Resolution Matrix-assisted Laser Desorption Ionization Research Articles

Single drop analysis of mercury ions by rational design of peptide coated gold nanoparticles integrated with MALDI-MS measurement

In this study, a novel and rapid method for specific identification and accurate quantification of Hg2+ in environmental water was developed by using laser cleavable cysteine containing peptides modified gold nanoparticles coupled with high resolution matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF MS) measurement. First, gold nanoparticles were prepared by the reduction of tetrachloroauric (III) acid (HAuCl4) solution. Various cysteine containing peptides, photolabile linkers, including mercury ion binding motif with a proper molecular mass and amino acids were synthesized by solid phase peptide synthesis (SPPS). Subsequently, thiol-containing peptides were coated onto the surface of gold nanoparticles via the formation of gold-thiol (Au–S) bond. The resulting cysteine containing peptides modified gold nanoparticles were designed to specifically capture Hg2+ in water samples. After conjugated complex formation, ions of Hg2+–peptide complex were directly liberated by ultraviolet laser radiation by way of MALDI-MS using α-Cyano-4-hydroxycinnamic acid (CHCA) as matrix. The linear dynamic range of Hg2+ concentration in this study was 1–100 pmol/μL with coefficient of determination 0.9987. The limit of detection (LOD) and limit of quantification (LOQ) were 0.19 and 0.63 pmol/μL, respectively. Notably, the developed method allows rapid quantification of Hg2+ in 5 min and the desired sample volume was down to few μL.

Multi-Class Cancer Subtyping in Salivary Gland Carcinomas with MALDI Imaging and Deep Learning.

Simple SummaryThe correct diagnosis of different salivary gland carcinomas is important for a prognosis. This diagnosis is imprecise if it is based only on clinical symptoms and histological methods. Mass spectrometry imaging can provide information about the molecular composition of sample tissues. Using a deep-learning method, we analyzed the mass spectrometry imaging data of 25 patients. Using this workflow we could accurately predict the tumor type in each patient sample.Salivary gland carcinomas (SGC) are a heterogeneous group of tumors. The prognosis varies strongly according to its type, and even the distinction between benign and malign tumor is challenging. Adenoid cystic carcinoma (AdCy) is one subgroup of SGCs that is prone to late metastasis. This makes accurate tumor subtyping an important task. Matrix-assisted laser desorption/ionization (MALDI) imaging is a label-free technique capable of providing spatially resolved information about the abundance of biomolecules according to their mass-to-charge ratio. We analyzed tissue micro arrays (TMAs) of 25 patients (including six different SGC subtypes and a healthy control group of six patients) with high mass resolution MALDI imaging using a 12-Tesla magnetic resonance mass spectrometer. The high mass resolution allowed us to accurately detect single masses, with strong contributions to each class prediction. To address the added complexity created by the high mass resolution and multiple classes, we propose a deep-learning model. We showed that our deep-learning model provides a per-class classification accuracy of greater than 80% with little preprocessing. Based on this classification, we employed methods of explainable artificial intelligence (AI) to gain further insights into the spectrometric features of AdCys.

Mapping the Lipids of Skin Sebaceous Glands and Hair Follicles by High Spatial Resolution MALDI Imaging Mass Spectrometry.

Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is a technology that utilizes the high sensitivity and specificity of mass spectrometry, combined with a high spatial resolution to characterize the molecular species present in skin tissue. In this article, we use MALDI IMS to map specific lipids characteristic of two important skin appendages in minipig skin: the sebaceous glands and hair follicles. A set of specific lipid markers linked to the synthesis of sebum, stages of sebum production, and the secretion of sebum for two different sebaceous gland subzones, the peripheral and central necrotic, were identified. Furthermore, biochemical pathway analysis of the identified markers provides potential drug-targeting strategies to reduce sebum overproduction in pathological conditions. In addition, specific lipid markers characteristic of the different layers in the hair follicle bulge area, including the outer root sheath, the inner root sheath, and the medulla that are associated with the growth cycles of the hair, were determined. This research highlights the ability of MALDI IMS to link a molecular distribution not only to the morphological features in skin tissue but to the physiological state as well. Thus, this platform can provide a basis for the investigation of biochemical pathways as well as the mechanisms of disease and pharmacology in the skin, which will ultimately be critical for drug discovery and the development of dermatology-related illnesses.

High Spatial Resolution MALDI Imaging Mass Spectrometry of Fresh-Frozen Bone.

Bone and bone marrow are vital to mammalian structure, movement, and immunity. These tissues are also commonly subjected to molecular alterations giving rise to debilitating diseases like rheumatoid arthritis and osteomyelitis. Technologies such as matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) facilitate the discovery of spatially resolved chemical information in biological tissue samples to help elucidate the complex molecular processes underlying pathology. Traditionally, preparation of osseous tissue for MALDI IMS has been difficult due to its mineralized composition and heterogeneous morphology, and compensation for these challenges with decalcification and fixation protocols can remove or delocalize molecular species. Here, sample preparation methods were advanced to enable multimodal MALDI IMS of undecalcified, fresh-frozen murine femurs, allowing the distribution of endogenous lipids to be linked to tissue structures and cell types. Adhesive-bound bone sections were mounted onto conductive glass slides with microscopy-compatible glue and freeze-dried to minimize artificial bone marrow damage. High spatial resolution (10 μm) MALDI IMS was employed to characterize lipid distributions, and use of complementary microscopy modalities aided tissue and cell assignments. For example, various phosphatidylcholines localize to the bone marrow, adipose tissue, marrow adipose tissue, and muscle. Further, sphingomyelin(42:1) was abundant in megakaryocytes, whereas sphingomyelin(42:2) was diminished in this cell type. These data reflect the vast molecular and cellular heterogeneity indicative of the bone marrow and the soft tissue surrounding the femur. Multimodal MALDI IMS has the potential to advance bone-related biomedical research by offering deep molecular coverage with spatial relevance in a preserved native bone microenvironment.

α-Cyano-4-hydroxycinnamic Acid and Tri-Potassium Citrate Salt Pre-Coated Silicon Nanopost Array Provides Enhanced Lipid Detection for High Spatial Resolution MALDI Imaging Mass Spectrometry.

We have developed a pre-coated substrate for matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) that enables high spatial resolution mapping of both phospholipids and neutral lipid classes in positive ion mode as metal cation adducts. The MALDI substrates are constructed by depositing a layer of α-cyano-4-hydroxycinnamic acid (CHCA) and potassium salts onto silicon nanopost arrays (NAPA) prior to tissue mounting. The matrix/salt pre-coated NAPA substrate significantly enhances all detected lipid signals allowing lipids to be detected at lower laser energies than bare NAPA. The improved sensitivity at lower laser energy enabled ion images to be generated at 10 μm spatial resolution from rat retinal tissue. Optimization of matrix pre-coated NAPA consisted of testing lithium, sodium, and potassium salts along with various matrices to investigate the increased sensitivity toward lipids for MALDI IMS experiments. It was determined that pre-coating NAPA with CHCA and potassium salts before thaw-mounting of tissue resulted in a signal intensity increase of at least 5.8 ± 0.1-fold for phospholipids and 2.0 ± 0.1-fold for neutral lipids compared to bare NAPA. Pre-coating NAPA with matrix and salt also reduced the necessary laser power to achieve desorption/ionization by ∼35%. This reduced the effective diameter of the ablation area from 13 ± 2 μm down to 8 ± 1 μm, enabling high spatial resolution MALDI IMS. Using pre-coated NAPA with CHCA and potassium salts offers a MALDI IMS substrate with broad molecular coverage of lipids in a single polarity that eliminates the need for extensive sample preparation after sectioning.

Rapid analysis of ketamine with in-house antibody conjugated boronic acid modified silver chip on MALDI-TOF MS measurement

An antibody conjugated boronic acid modified silver chip (ABAS ship) is fabricated as a simple, rapid, accurate, sensitive and cost-effective sample preparation method for abused drug quantification in human urine. Ketamine, one common abused drug, was applied as proof of concept for ABAS chip with high resolution matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF MS) analysis. The overall testing process required 10 min at part per billion (ppb) sensitivity level, where current drug testing method necessitated several hours with similar sensitivity. The ABAS chip manufacture process started with slide glass by way of silver mirror reaction to form silver conductive glass for further chemical conjugation. Boronic acid functional group was decorated on silver conductive glass through the formation of silver-thiol (Ag–S) bond. Anti-ketamine antibody was covalently conjugated to boronic acid modified silver conductive glass through the formation of cyclic boronate ester between the boronic acid and the cis-diol groups on the glycans of antibody, which maintain the correct orientation to maximally capture its antigen. The resulting ABAS chip were designed to specifically capture ketamine in human urine samples, that could be directly analyzed by addition of MALDI α-Cyano-4-hydroxycinnamic acid (CHCA) matrix solution. The linear dynamic range of concentration in this method was 10–500 ng/mL with coefficient of determination 0.996. The limit of detection (LOD) and limit of quantification (LOQ) were 2.0 and 7.0 ng/mL, respectively. Importantly, the proposed method allows rapid and accurate quantification of ketamine from suspects’ urine samples in 10 min and small sample volume of 1 μL was required. The resulting data were consistent with traditional gas chromatography-mass spectrometry (GC-MS) analysis. Our homemade ABAS chip could thus provide a powerful tool not only for forensic science but also for most clinical diagnosis of disease as many expression antibodies for the occurrence of diverse diseases could be simply produced and purchased.

Combining Salt Doping and Matrix Sublimation for High Spatial Resolution MALDI Imaging Mass Spectrometry of Neutral Lipids.

The combination of sodium salt doping of a tissue section along with the sublimation of the matrix 2,5-dihydrobenzoic acid (DHB) was found to be an effective coating for the simultaneous detection of neutral lipids and phospholipids using matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry in positive ionization mode. Lithium, sodium, and potassium acetate were initially screened for their ability to cationize difficult to analyze neutral lipids such as cholesterol esters, cerebrosides, and triglycerides directly from a tissue section. The combination of sodium salt and DHB sublimation was found to be an effective cation/matrix combination for detection of neutral lipids. Further experimental optimizations revealed that sodium carbonate or sodium phosphate followed by DHB sublimation increases the signal intensity of the neutral lipids studied depending on the specific lipid family and tissue type by 10-fold to 140-fold compared with that of previously published methods. Application of sodium carbonate tissue doping and DHB sublimation resulted in crystal sizes ≤2 μm. We were thus able to image a mouse brain cerebellum at a high spatial resolution and detected 37 cerebrosides in a single run using a MALDI-TOF instrument. The combination of sodium doping and DHB sublimation offer a targeted and sensitive approach for the detection of neutral lipids that do not typically ionize well under normal MALDI conditions.

Automatic 3D Nonlinear Registration of Mass Spectrometry Imaging and Magnetic Resonance Imaging Data.

Multimodal integration between mass spectrometry imaging (MSI) and radiology-established modalities such as magnetic resonance imaging (MRI) would allow the investigations of key questions in complex biological systems such as the central nervous system. Such integration would provide complementary multiscale data to bridge the gap between molecular and anatomical phenotypes, potentially revealing new insights into molecular mechanisms underlying anatomical pathologies presented on MRI. Automatic coregistration between 3D MSI/MRI is a computationally challenging process due to dimensional complexity, MSI data sparsity, lack of direct spatial-correspondences, and nonlinear tissue deformation. Here, we present a new computational approach based on stochastic neighbor embedding to nonlinearly align 3D MSI to MRI data, identify and reconstruct biologically relevant molecular patterns in 3D, and fuse the MSI datacube to the MRI space. We demonstrate our method using multimodal high-spectral resolution matrix-assisted laser desorption ionization (MALDI) 9.4 T MSI and 7 T in vivo MRI data, acquired from a patient-derived, xenograft mouse brain model of glioblastoma following administration of the EGFR inhibitor drug of Erlotinib. Results show the distribution of some identified molecular ions of the EGFR inhibitor erlotinib, a phosphatidylcholine lipid, and cholesterol, which were reconstructed in 3D and mapped to the MRI space. The registration quality was evaluated on two normal mouse brains using the Dice coefficient for the regions of brainstem, hippocampus, and cortex. The method is generic and can therefore be applied to hyperspectral images from different mass spectrometers and integrated with other established in vivo imaging modalities such as computed tomography (CT) and positron emission tomography (PET).

Defining Changes in the Spatial Distribution and Composition of Brain Lipids in the Shiverer and Cuprizone Mouse Models of Myelin Disease.

Myelin is composed primarily of lipids and diseases affecting myelin are associated with alterations in its lipid composition. However, correlation of the spatial (in situ) distribution of lipids with the disease-associated compositional and morphological changes is not well defined. Herein we applied high resolution matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS), immunohistochemistry (IHC), and liquid chromatography–electrospray ionization–mass spectrometry (LC-ESI-MS) to evaluate brain lipid alterations in the dysmyelinating shiverer (Shi) mouse and cuprizone (Cz) mouse model of reversible demyelination. MALDI-IMS revealed a decrease in the spatial distribution of sulfatide (SHexCer) species, SHexCer (d42:2), and a phosphatidylcholine (PC) species, PC (36:1), in white matter regions like corpus callosum (CC) both in the Shi mouse and Cz mouse model. Changes in these lipid species were restored albeit not entirely upon spontaneous remyelination after demyelination in the Cz mouse model. Lipid distribution changes correlated with the local morphological changes as confirmed by IHC. LC-ESI-MS analyses of CC extracts confirmed the MALDI-IMS derived reductions in SHexCer and PC species. These findings highlight the role of SHexCer and PC in preserving the normal myelin architecture and our experimental approaches provide a morphological basis to define lipid abnormalities relevant to myelin diseases.

Rapid and accurate quantification of amphetamine and methamphetamine in human urine by antibody decorated magnetite nanoparticles coupled with matrix-assisted laser desorption ionization time-of-flight mass spectrometer analysis

In this study, a novel method for the simultaneous determination and accurate quantification of abused drugs in human urine was developed. Antibody conjugated boronic acid modified magnetite nanoparticles (Fe3O4, MNPs) were prepared for the selectively purification of illicit drugs in combination with high resolution matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF MS) analysis. Illicit drugs, amphetamine (AM) and methamphetamine (MA), were used as model analytes to demonstrate the feasibility of our strategy. Boronic acid functionalized MNPs were first prepared via one-pot synthesis to simplify and improve the efficiency of a chemical reaction. Anti-amphetamine antibody (anti-AM antibody) and anti-methamphetamine antibody (anti-MA antibody) was conjugated onto boronic acid modified MNPs, respectively, through the formation of boronate ester bond that could maintain the correct orientation to maximally capture their antigens. The capacity of antibody conjugation to boronic acid modified MNPs was at least 24 μg antibody/mg MNPs. Antibody-conjugated MNPs were designed to specifically capture AM and MA in human urine samples, both of which can be directly eluted to MALDI target plate by adding MALDI CHCA matrix solution for the following MALDI-MS analysis. The linear range of detection of the proposed method were 25–400 ng/mL and 25–1000 ng/mL with coefficients of determination between 0.9923 and 0.9997 for AM and MA, respectively. The lowest detectable concentrations of AM and MA were 1.87 and 3.75 ng/mL, respectively. More importantly, the proposed method allows rapid and accurate quantification of AM and MA from three suspects' urine samples. The obtained results are consistent with traditional GC/MS analysis. Antibody-conjugated MNPs could thus prove to be powerful tools for important applications such as forensic science, food safety and clinical diagnosis of disease.

A high resolution atmospheric pressure matrix-assisted laser desorption/ionization-quadrupole-orbitrap MS platform enables in situ analysis of biomolecules by multi-mode ionization and acquisition

Introduced in 2000, atmospheric pressure (AP)/matrix-assisted laser desorption/ionization (MALDI) has attracted substantial attention in the mass spectrometry community due to its ease of sample introduction and handling, interchangeability with ESI source and capability of analyzing volatile species. In this study, an AP/MALDI source with ultra-high spatial resolution was coupled to a Q Exactive HF orbitrap mass spectrometer for high resolution in situ analysis by MALDI, laserspray ionization (LSI) and matrix assisted ionization (MAI) without instrument modification. LSI and MAI generated multiply charged ions, which expanded the mass detection range and improved fragmentation efficiency. Full MS, targeted MS/MS, data dependent acquisition (DDA) and parallel reaction monitoring (PRM) acquisitions were performed on peptide and protein standards, tissue extracts and tissue sections for in depth characterization of various biomolecules. High resolution full MS and MS/MS images were obtained from crustacean and rat tissues with pixel size less than 30 μm. Overall, AP/MALDI-Q-Orbitrap is a fast scanning instrument that is capable of performing multiple types of ionization and multiple acquisition modes without instrument modification. This instrument platform provides an attractive alternative to other high resolution MALDI instruments.

Cellular and Subcellular Level Localization of Maize Lipids and Metabolites Using High-Spatial Resolution MALDI Mass Spectrometry Imaging.

Recent technological advances have pushed the achievable spatial resolution for mass spectrometry imaging (MSI) to cellular and subcellular levels. Direct visualization of maize tissues by this tool has provided key insights into the localization of metabolites and lipids. This chapter outlines methodology for sample preparation, data acquisition, and data analysis of maize tissue sections using high-spatial resolution matrix-assisted laser desorption ionization (MALDI)-MSI, as well as the incorporation of a multi-resolution optical system, which allows for simple inter-conversion between different resolution setups (5, 10, and 50μm imaging).

Mass Spectrometry Imaging proves differential absorption profiles of well-characterised permeability markers along the crypt-villus axis

Knowledge about the region-specific absorption profiles from the gastrointestinal tract of orally administered drugs is a critical factor guiding dosage form selection in drug development. We have used a novel approach to study three well-characterized permeability and absorption marker drugs in the intestine. Propranolol and metoprolol (highly permeable compounds) and atenolol (low-moderate permeability compound) were orally co-administered to rats. The site of drug absorption was revealed by high spatial resolution matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) and complemented by quantitative measurement of drug concentration in tissue homogenates. MALDI-MSI identified endogenous molecular markers that illustrated the villi structures and confirmed the different absorption sites assigned to histological landmarks for the three drugs. Propranolol and metoprolol showed a rapid absorption and shorter transit distance in contrast to atenolol, which was absorbed more slowly from more distal sites. This study provides novel insights into site specific absorption for each of the compounds along the crypt-villus axis, as well as confirming a proximal-distal absorption gradient along the intestine. The combined analytical approach allowed the quantification and spatial resolution of drug distribution in the intestine and provided experimental evidence for the suggested absorption behaviour of low and highly permeable compounds.

Top-down Metabolomic Approaches for Nitrogen-Containing Metabolites.

Streamlining the processes that reveal heteroatom-containing metabolites and their biosynthetic genes is essential in integrated metabolomics studies. These metabolites are especially targeted for their potential pharmaceutical activities. By using a Fourier-transform ion cyclotron resonance-mass spectrometry (FTICR-MS) instrument, we provide top-down targeted metabolomic analyses using ultrahigh-resolution liquid chromatography-mass spectrometry (LC-MS), high-resolution matrix-assisted laser desorption/ionization (MALDI), and high-resolution imaging mass spectrometry (IMS) with 15N labeling of nitrogen-containing metabolites. In this study, we efficiently extract known and unknown chemicals and spatial information from the medicinal plant Catharanthus roseus, which sources several cancer drugs. The ultrahigh-resolution LC-MS analysis showed that the molecular formula of 65 N-metabolites were identified using the petals, peduncles, leaves, petioles, stems, and roots of the non- and 15N-labeled Catharanthus plants. The high resolution MALDI analysis showed the molecular formula of 64 N-metabolites using the petals, leaves, and stems of the non- and 15N-labeled Catharanthus. The chemical assignments using molecular formulas stored in databases identified known and unknown metabolites. The comparative analyses using the assigned metabolites revealed that most of the organ-specific ions are derived from unknown N-metabolites. The high-resolution IMS analysis characterized the spatial accumulation patterns of 32 N-metabolites using the buds, leaves, stems, and roots in Catharanthus. The comparative analysis using the non- and 15N-labeled IMS data showed the same spatial accumulation patterns of a non- and 15N-labeled metabolite in the organs, showing that top-down analysis can be performed even in IMS analysis.

Five Micron High Resolution MALDI Mass Spectrometry Imaging with Simple, Interchangeable, Multi-Resolution Optical System.

High-spatial resolution mass spectrometry imaging (MSI) is crucial for the mapping of chemical distributions at the cellular and subcellular level. In this work, we improved our previous laser optical system for matrix-assisted laser desorption ionization (MALDI)-MSI, from ~9 μm practical laser spot size to a practical laser spot size of ~4 μm, thereby allowing for 5 μm resolution imaging without oversampling. This is accomplished through a combination of spatial filtering, beam expansion, and reduction of the final focal length. Most importantly, the new laser optics system allows for simple modification of the spot size solely through the interchanging of the beam expander component. Using 10×, 5×, and no beam expander, we could routinely change between ~4, ~7, and ~45 μm laser spot size, in less than 5 min. We applied this multi-resolution MALDI-MSI system to a single maize root tissue section with three different spatial resolutions of 5, 10, and 50 μm and compared the differences in imaging quality and signal sensitivity. We also demonstrated the difference in depth of focus between the optical systems with 10× and 5× beam expanders. Graphical Abstract ᅟ.

Multi-stage Mass Spectrometry of Poly(vinyl pyrrolidone) and Its Vinyl Succinimide Copolymer Formed upon Exposure to Sodium Hypochlorite.

The degradation routes of poly(vinyl pyrrolidone) (PVP) exposed to sodium hypochlorite (bleach) have been previously investigated using chemical analyses such as infrared spectroscopy. So far, no reports have proposed mass spectrometry (MS) as an alternative tool despite its capability to provide molecular and structural information using its single stage electrospray (ESI) or matrix assisted laser desorption ionization (MALDI) and multi stage (MS n ) configurations, respectively. The present study thus reports on the characterization of PVP after its exposure to bleach by high resolution MALDI spiralTOF-MS and Kendrick mass defect analysis providing clues as to the formation of a vinyl pyrrolidone/vinyl succinimide copolymeric degradation product. A thorough investigation of the fragmentation pathways of PVP adducted with sodium and proton allows one main route to be described-namely the release of the pyrrolidone pendant group in a charge remote and charge driven mechanism, respectively. Extrapolating this fragmentation pathway, the oxidation of vinyl pyrrolidone into vinyl succinimide hypothesized from the single stage MS is validated by the detection of an alternative succinimide neutral loss in lieu of the pyrrolidone release in the ESI-MS n spectra of the aged PVP sample. It constitutes an example of application of multi-stage mass spectrometry for the characterization of the degradation of polymeric samples at a molecular level.

High Throughput In Situ DDA Analysis of Neuropeptides by Coupling Novel Multiplex Mass Spectrometric Imaging (MSI) with Gas-Phase Fractionation.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometric imaging (MSI) is a powerful tool to map the spatial distribution of biomolecules on tissue sections. Recent developments of hybrid MS instruments allow combination of different types of data acquisition by various mass analyzers into a single MSI analysis, which reduces experimental time and sample consumptions. Here, using the well-characterized crustacean nervous system as a test-bed, we explore the utility of high resolution and accurate mass (HRAM) MALDI Orbitrap platform for enhanced in situ characterization of the neuropeptidome with improved chemical information. Specifically, we report on a multiplex-MSI method, which combines HRAM MSI with data dependent acquisition (DDA) tandem MS analysis in a single experiment. This method enables simultaneous mapping of neuropeptide distribution, sequence validation, and novel neuropeptide discovery in crustacean neuronal tissues. To enhance the dynamic range and efficiency of in situ DDA, we introduced a novel approach of fractionating full m/z range into several sub-mass ranges and embedding the setup using the multiplex-DDA-MSI scan events to generate pseudo fractionation before MS/MS scans. The division of entire m/z into multiple segments of m/z sub-ranges for MS interrogation greatly decreased the complexity of molecular species from tissue samples and the heterogeneity of the distribution and variation of intensities of m/z peaks. By carefully optimizing the experimental conditions such as the dynamic exclusion, the multiplex-DDA-MSI approach demonstrates better performance with broader precursor coverage, less biased MS/MS scans towards high abundance molecules, and improved quality of tandem mass spectra for low intensity molecular species. Graphical Abstract ᅟ.

Sublimation of New Matrix Candidates for High Spatial Resolution Imaging Mass Spectrometry of Lipids: Enhanced Information in Both Positive and Negative Polarities after 1,5-Diaminonapthalene Deposition

Matrix sublimation has demonstrated to be a powerful approach for high-resolution matrix-assisted laser desorption ionization (MALDI) imaging of lipids, providing very homogeneous solvent-free deposition. This work presents a comprehensive study aiming to evaluate current and novel matrix candidates for high spatial resolution MALDI imaging mass spectrometry of lipids from tissue section after deposition by sublimation. For this purpose, 12 matrices including 2,5-dihydroxybenzoic acid (DHB), sinapinic acid (SA), α-cyano-4-hydroxycinnamic acid (CHCA), 2,6-dihydroxyacetphenone (DHA), 2',4',6'-trihydroxyacetophenone (THAP), 3-hydroxypicolinic acid (3-HPA), 1,8-bis(dimethylamino)naphthalene (DMAN), 1,8,9-anthracentriol (DIT), 1,5-diaminonapthalene (DAN), p-nitroaniline (NIT), 9-aminoacridine (9-AA), and 2-mercaptobenzothiazole (MBT) were investigated for lipid detection efficiency in both positive and negative ionization modes, matrix interferences, and stability under vacuum. For the most relevant matrices, ion maps of the different lipid species were obtained from tissue sections at high spatial resolution and the detected peaks were characterized by matrix-assisted laser desorption ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry. First proposed for imaging mass spectrometry (IMS) after sublimation, DAN has demonstrated to be of high efficiency providing rich lipid signatures in both positive and negative polarities with high vacuum stability and sub-20 μm resolution capacity. Ion images from adult mouse brain were generated with a 10 μm scanning resolution. Furthermore, ion images from adult mouse brain and whole-body fish tissue sections were also acquired in both polarity modes from the same tissue section at 100 μm spatial resolution. Sublimation of DAN represents an interesting approach to improve information with respect to currently employed matrices providing a deeper analysis of the lipidome by IMS.

High-Spatial Resolution Matrix-Assisted Laser Desorption Ionization Imaging Analysis of Glucosylceramide in Spleen Sections from a Mouse Model of Gaucher Disease

MALDI mass spectrometric imaging (MSI) enables spatially resolved mass and intensity information to be obtained directly from tissue sections, thereby illustrating how analytes are distributed within these sections. Here we have used an oversampling technique on a commercially available MALDI orthogonal acceleration TOF mass spectrometer with ion mobility separation capability to produce high spatial resolution images of the glycosphingolipid, glucosylceramide (GC). To exemplify the biological application of our approach, GC was imaged in spleen sections from a conditional knockout mouse model of type 1 Gaucher disease in which the catabolism of this glycosphingolipid is impaired. The laser was continually fired at one position until no more ions were observed and then the sample was moved by 15 microm (laser diameter approximately 150 microm). Ions were generated from only the unirradiated surface at each of these positions achieving an effective spacing of 15 microm. At 15 microm laser step-size, it was possible to visualize macrophages enriched in GC, which could be distinguished from other cell types in the spleen. Current MALDI MSI spatial resolution is typically limited by the diameter of the laser spot-size, which is usually between 50 and 100 microm, covering an area equivalent to tens of mammalian cells.

Fast and automated large-area imaging MALDI mass spectrometry in microprobe and microscope mode

Since the introduction of matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS), numerous instrumental developments have been presented. The introduction of microscope MALDI IMS was a major breakthrough, making micron-range resolution MALDI imaging of kiloDalton mass species possible. We discuss new developments that makes large (cm range) field of view, high resolution (μm range) microscope mode imaging mass spectrometry (IMS) possible in a single experiment, using three different data acquisition approaches simultaneously. We demonstrate how a combination of these acquisition approaches is used to correlate mass spectral and high resolution imaging data.