directly from TMA sections after on-tissue tryptic digestion. A novel approach that combines MALDI-IMS-MSI and principal component analysis-discriminant analysis (PCA-DA) is described, which has the aim of generating tumour classification models based on protein profile patterns. The molecular classification models obtained by PCA-DA have been validated by applying the same statistical analysis to other tissue cores and patient samples. The ability to correlate proteomic information obtained from samples with known and/or unknown clinical outcome by statistical analysis is of great importance, since it may lead to a better understanding of tumour progression and aggressiveness and hence improve diagnosis, prognosis as well as therapeutic treatments. The selectivity, robustness and current limitations of the methodology are discussed.
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Adenocarcinoma/patologia , Neoplasias Pancreáticas/patologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Análise Serial de Tecidos/métodos , Sequência de Aminoácidos , Humanos , Dados de Sequência Molecular , Proteínas/análiseRESUMO
Matrix-Assisted Laser Desorption Ionization (MALDI) and Desorption Electrospray Ionization (DESI) are two complementary ionization techniques that have transformed the field of biomolecular analysis, enabling the measurement of a wide range of biomolecules by mass spectrometry. These techniques have also been applied to imaging mass spectrometry where the spatial localization of molecules is determined. Coupling this with Ion Mobility Spectrometry (IM) allows an additional level of separation and specificity to be obtained. Here, we describe the coupling of the technologies and the practical advantages of these combinations, highlighting specific examples.
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Espectrometria de Mobilidade Iônica , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Mobilidade Iônica/métodos , Lipídeos/análise , Peptídeos/análise , Polissacarídeos/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
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.
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Glioblastoma/diagnóstico por imagem , Imageamento Tridimensional/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Animais , Diagnóstico por Imagem/métodos , Modelos Animais de Doenças , Feminino , Xenoenxertos/diagnóstico por imagem , Metabolismo dos Lipídeos/fisiologia , Lipídeos/química , Camundongos , Camundongos Nus , Estudo de Prova de Conceito , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
The identification of proteins involved in tumour progression or which permit enhanced or novel therapeutic targeting is essential for cancer research. Direct MALDI analysis of tissue sections is rapidly demonstrating its potential for protein imaging and profiling in the investigation of a range of disease states including cancer. MALDI-mass spectrometry imaging (MALDI-MSI) has been used here for direct visualisation and in situ characterisation of proteins in breast tumour tissue section samples. Frozen MCF7 breast tumour xenograft and human formalin-fixed paraffin-embedded breast cancer tissue sections were used. An improved protocol for on-tissue trypsin digestion is described incorporating the use of a detergent, which increases the yield of tryptic peptides for both fresh frozen and formalin-fixed paraffin-embedded tumour tissue sections. A novel approach combining MALDI-MSI and ion mobility separation MALDI-tandem mass spectrometry imaging for improving the detection of low-abundance proteins that are difficult to detect by direct MALDI-MSI analysis is described. In situ protein identification was carried out directly from the tissue section by MALDI-MSI. Numerous protein signals were detected and some proteins including histone H3, H4 and Grp75 that were abundant in the tumour region were identified.