Stretched tissue mounting for MALDI mass spectrometry imaging

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) combines information-rich chemical detection with spatial localization of analytes. For a given instrumental platform and analyte class, the data acquired can represent a compromise between analyte extraction and spatial information. Here, we introduce an improvement to the spatial resolution achievable with MALDI MSI conducted with standard mass spectrometric systems that also reduces analyte migration during matrix application. Tissue is placed directly on a stretchable membrane that, when stretched, fragments the tissue into micrometer-sized pieces. Scanning electron microscopy analysis shows that this process produces fairly homogeneous distributions of small tissue fragments separated and surrounded by areas of hydrophobic membrane surface. MALDI matrix is then applied by either a robotic microspotter or an artist's airbrush. Rat spinal cord samples imaged with an instrumental resolution of 50-250 μm demonstrate lipid distributions with a 5-fold high spatial resolution (a 25-fold increase in pixel density) after stretching compared to tissues that were not stretched.     

Matrix solution fixation: histology-compatible tissue preparation for MALDI mass spectrometry imaging

Mass spectrometry imaging (MSI) acquires a grid of spatially resolved mass spectra and provides a molecular landscape of a tissue. This can have a myriad of uses: from basic tissue characterization to a comprehensive pathological diagnosis. We have developed a fast, inexpensive, histology-compatible tissue preparation method for matrix-assisted laser desorption/ionization (MALDI)-MSI, which overcomes current sample preparation-imposed limitations in image resolution. Tissue sections are prepared via simultaneous fixation and matrix deposition. This is accomplished by incorporating the MALDI matrix into solvents that preserve tissue integrity when applied according to standard histology procedures. This concept was expanded to include multiple histology protocols, thereby enabling analysis to be tailored to a variety of biomolecules and tissue types.     

Effect of local matrix crystal variations in matrix-assisted ionization techniques for mass spectrometry

Intense intact molecular ion signals have been obtained from phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidyiinositol using matrix-enhanced secondary ion mass spectrometry (ME-SIMS). It was found that the high-mass (m/z >500) regions of the ME-SIMS spectra closely resembled those obtained using matrix-assisted laser desorption/ionization (MALDI). Using high spatial resolution SIMS, a detailed investigation of dried-droplet samples was performed. Based on the detected Na+ and 2,5-DHB matrix signal intensities, different crystal types were distinguished, in addition to different sizes of crystals. Spatially mapping the pseudomolecular and fragment ions of the phospholipids revealed that the nature of the pseudomolecular ions formed, as well as the ratio of intact molecular to fragment ion, was dependent on the type and surface composition of the crystal. The observed chemical bias effects due to crystal heterogeneity and the resulting variation in desorption/ionization efficiency will complicate the interpretation of data obtained from matrix-assisted mass spectrometric (imaging) techniques and is an important factor in the "hot spot" phenomenon frequently encountered in MALDI experiments. In this respect, imaging SIMS was found to be a versatile tool to investigate the effects of the local physicochemical conditions on the detected molecular species.     

Single cell matrix-assisted laser desorption/ionization mass spectrometry imaging

Application of mass spectrometry imaging (MS imaging) analysis to single cells was so far restricted either by spatial resolution in the case of matrix-assisted laser desorption/ionization (MALDI) or by mass resolution/mass range in the case of secondary ion mass spectrometry (SIMS). In this study we demonstrate for the first time the combination of high spatial resolution (7 μm pixel), high mass accuracy (<3 ppm rms), and high mass resolution (R = 100?000 at m/z = 200) in the same MS imaging measurement of single cells. HeLa cells were grown directly on indium tin oxide (ITO) coated glass slides. A dedicated sample preparation protocol was developed including fixation with glutaraldehyde and matrix coating with a pneumatic spraying device. Mass spectrometry imaging measurements with 7 μm pixel size were performed with a high resolution atmospheric-pressure matrix-assisted laser desorption/ionization (AP-MALDI) imaging source attached to an Exactive Orbitrap mass spectrometer. Selected ion images were generated with a bin width of Δm/z = ±0.005. Selected ion images and optical fluorescence images of HeLa cells showed excellent correlation. Examples demonstrate that a lower mass resolution and a lower spatial resolution would result in a significant loss of information. High mass accuracy measurements of better than 3 ppm (root-mean-square) under imaging conditions provide confident identification of imaged compounds. Numerous compounds including small metabolites such as adenine, guanine, and cholesterol as well as different lipid classes such as phosphatidylcholine, sphingomyelin, diglycerides, and triglycerides were detected and identified based on a mass spectrum acquired from an individual spot of 7 μm in diameter. These measurements provide molecularly specific images of larger metabolites (phospholipids) in native single cells. The developed method can be used for a wide range of detailed investigations of metabolic changes in single cells.     

High-reactivity matrices increase the sensitivity of matrix enhanced secondary ion mass spectrometry

Secondary ion mass spectrometry (SIMS) is a desorption/ionization method in which ions are generated by the impact of a primary ion beam on a sample. Classic matrix assisted laser desorption and ionization (MALDI) matrices can be used to increase secondary ion yields and decrease fragmentation in a SIMS experiment, which is referred to as matrix enhanced SIMS (ME-SIMS). Contrary to MALDI, the choice of matrices for ME-SIMS is not constrained by their photon absorption characteristics. This implies that matrix compounds that exhibit an insufficient photon absorption coefficient have the potential of working well with ME-SIMS. Here, we evaluate a set of novel derivatives of the classical MALDI matrices α-cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB) for usability in ME-SIMS. This evaluation was carried out using peptide mixtures of different complexity and demonstrates significant improvements in signal intensity for several compounds with insufficient UV absorption at the standard MALDI laser wavelengths. Our study confirms that the gas-phase proton affinity of a matrix compound is a key physicochemical characteristic that determines its performance in a ME-SIMS experiment. As a result, these novel matrices improve the performance of matrix enhanced secondary ion mass spectrometry experiments on complex peptide mixtures.     

An analytical pipeline for MALDI in-source decay mass spectrometry imaging

In-source decay (ISD) fragmentation as combined with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry allows protein sequencing directly from mass spectra. Acquisition of MALDI-ISD mass spectra from tissue samples is achieved using an appropriate MALDI matrix, such as 1,5-diaminonaphthalene (DAN). Recent efforts have focused on combining MALDI-ISD with mass spectrometry imaging (MSI) to provide simultaneous sequencing and localization of proteins over a thin tissue surface. Successfully coupling these approaches requires the development of new data analysis tools, but first, investigating the properties of MALDI-ISD as applied to mixtures of protein standards reveals a high sensitivity to the relative protein ionization efficiency. This finding translates to the protein mixtures found in tissues and is used to inform the development of an analytical pipeline for data analysis in MALDI-ISD MS imaging, including software to identify the most pertinent spectra, to sequence protein mixtures, and to generate ion images for comparison with tissue morphology. The ability to simultaneously identify and localize proteins is demonstrated by using the analytical pipeline on three tissue sections from porcine eye lens, resulting in localizations for crystallins and cytochrome c. The variety of protein identifications provided by MALDI-ISD-MSI between tissue sections creates a discovery tool, and the analytical pipeline makes this process more efficient.     

C60 secondary ion mass spectrometry with a hybrid-quadrupole orthogonal time-of-flight mass spectrometer

A hybrid quadrupole orthogonal time-of-flight mass spectrometer optimized for matrix-assisted laser desorption ionization (MALDI) and electrospray ionization has been equipped with a C 60 cluster ion source. This configuration is shown to exhibit a number of characteristics that improve the performance of traditional time-of-flight secondary ion mass spectrometry (TOF-SIMS) experiments for the analysis of complex organic materials and, potentially, for chemical imaging. Specifically, the primary ion beam is operated as a continuous rather than a pulsed beam, resulting in up to 4 orders of magnitude greater ion fluence on the target. The secondary ions are extracted at very low voltage into 8 mTorr of N 2 gas introduced for collisional focusing and cooling purposes. This extraction configuration is shown to yield secondary ions that rapidly lose memory of the mechanism of their birth, yielding tandem mass spectra that are identical for SIMS and MALDI. With implementation of ion trapping, the extraction efficiency is shown to be equivalent to that found in traditional TOF-SIMS machines. Examples are given, for a variety of substrates that illustrate mass resolution of 12,000-15,600 with a mass range for inorganic compounds to m/ z 40,000. Preliminary chemical mapping experiments show that with added sensitivity, imaging in the MS/MS mode of operation is straightforward. In general, the combination of MALDI and SIMS is shown to add capabilities to each technique, providing a robust platform for TOF-SIMS experiments that already exists in a large number of laboratories.     

Role of electrons in laser desorption/ionization mass spectrometry

A nonmetallic sample support for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry enhances the positive ion yield by 2 orders of magnitude and generally affects the charge balance in the desorption plume. We interpret the effects of the target material and of the sample preparation on MALDI mass spectra as a result of photoelectrons emitted upon laser irradiation of a metal target covered by a thin sample layer. These electrons are shown to play an important role in MALDI and laser desorption/ionization because they decrease the yield of positive ions, reduce ions with higher oxidation states, and affect the ion velocity distribution as well as the mass resolution. Understanding the role of these photoelectrons helps to clarify previously obscure aspects of the ion formation mechanism in MALDI.     

Infrared laser ablation sample transfer for MALDI imaging

An infrared laser was used to ablate material from tissue sections under ambient conditions for direct collection on a matrix assisted laser desorption ionization (MALDI) target. A 10 μm thick tissue sample was placed on a microscope slide and was mounted tissue-side down between 70 and 450 μm from a second microscope slide. The two slides were mounted on a translation stage, and the tissue was scanned in two dimensions under a focused mid-infrared (IR) laser beam to transfer material to the target slide via ablation. After the material was transferred to the target slide, it was analyzed using MALDI imaging using a tandem time-of-flight mass spectrometer. Images were obtained from peptide standards for initial optimization of the system and from mouse brain tissue sections using deposition either onto a matrix precoated target or with matrix addition after sample transfer and compared with those from standard MALDI mass spectrometry imaging. The spatial resolution of the transferred material is approximately 400 μm. Laser ablation sample transfer provides several new capabilities not possible with conventional MALDI imaging including (1) ambient sampling for MALDI imaging, (2) area to spot concentration of ablated material, (3) collection of material for multiple imaging analyses, and (4) direct collection onto nanostructure assisted laser desorption ionization (NALDI) targets without blotting or ultrathin sections.     

Robust data processing and normalization strategy for MALDI mass spectrometric imaging

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) provides localized information about the molecular content of a tissue sample. To derive reliable conclusions from MSI data, it is necessary to implement appropriate processing steps in order to compare peak intensities across the different pixels comprising the image. Here, we review commonly used normalization methods, and propose a rational data processing strategy, for robust evaluation and modeling of MSI data. The approach includes newly developed heuristic methods for selecting biologically relevant peaks and pixels to reduce the size of a data set and remove the influence of the applied MALDI matrix. The methods are demonstrated on a MALDI MSI data set of a sagittal section of rat brain (4750 bins, m/z = 50-1000, 111 × 185 pixels) and the proposed preferred normalization method uses the median intensity of selected peaks, which were determined to be independent of the MALDI matrix. This was found to effectively compensate for a range of known limitations associated with the MALDI process and irregularities in MS image sampling routines. This new approach is relevant for processing of all MALDI MSI data sets, and thus likely to have impact in biomarker profiling, preclinical drug distribution studies, and studies addressing underlying molecular mechanisms of tissue pathology.     

High dynamic range bio-molecular ion microscopy with the Timepix detector

Highly parallel, active pixel detectors enable novel detection capabilities for large biomolecules in time-of-flight (TOF) based mass spectrometry imaging (MSI). In this work, a 512 × 512 pixel, bare Timepix assembly combined with chevron microchannel plates (MCP) captures time-resolved images of several m/z species in a single measurement. Mass-resolved ion images from Timepix measurements of peptide and protein standards demonstrate the capability to return both mass-spectral and localization information of biologically relevant analytes from matrix-assisted laser desorption ionization (MALDI) on a commercial ion microscope. The use of a MCP-Timepix assembly delivers an increased dynamic range of several orders of magnitude. The Timepix returns defined mass spectra already at subsaturation MCP gains, which prolongs the MCP lifetime and allows the gain to be optimized for image quality. The Timepix peak resolution is only limited by the resolution of the in-pixel measurement clock. Oligomers of the protein ubiquitin were measured up to 78 kDa.     

Quantitative tandem mass spectrometric imaging of endogenous acetyl-L-carnitine from piglet brain tissue using an internal standard

Matrix-assisted laser desorption/ionization (MALDI) based mass spectrometric imaging (MSI) is increasingly being used as an analytical tool to evaluate the molecular makeup of tissue samples. From the direct analysis of a tissue section, the physical integrity of sample is preserved; thus, spatial information of a compound's distribution may be determined. One limitation of the technique, however, has been the inability to determine the absolute concentration from a tissue sample. Here we report the development of a quantitative MSI technique in which the distribution of acetyl-L-carnitine (AC) in a piglet brain sample is quantified with MALDI MSI. An isotopically labeled internal standard was applied uniformly beneath the tissue section, and wide-isolation tandem mass spectrometry was performed. Normalizing the analyte ion signal by the internal standard ion signal resulted in significant improvements in MS images, signal reproducibility, and calibration curve linearity. From the improved MS images, the concentration of AC was determined and plotted producing a concentration-scaled image of the distribution of AC in the piglet brain section.     

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.     

Qualitative and quantitative MALDI imaging of the positron emission tomography ligands raclopride (a D2 dopamine antagonist) and SCH 23390 (a D1 dopamine antagonist) in rat brain tissue sections using a solvent-free dry matrix application method

The distributions of positron emission tomography (PET) ligands in rat brain tissue sections were analyzed by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI). The detection of the PET ligands was possible following the use of a solvent-free dry MALDI matrix application method employing finely ground dry α-cyano-4-hydroxycinnamic acid (CHCA). The D2 dopamine receptor antagonist 3,5-dichloro-N-{[(2S)-1-ethylpyrrolidin-2-yl]methyl}-2-hydroxy-6-methoxybenzamide (raclopride) and the D1 dopamine receptor antagonist 7-chloro-3-methyl-1-phenyl-1,2,4,5-tetrahydro-3-benzazepin-8-ol (SCH 23390) were both detected at decreasing abundance at increasing period postdosing. Confirmation of the compound identifications and distributions was achieved by a combination of mass-to-charge ratio accurate mass, isotope distribution, and MS/MS fragmentation imaging directly from tissue sections (performed using MALDI TOF/TOF, MALDI q-TOF, and 12T MALDI-FT-ICR mass spectrometers). Quantitative data was obtained by comparing signal abundances from tissues to those obtained from quantitation control spots of the target compound applied to adjacent vehicle control tissue sections (analyzed during the same experiment). Following a single intravenous dose of raclopride (7.5 mg/kg), an average tissue concentration of approximately 60 nM was detected compared to 15 nM when the drug was dosed at 2 mg/kg, indicating a linear response between dose and detected abundance. SCH 23390 was established to have an average tissue concentration of approximately 15 μM following a single intravenous dose at 5 mg/kg. Both target compounds were also detected in kidney tissue sections when employing the same MSI methodology. This study illustrates that a MSI may well be readily applied to PET ligand research development when using a solvent-free dry matrix coating.     

Gold-enhanced biomolecular surface imaging of cells and tissue by SIMS and MALDI mass spectrometry

Surface metallization by plasma coating enhances desorption/ionization of membrane components such as lipids and sterols in imaging time-of-flight secondary ion mass spectrometry (TOF-SIMS) of tissues and cells. High-resolution images of cholesterol and other membrane components were obtained for neuroblastoma cells and revealed subcellular details (resolving power 1.5 mum). Alternatively, in matrix-enhanced SIMS, 2,5-dihydroxybenzoic acid electrosprayed on neuroblastoma cells allowed intact molecular ion imaging of phosphatidylcholine and sphingomyelin at the cellular level. Gold deposition on top of matrix-coated rat brain tissue sections strongly enhanced image quality and signal intensity in stigmatic matrix-assisted laser desorption/ionization imaging mass spectrometry. High-quality total ion count images were acquired, and the neuropeptide vasopressin was localized in the rat brain tissue section at the hypothalamic area around the third ventricle. Although the mechanism of signal enhancement by gold deposition is under debate, the results we have obtained for cells and tissue sections illustrate the potential of this sample preparation technique for biomolecular surface imaging by mass spectrometry.     

Dual desorption electrospray ionization-laser desorption ionization mass spectrometry on a common nanoporous alumina platform for enhanced shotgun proteomic analysis

A gold coated nanoporous alumina surface was used for dual ionization mode mass spectrometric analysis using desorption electrospray ionization (DESI) and laser desorption ionization (LDI). DESI and LDI mass spectrometry (MS) from the nanoporous alumina surface were compared with conventional electrospray ionization (ESI) mass spectrometry and matrix assisted laser desorption ionization (MALDI) for analysis of tryptic digests of proteins. Combined use of DESI and LDI offer greater peptide coverage than either method alone and comparable peptide coverage as with dual MALDI and ESI. This dual ionization technique using a common platform with same sample spot demonstrates a potential time and cost-effective tool for improved shotgun proteomic analysis.     

Manganese oxide nanoparticle-assisted laser desorption/ionization mass spectrometry for medical applications

Abstract

We prepared and characterized manganese oxide magnetic nanoparticles (d =5.6 nm) and developed nanoparticle-assited laser desorption/ionization (nano-PALDI) mass spectrometry. The nanoparticles had MnO₂ and Mn₂O₃ cores conjugated with hydroxyl and amino groups, and showed paramagnetism at room temperature. The nanoparticles worked as an ionization assisting reagent in mass spectroscopy. The mass spectra showed no background in the low m/z. The nanoparticles could ionize samples of peptide, drug and proteins (approx. 5000 Da) without using matrix, i.e., 2,5-dihydroxybenzoic acid (DHB), 4-hydroxy-α-cinnamic acid (CHCA) and liquid matrix, as conventional ionization assisting reagents. Post source decay spectra by nano-PALDI mass spectrometry will yield information of the chemical structure of analytes.     

Two-step matrix application for the enhancement and imaging of latent fingermarks

Matrix deposition is a crucial aspect for successful matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) analysis. The search for more efficient protocols over the years has resulted in the devising of "dry matrix methods" in which the matrix is solely or preliminarily deposited as powder and acts in most cases as a seeding agent. Although not fully embraced by the MALDI MSI community, these methods have proven to be more efficient in terms of ion intensity, ion abundance, and ion images in the experimental circumstances they were employed. Here we report a novel two-step matrix application method, that we have named the "dry-wet" method, where the matrix is dusted onto the sample followed by solvent spray using a robotic device. The new method has been successfully applied to the detection and mapping of several analyte classes within latent fingermarks. Dusting the matrix generated the added advantage of enhancing the latent fingermarks which are invisible. This allows not only for an optical image to be taken of the fingermark in situ but also bridges the gap in the application of MALDI MSI technology in this field; with the use of the methodology reported, fingermark enhancement, recovery, and analysis from different surfaces is now compatible with subsequent MALDI MSI analysis thus allowing visual and chemical information to be obtained simultaneously.     

Characterizing the phospholipid profiles in mammalian tissues by MALDI FTMS

Discussed here is an analytical method for profiling lipids and phospholipids directly from mammalian tissues excised from Mus musculus (house mouse). Biochemical analysis was accomplished through the use of matrix-assisted laser desorption/ionization (MALDI) Fourier transform mass spectrometry, where whole tissue sections of mouse brain, heart, and liver were investigated. Lipid and phospholipid ions create complex MALDI mass spectra containing multiple ions with different m/z values corresponding to the same fundamental chemical species. When a computational sorting approach is used to group these ions, the standard deviation for observed relative chemical abundance can be reduced to 6.02%. Relative standard deviations of 10% are commonly accepted for standard chromatographic phospholipid analyses. Average mass measurement accuracy for 232 spectra representing three tissue types from 12 specimens was calculated to be 0.0053 Da. Further it is observed, that the data and the analysis between all the animals have near-identical phospholipid contents in their brain, heart, and liver tissues, respectively. In addition to the need to accurately measure relative abundances of phospholipid species, it is essential to have adequate mass resolution for complete and accurate overall analysis. It is reasonable to make mass composition assignments with spectral resolving power greater than 8000. However, results from the present study reveal 14 instances (C12 carbon isotope) of multiple m/z ions having the same nominal value that require greater resolution in order that overlap will not occur. Spectra measured here have an average resolving power of 12 000. It is established that high mass resolution and mass accuracy coupled with MALDI ionization provide for rapid and accurate phospholipid analysis of mammalian tissue sections.     

Matrix-enhanced secondary ion mass spectrometry: a method for molecular analysis of solid surfaces

A new methodology, matrix-enhanced secondary ion mass spectrometry (ME-SIMS), is reported for the molecular analysis of biomaterials. The technique applies static secondary ion mass spectrometry (SSIMS) techniques to samples prepared in a solid organic matrix similar to sample preparations used in matrix-assisted laser desorption/ionization (MALDI). Molecular ions are observed in this ion beam sputtering of organic mixtures for peptides and oligonucleotides up to masses on the order of 10?000 Da. This matrix-enhanced SIMS exhibits substantial increases in the ionization efficiency of selected analyte molecules compared to conventional SSIMS processes. Thus, higher mass peptides, proteins, and nucleic acids become accessible to near-surface analysis by ion beam techniques, and subpicomole sensitivity has been demonstrated. A number of matrices were examined for their efficiency in ME-SIMS applications, and these initial matrix studies focused on common MALDI matrices and their isomers. The results of this survey indicate that 2,5-dihydroxybenzoic acid provides the best general enhancement of molecular secondary ions emitted from analyte/matrix mixtures.