Matrix assisted laser desorption ionization imaging mass spectrometry (MALDI IMS) has the ability to provide an enormous amount of information on the abundances and spatial distributions of molecules within biological tissues. The rapid progress in the development of this technology significantly improves our ability to analyze smaller and smaller areas and features within tissues. The mammalian eye has evolved over millions of years to become an essential asset for survival, providing important sensory input of an organism’s surroundings. The highly complex sensory retina of the eye is comprised of numerous cell types organized into specific layers with varying dimensions, the thinnest of which is the 10 μm retinal pigment epithelium (RPE). This single cell layer and the photoreceptor layer contain the complex biochemical machinery required to convert photons of light into electrical signals that are transported to the brain by axons of retinal ganglion cells. Diseases of the retina, including age-related macular degeneration (AMD), retinitis pigmentosa, and diabetic retinopathy, occur when the functions of these cells are interrupted by molecular processes that are not fully understood. In this report, we demonstrate the use of high spatial resolution MALDI IMS and FT-ICR tandem mass spectrometry in the Abca4–/– knockout mouse model of Stargardt disease, a juvenile onset form of macular degeneration. The spatial distributions and identity of lipid and retinoid metabolites are shown to be unique to specific retinal cell layers.
We describe an easy and inexpensive way to provide a highly defined Gaussian shaped laser spot on target of 5 μm diameter for imaging mass spectrometry using a commercial MALDI TOF instrument that is designed to produce a 20 μm diameter laser beam on target at its lowest setting. A 25 μm pinhole filter on a swivel arm was installed in the laser beam optics outside the vacuum ion source chamber so it is easily flipped into or out of the beam as desired by the operator. The resulting ion images at 5 μm spatial resolution are sharp since the satellite secondary laser beam maxima have been removed by the filter. Ion images are shown to demonstrate the performance and are compared with the method of oversampling to achieve higher spatial resolution when only a larger laser beam spot on target is available.
Patient-derived colorectal tumor organoids (CTOs) closely recapitulate the complex morphological, phenotypic, and genetic features observed in in vivo tumors. Therefore, evaluation of drug distribution and metabolism in this model system can provide valuable information to predict the clinical outcome of a therapeutic response in individual patients. In this report, we applied matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to examine the spatial distribution of the drug irinotecan and its metabolites in CTOs from two patients. Irinotecan is a prodrug and is often prescribed as part of therapeutic regimes for patients with advanced colorectal cancer. Irinotecan shows a time-dependent and concentration-dependent permeability and metabolism in the CTOs. More interestingly, the active metabolite SN-38 does not co-localize well with the parent drug irinotecan and the inactive metabolite SN-38G. The phenotypic effect of irinotecan metabolism was also confirmed by a viability study showing significantly reduced proliferation in the drug treated CTOs. MALDI-MSI can be used to investigate various pharmaceutical compounds in CTOs derived from different patients. By analyzing multiple CTOs from a patient, this method could be used to predict patient-specific drug responses and help to improve personalized dosing regimens.
Uniform matrix deposition on tissue samples for matrix-assisted laser desorption/ionization (MALDI) is key for reproducible
analyte ion signals. Current methods often result in nonhomogenous matrix deposition, and take time and effort to produce
acceptable ion signals. Here we describe a fully-automated method for matrix deposition using an enclosed spray chamber and
spray nozzle for matrix solution delivery. A commercial air-atomizing spray nozzle was modified and combined with solenoid
controlled valves and a Programmable Logic Controller (PLC) to control and deliver the matrix solution. A spray chamber was
employed to contain the nozzle, sample, and atomized matrix solution stream, and to prevent any interference from outside
conditions as well as allow complete control of the sample environment. A gravity cup was filled with MALDI matrix solutions,
including DHB in chloroform/methanol (50:50) at concentrations up to 60 mg/mL. Various samples (including rat brain tissue
sections) were prepared using two deposition methods (spray chamber, inkjet). A linear ion trap equipped with an intermediate-pressure
MALDI source was used for analyses. Optical microscopic examination showed a uniform coating of matrix crystals across the
sample. Overall, the mass spectral images gathered from tissues coated using the spray chamber system were of better quality
and more reproducible than from tissue specimens prepared by the inkjet deposition method. 相似文献
Mixtures of pollen grains of three different species (Corylus avellana, Alnus cordata, and Pinus sylvestris) were investigated by matrix-assisted laser desorption/ionization time-of-flight imaging mass spectrometry (MALDI-TOF imaging MS). The amount of pollen grains was reduced stepwise from >?10 to single pollen grains. For sample pretreatment, we modified a previously applied approach, where any additional extraction steps were omitted. Our results show that characteristic pollen MALDI mass spectra can be obtained from a single pollen grain, which is the prerequisite for a reliable pollen classification in practical applications. MALDI imaging of laterally resolved pollen grains provides additional information by reducing the complexity of the MS spectra of mixtures, where frequently peak discrimination is observed. Combined with multivariate statistical analyses, such as principal component analysis (PCA), our approach offers the chance for a fast and reliable identification of individual pollen grains by mass spectrometry.
EphrinA1 is a tyrosine kinase receptor localized in the cellular membrane of healthy cardiomyocytes, the expression of which is lost upon myocardial infarction (MI). Intra-cardiac injection of the recombinant form of ephrinA1 (ephrinA1-Fc) at the time of ligation in mice has shown beneficial effects by reducing infarct size and myocardial necrosis post-MI. To date, immunohistochemistry and Western blotting comprise the only experimental approaches utilized to localize and quantify relative changes of ephrinA1 in sections and homogenates of whole left ventricle, respectively. Herein, we used matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) coupled with a time-of-flight mass spectrometer (MALDI/TOF MS) to identify intact as well as tryptic fragments of ephrinA1 in healthy controls and acutely infarcted murine hearts. The purpose of the present study was 3-fold: (1) to spatially resolve the molecular distribution of endogenous ephrinA1, (2) to determine the anatomical expression profile of endogenous ephrinA1 after acute MI, and (3) to identify molecular targets of ephrinA1-Fc action post-MI. The tryptic fragments detected were identified as the ephrinA1-isoform with 38% and 34% sequence coverage and Mascot scores of 25 for the control and MI hearts, respectively. By using MALDI-MSI, we have been able to simultaneously measure the distribution and spatial localization of ephrinA1, as well as additional cardiac proteins, thus offering valuable information for the elucidation of molecular partners, mediators, and targets of ephrinA1 action in cardiac muscle.
Matrix-Assisted Laser Desorption/Ionization (MALDI) allows the identification of repeat units and end groups, the structural analysis of linear and cyclic oligomers, and the estimate of composition and sequence for copolymers. MALDI has also been applied to the measurement of molar mass distributions in polymers and to the study of thermal and oxidative processes in polymers. This paper illustrates the detection of self-association in macromolecules made by coupling MALDI and Size Exclusion Chromatography (SEC), the investigation of polymer oxidation phenomena, and the characterization of copolymers formed in the processing of reactive polymer blends. 相似文献
Pancreatic ductal adenocarcinoma and cholangiocarcinoma constitute two aggressive tumor types that originate from the epithelial lining of the excretory ducts of the pancreatobiliary tract. Given their close histomorphological resemblance, a correct diagnosis can be challenging and almost impossible without clinical information. In this study, we investigated whether mass spectrometric peptide features could be employed to distinguish pancreatic ductal adenocarcinoma from cholangiocarcinoma. Three tissue microarrays of formalin-fixed and paraffin-embedded material (FFPE) comprising 41 cases of pancreatic ductal adenocarcinoma and 41 cases of cholangiocarcinoma were analyzed by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). The derived peptide features and respective intensities were used to build different supervised classification algorithms: gradient boosting (GB), support vector machine (SVM), and k-nearest neighbors (KNN). On a pixel-by-pixel level, a classification accuracy of up to 95% could be achieved. The tentative identification of discriminative tryptic peptide signatures revealed proteins that are involved in the epigenetic regulation of the genome and tumor microenvironment. Despite their histomorphological similarities, mass spectrometry imaging represents an efficient and reliable approach for the distinction of PDAC from CC, offering a promising complementary or alternative approach to the existing tools used in diagnostics such as immunohistochemistry. 相似文献
We report using MALDI-ISD (in-source decay) mass spectrometry (MS) to characterize highly branched synthetic polymers of polyamidoamine (PAMAM) dendrimer. This inherently monodisperse polymer possesses dendritic branches networked by tertiary amines and an amide functionality in each repeating unit. Among various ISD matrices examined, 2,5-DHB was the most efficient, yielding 33 fragments produced by single- or multiple-bond cleavages. Detailed analysis revealed that cleavages at tertiary amine sites (S- and E-type fragments) were the most pronounced, with various other cleavages around amide groups. The fragmentation mechanism appeared to follow the radical-induced dissociation pathway. In addition, the matrix dependence of PAMAM MALDI-ISD differed from that of peptides/proteins. The observed fragments provided rich structural information, which was suitable to characterize dendritic polymers. 相似文献
Methods to visualize the two-dimensional (2D) distribution of molecules by mass spectrometric imaging evolve rapidly and yield
novel applications in biology, medicine, and material surface sciences. Most mass spectrometric imagers acquire high mass
resolution spectra spot-by-spot and thereby scan the object’s surface. Thus, imaging is slow and image reconstruction remains
cumbersome. Here we describe an imaging mass spectrometer that exploits the true imaging capabilities by ion optical means
for the time of flight mass separation. The mass spectrometer is equipped with the ASIC Timepix chip as an array detector
to acquire the position, mass, and intensity of ions that are imaged by matrix-assisted laser desorption/ionization (MALDI)
directly from the target sample onto the detector. This imaging mass spectrometer has a spatial resolving power at the specimen
of (84 ± 35) μm with a mass resolution of 45 and locates atoms or organic compounds on a surface area up to ~2 cm2. Extended laser spots of ~5 mm2 on structured specimens allows parallel imaging of selected masses. The digital imaging mass spectrometer proves high hit-multiplicity,
straightforward image reconstruction, and potential for high-speed readout at 4 kHz or more. This device demonstrates a simple
way of true image acquisition like a digital photographic camera. The technology may enable a fast analysis of biomolecular
samples in near future. 相似文献
A possibility of using tryptamine as a reactive matrix for the analysis of non-polar carbonyl compounds by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry has been shown. Presence of a terminal primary amine group in the tryptamine molecule predetermines the formation of Schiff bases from aliphatic and alicyclic carbonyl compounds. No additional matrix compounds are necessary to register MALDI mass spectra, because the excess of the derivatization agent plays the role of a matrix. MALDI mass spectra demonstrate high efficiency of desorption/ionization of the derivatives. To discover reactive matrices, a set of aromatic primary amines (mainly substituted anilines) has been tested, but they have not demonstrated matrix properties. 相似文献
The large‐scale and label‐free molecular characterization of single cells in their natural tissue habitat remains a major challenge in molecular biology. We present a method that integrates morphometric image analysis to delineate and classify individual cells with their single‐cell‐specific molecular profiles. This approach provides a new means to study spatial biological processes such as cancer field effects and the relationship between morphometric and molecular features. 相似文献
Isoniazid (INH) is an important component of front-line anti-tuberculosis therapy with good serum pharmacokinetics but unknown
ability to penetrate tuberculous lesions. However, endogenous background interferences hinder our ability to directly analyze
INH in tissues. Chemical derivatization has been successfully used to measure isoniazid directly from tissue samples using
matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS). MALDI targets were pretreated with trans-cinnamaldehyde (CA) prior to mounting tissue slices. Isoniazid present in the tissues was efficiently derivatized and the
INH-CA product measured by MS/MS. Precoating of MALDI targets allows the tissues to be directly thaw-mounted and derivatized,
thus simplifying the preparation. A time-course series of tissues from tuberculosis infected/INH dosed animals were assayed
and the MALDI MS/MS response correlates well with the amount of INH determined to be in the tissues by high-performance liquid
chromatography (HPLC)-MS/MS. 相似文献
A high performance liquid chromatography and a mass spectrometry matrix-assisted laser desorption/ionization (MALDI) methods were used to select the conditions of both successful separation and to identify amino acids and peptides on the carbon sorbents. For the first time the graphitized and nongraphitized carbon blacks were used for the identification of amino acids and peptides by the MALDI method. It was shown that an increased surface area of carbon blacks led to a sharp increase in the intensities of the detected peaks of protonated molecular ions and adducts with alkali metal cations of the amino acids and peptides under study. 相似文献
Native mass spectrometry (MS) encompasses methods to keep noncovalent interactions of biomolecular complexes intact in the gas phase throughout the instrument and to measure the mass-to-charge ratios of supramolecular complexes directly in the mass spectrometer. Electrospray ionization (ESI) in nondenaturing conditions is now an established method to characterize noncovalent systems. Matrix-assisted laser desorption/ionization (MALDI), on the other hand, consumes low quantities of samples and largely tolerates contaminants, making it a priori attractive for native MS. However, so-called native MALDI approaches have so far been based on solid deposits, where the rapid transition of the sample through a solid state can engender the loss of native conformations. Here we present a new method for native MS based on liquid deposits and MALDI ionization, unambiguously detecting intact noncovalent protein complexes by direct desorption from a liquid spot for the first time. To control for aggregation, we worked with HUαβ, a heterodimer that does not spontaneously rearrange into homodimers in solution. Screening through numerous matrix solutions to observe first the monomeric protein, then the dimer complex, we settled on a nondenaturing binary matrix solution composed of acidic and basic organic matrices in glycerol, which is stable in vacuo. The role of temporal and spatial laser irradiation patterns was found to be critical. Both a protein-protein and a protein-ligand complex could be observed free of aggregation. To minimize gas-phase dissociation, source parameters were optimized to achieve a conservation of complexes above 50% for both systems.
