Direct imaging of single cells and tissue at sub‐cellular spatial resolution using transmission geometry MALDI MS

The need of cellular and sub‐cellular spatial resolution in laser desorption ionization (LDI)/matrix‐assisted LDI (MALDI) imaging mass spectrometry (IMS) necessitates micron and sub‐micron laser spot sizes at biologically relevant sensitivities, introducing significant challenges for MS technology. To this end, we have developed a transmission geometry vacuum ion source that allows the laser beam to irradiate the back side of the sample. This arrangement obviates the mechanical/ion optic complications in the source by completely separating the optical lens and ion optic structures. We have experimentally demonstrated the viability of transmission geometry MALDI MS for imaging biological tissues and cells with sub‐cellular spatial resolution. Furthermore, we demonstrate that in conjunction with new sample preparation protocols, the sensitivity of this instrument is sufficient to obtain molecular images at sub‐micron spatial resolution. Copyright © 2012 John Wiley & Sons, Ltd.     

A concise review of mass spectrometry imaging

Mass spectrometric imaging allows the investigation of the spatial distribution of molecules at complex surfaces. The combination of molecular speciation with local analysis renders a chemical microscope that can be used for the direct biomolecular characterization of histological tissue surfaces. MS based imaging advantageously allows label-free detection and mapping of a wide-range of biological compounds whose presence or absence can be the direct result of disease pathology. Successful detection of the analytes of interest at the desired spatial resolution requires careful attention to several steps in the mass spectrometry imaging protocol. This review will describe and discuss a selected number of crucial developments in ionization, instrumentation, and application of this innovative technology. The focus of this review is on the latest developments in imaging MS. Selected biological applications are employed to illustrate some of the novel features discussed. Two commonly used MS imaging techniques, secondary ion mass spectrometric (SIMS) imaging and matrix-assisted laser desorption ionization (MALDI) mass spectrometric imaging, center this review. New instrumental developments are discussed that extend spatial resolution, mass resolving power, mass accuracy, tandem-MS capabilities, and offer new gas-phase separation capabilities for both imaging techniques. It will be shown how the success of MS imaging is crucially dependent on sample preparation protocols as they dictate the nature and mass range of detected biomolecules that can be imaged. Finally, developments in data analysis strategies for large imaging datasets will be briefly discussed.     

Enhancement of protein sensitivity for MALDI imaging mass spectrometry after chemical treatment of tissue sections

MALDI imaging mass spectrometry (IMS) has become a valuable tool for the investigation of the content and distribution of molecular species in tissue specimens. Numerous methodological improvements have been made to optimize tissue section preparation and matrix deposition protocols, as well as MS data acquisition and processing. In particular for proteomic analyses, washing the tissue sections before matrix deposition has proven useful to improve spectral qualities by increasing ion yields and the number of signals observed. We systematically explore here the effects of several solvent combinations for washing tissue sections. To minimize experimental variability, all of the measurements were performed on serial sections cut from a single mouse liver tissue block. Several other key steps of the process such as matrix deposition and MS data acquisition and processing have also been automated or standardized. To assess efficacy, after each washing procedure the total ion current and number of peaks were counted from the resulting protein profiles. These results were correlated to on-tissue measurements obtained for lipids. Using similar approaches, several selected washing procedures were also tested for their ability to extend the lifetime as well as revive previously cut tissue sections. The effects of these washes on automated matrix deposition and crystallization behavior as well as their ability to preserve tissue histology were also studied. Finally, in a full-scale IMS study, these washing procedures were tested on a human renal cell carcinoma biopsy.     

Subcellular imaging mass spectrometry of brain tissue

Imaging mass spectrometry provides both chemical information and the spatial distribution of each analyte detected. Here it is demonstrated how imaging mass spectrometry of tissue at subcellular resolution can be achieved by combining the high spatial resolution of secondary ion mass spectrometry (SIMS) with the sample preparation protocols of matrix-assisted laser desorption/ionization (MALDI). Despite mechanistic differences and sampling 10(5) times less material, matrix-enhanced (ME)-SIMS of tissue samples yields similar results to MALDI (up to m/z 2500), in agreement with previous studies on standard compounds. In this regard ME-SIMS represents an attractive alternative to polyatomic primary ions for increasing the molecular ion yield. ME-SIMS of whole organs and thin sections of the cerebral ganglia of Lymnaea stagnalis demonstrate the advantages of ME-SIMS for chemical imaging mass spectrometry. Subcellular distributions of cellular analytes are clearly obtained, and the matrix provides an in situ height map of the tissue, allowing the user to identify rapidly regions prone to topographical artifacts and to deconvolute topographical losses in mass resolution and signal-to-noise ratio.     

Distribution study of atorvastatin and its metabolites in rat tissues using combined information from UHPLC/MS and MALDI-Orbitrap-MS imaging

The combination of ultrahigh-resolution mass spectrometry imaging (UHRMSI) and ultrahigh-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC/MS/MS) was used for the identification and the spatial localization of atorvastatin (AT) and its metabolites in rat tissues. Ultrahigh-resolution and high mass accuracy measurements on a matrix-assisted laser desorption/ionization (MALDI)-Orbitrap mass spectrometer allowed better detection of desired analytes in the background of matrix and endogenous compounds. Tandem mass spectra were also used to confirm the identification of detected metabolites in complex matrices. The optimization of sample preparation before imaging experiments included the tissue cryogenic sectioning (thickness 20 μm), the transfer to stainless steel or glass slide, and the selection of suitable matrix and its homogenous deposition on the tissue slice. Thirteen matrices typically used for small molecule analysis, e.g., 2,5-dihydroxybenzoic acid (DHB), 1,5-diaminonaphthalene (DAN), 9-aminoacridine (AA), etc., were investigated for the studied drug and its metabolite detection efficiency in both polarity modes. Particular matrices were scored based on the strength of extracted ion current (EIC), relative ratio of AT molecular adducts, and fragment ions. The matrix deposition on the tissue for the most suitable matrices was done by sublimation to obtain the small crystal size and to avoid local variations in the ionization efficiency. UHPLC/MS profiling of drug metabolites in adjacent tissue slices with the previously optimized extraction was performed in parallel to mass spectrometry imaging (MSI) measurements to obtain more detailed information on metabolites in addition to the spatial information from MSI. The quantitation of atorvastatin in rat liver, serum, and feces was also performed.

Analysis of chloroquine and metabolites directly from whole‐body animal tissue sections by liquid extraction surface analysis (LESA) and tandem mass spectrometry

The rapid and direct analysis of the amount and spatial distribution of exogenous chloroquine (CHQ) and CHQ metabolites from tissue sections by liquid extraction surface sampling analysis coupled with tandem mass spectrometry (LESA‐MS/MS) was demonstrated. LESA‐MS/MS results compared well with previously published CHQ quantification data collected by organ excision, extraction and fluorescent detection. The ability to directly sample and analyze spatially resolved exogenous molecules from tissue sections with minimal sample preparation and analytical method development has the potential to facilitate the assessment of target tissue penetration of pharmaceutical compounds, to establish pharmacokinetic/pharmacodynamic relationships, and to complement established pharmacokinetic methods used in the drug discovery process during tissue distribution assessment. Copyright © 2012 John Wiley & Sons, Ltd.     

Imaging of atomic and molecular species in tissue with laser‐SNMS for pharmaceutical studies

Successful anticancer therapies will have the ability to selectively deliver compounds to target cells while sparing normal tissue. Currently, methods to determine the distribution of compounds with very high sensitivity and subcellular resolution are still unavailable. Laser secondary neutral mass spectrometry (laser‐SNMS) and time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS) are capable of detecting atoms and molecules with high sensitivity and a spatial resolution of up to 80 nm. The use of such methods requires special preparation techniques that preserve the morphological and chemical integrity of living cells. In this paper, the ability of laser‐SNMS to study transportation processes in animals of boron‐containing compounds for boron neutron capture therapy will be discussed. The data show that with laser‐SNMS it is possible to measure the distribution of these compounds in tissues with subcellular resolution, and that laser‐SNMS is a very powerful tool for locating anticancer drugs in tissues. Copyright © 2006 John Wiley & Sons, Ltd.     

Independent assessment of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) sample preparation quality: A novel statistical approach for quality scoring

Preparation of samples according to an optimized method is crucial for accurate determination of polymer sample characteristics by Matrix-Assisted Laser Desorption Ionization (MALDI) analysis. Sample preparation conditions such as matrix choice, cationization agent, deposition technique or even the deposition volume should be chosen to suit the sample of interest. Many sample preparation protocols have been developed and employed, yet finding the optimal sample preparation protocol remains a challenge. Because an objective comparison between the results of diverse protocols is not possible, “gut-feeling” or “good enough” is often decisive in the search for an optimum. This implies that sub-optimal protocols are used, leading to a loss of mass spectral information quality. To address this problem a novel analytical strategy based on MALDI imaging and statistical data processing was developed in which eight parameters were formulated to objectively quantify the quality of sample deposition and optimal MALDI matrix composition and finally sum up to an overall quality score of the sample deposition. These parameters can be established in a fully automated way using commercially available mass spectrometry imaging instruments without any hardware adjustments. With the newly developed analytical strategy the highest quality MALDI spots were selected, resulting in more reproducible and more valuable spectra for PEG in a variety of matrices. Moreover, our method enables an objective comparison of sample preparation protocols for any analyte and opens up new fields of investigation by presenting MALDI performance data in a clear and concise way.     

Mass spectrometry imaging with high resolution in mass and space (HR<Superscript>2</Superscript> MSI) for reliable investigation of drug compound distributions on the cellular level

Mass spectrometry (MS) imaging is a versatile method to analyze the spatial distribution of analytes in tissue sections. It provides unique features for the analysis of drug compounds in pharmacokinetic studies such as label-free detection and differentiation of compounds and metabolites. We have recently introduced a MS imaging method that combines high mass resolution and high spatial resolution in a single experiment, hence termed HR² MS imaging. In the present study, we applied this method to analyze the spatial distribution of the anti-cancer drugs imatinib and ifosfamide in individual mouse organs. The whole kidney of an animal dosed with imatinib was measured at 35 μm spatial resolution. Imatinib showed a well-defined distribution in the outer stripe of the outer medulla. This area was analyzed in more detail at 10 μm step size, which constitutes a tenfold increase in effective spatial resolution compared to previous studies of drug compounds. In parallel, ion images of phospholipids and heme were used to characterize the histological features of the tissue section and showed excellent agreement with histological staining of the kidney after MS imaging. Ifosfamide was analyzed in mouse kidney at 20 μm step size and was found to be accumulated in the inner medulla region. The identity of imatinib and ifosfamide was confirmed by on-tissue MS/MS measurements. All measurements including mass spectra from 10 μm pixels featured accurate mass (≤2 ppm root mean square) and mass resolving power of R = 30,000. Selected ion images were generated with a bin size of ∆m/z = 0.01 ensuring highly specific information. The ability of the method to cover larger areas was demonstrated by imaging a compound in the intestinal tract of a rat whole-body tissue section at 200 μm step size. The described method represents a major improvement in terms of spatial resolution and specificity for the analysis of drug compounds in tissue sections.     

Imaging of peptides in the rat brain using MALDI-FTICR mass spectrometry

Analytical methods are pursued to measure the identity and location of biomolecules down to the subcellular (microm) level. Available mass spectrometric imaging methods either compromise localization accuracy or identification accuracy in their analysis of surface biomolecules. In this study, imaging FTICR-MS is applied for the spatially resolved mass analysis of rat brain tissue with the aim to optimize protein identification by the high mass accuracy and online MS/MS capabilities of the technique. Mass accuracies up to 6 ppm were obtained in the direct MALDI-analysis of the tissue together with a spatial resolution of 200 microm. The spatial distributions of biomolecules differing in mass by less than 0.1 Da could be resolved, and are shown to differ significantly. Online MS/MS analysis of selected ions was demonstrated. A comparison of the FTICR-MS imaging results with stigmatic TOF imaging on the same sample is presented. To reduce the extended measuring times involved, it is recommended to restrict the FTICR-MS analyses to areas of interest as can be preselected by other, faster imaging methods.     

Mobility-Modulated Sequential Dissociation Analysis Enables Structural Lipidomics in Mass Spectrometry Imaging

Spatial lipidomics based on mass spectrometry imaging (MSI) is a powerful tool for fundamental biology studies and biomarker discovery. But the structure-resolving capability of MSI is limited because of the lack of multiplexed tandem mass spectrometry (MS/MS) method, primarily due to the small sample amount available from each pixel and the poor ion usage in MS/MS analysis. Here, we report a mobility-modulated sequential dissociation (MMSD) strategy for multiplex MS/MS imaging of distinct lipids from biological tissues. With ion mobility-enabled data-independent acquisition and automated spectrum deconvolution, MS/MS spectra of a large number of lipid species from each tissue pixel are acquired, at no expense of imaging speed. MMSD imaging is highlighted by MS/MS imaging of 24 structurally distinct lipids in the mouse brain and the revealing of the correlation of a structurally distinct phosphatidylethanolamine isomer (PE 18 : 1_18 : 1) from a human hepatocellular carcinoma (HCC) tissue. Mapping of structurally distinct lipid isomers is now enabled and spatial lipidomics becomes feasible for MSI.     

Reagent Precoated Targets for Rapid In-Tissue Derivatization of the Anti-Tuberculosis Drug Isoniazid Followed by MALDI Imaging Mass Spectrometry

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.     

Development of a MALDI two-layer volume sample preparation technique for analysis of colored conidia spores of <Emphasis Type="Italic">Fusarium</Emphasis> by MALDI linear TOF mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS) has been proved to be a powerful tool for the identification and characterization of microorganisms based on their surface peptide/protein pattern. Because of the complexity of microorganisms, there are no standardized protocols to acquire reproducible peptide/protein profiles for a broad range of microorganisms and for fungi in particular. Small variations during MALDI MS sample preparation affect the quality of mass spectra quite often. In this study, we were aiming to develop a sample preparation method for the analysis of colored, a quite often observed phenomenon, and mycotoxin-producing Fusarium conidia spores using MALDI–TOF MS. Different washing solvent systems for light- and deep-colored (from slightly orange to red-brown) conidia spores and connected sample deposition techniques were evaluated based on MS reproducibility and number and intensities of peaks. As a method of choice for generation of reproducible and characteristic MALDI–TOF mass spectra, the use of a washing process for colored Fusarium conidia spores with acetonitrile/0.5% formic acid (7/3) was found and subsequently combined with two-layer volume technique (spores/matrix (ferulic acid) solution was deposited onto a MALDI target, and after solvent evaporation, a second matrix layer was deposited). With the application of this sample preparation method, for deep-colored Fusarium species, 19 abundant molecular ions in the m/z range 2,000–10,000 were always detected with an S/N ratio of 3:1 or better. Finally this optimized sample preparation for the first time provided mass spectrometric fingerprints of strongly colored Fusarium conidia spores resulting in the possibility of differentiation of such spores at the species level.      

Combined Fourier-transform infrared imaging and desorption electrospray-ionization linear ion-trap mass spectrometry for analysis of counterfeit antimalarial tablets

This paper reports use of a combination of Fourier-transform infrared (FTIR) spectroscopic imaging and desorption electrospray ionization linear ion-trap mass spectrometry (DESI MS) for characterization of counterfeit pharmaceutical tablets. The counterfeit artesunate antimalarial tablets were analyzed by both techniques. The results obtained revealed the ability of FTIR imaging in non-destructive micro-attenuated total reflection (ATR) mode to detect the distribution of all components in the tablet, the identities of which were confirmed by DESI MS. Chemical images of the tablets were obtained with high spatial resolution. The FTIR spectroscopic imaging method affords inherent chemical specificity with rapid acquisition of data. DESI MS enables high-sensitivity detection of trace organic compounds. Combination of these two orthogonal surface-characterization methods has great potential for detection and analysis of counterfeit tablets in the open air and without sample preparation.     

Silver spray deposition for AgLDI imaging MS of cholesterol and other olefins on thin tissue sections

Olefins such as cholesterol and unsaturated fatty acids play important biological roles. Silver‐assisted laser desorption ionization (AgLDI) takes advantage of the strong affinity of silver to conjugate with double bonds to selectively ionize these molecules for imaging mass spectrometry (IMS) experiments. For IMS studies, two main approaches for silver deposition have been described in the literature: fine coating by silver sputtering and spray deposition of silver nanoparticles. While these approaches allow for extremely high resolution IMS experiments to be conducted, they are not readily available to all laboratories. Herein, we present a silver nitrate spray deposition approach as an alternative to silver sputtering and nanoparticle deposition for routine IMS analysis. The silver nitrate spray has the same level of specificity and sensitivity for olefins, particularly cholesterol, and has shown to be capable of IMS experiments down to 10‐μm spatial resolution. Minimal sample preparation and the affordability of silver nitrate make this a convenient and accessible technique worth considering.     

Mapping the fly Malpighian tubule lipidome by imaging mass spectrometry

Matrix‐assisted laser/desorption ionization imaging mass spectrometry (MALDI IMS) is an analytical technique for understanding the spatial distribution of biomolecules across a sample surface. Originally employed for mammalian tissues, this technology has been adapted to study specimens as diverse as microbes and cell cultures, food such as strawberries, and invertebrates including the vinegar fly Drosophila melanogaster. As an ideal model organism, Drosophila has brought greater understanding about conserved biological processes, organism development, and diseased states and even informed management practices of agriculturally and environmentally important species. Drosophila displays anatomically separated renal (Malpighian) tubules that are the physiological equivalent to the vertebrate nephron. Insect Malpighian tubules are also responsible for pesticide detoxification. In this article, we first describe an effective workflow and sample preparation method to study the phospholipid distribution of the Malpighian tubules that initially involves the manual microdissection of the tubules in saline buffer followed by a series of washes to remove excess salt and enhances the phospholipid signals prior to matrix deposition and IMS at 25‐μm spatial resolution. We also established a complementary methodology for lipid IMS analysis of whole‐body fly sections using a dual‐polarity data acquisition approach at the same spatial resolution after matrix deposition by sublimation. Both procedures yield rich signal profiles from the major phospholipid classes. The reproducibility and high‐quality results offered by these methodologies enable cohort studies of Drosophila through MALDI IMS.     

Organic ion imaging of biological tissue with secondary ion mass spectrometry and matrix-assisted laser desorption/ionization

Organic secondary ion mass spectrometry (SIMS) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry can be used to produce molecular images of samples. This is achieved through ionization from a clearly identified point on a flat sample, and performing a raster of the sample by moving the point of ionization over the sample surface. The unique analytical capabilities of mass spectrometry for mapping a variety of biological samples at the tissue level are discussed. SIMS provides information on the spatial distribution of the elements and low molecular mass compounds as well as molecular structures on these compounds, while MALDI yields spatial information about higher molecular mass compounds, including their distributions in tissues at very low levels, as well as information on the molecular structures of these compounds. Application of these methods to analytical problems requires appropriate instrumentation, sample preparation methodology, and a data presentation usually in a three-coordinate plot where x and y are physical dimensions of the sample and z is the signal amplitude. The use of imaging mass spectrometry is illustrated with several biological systems.     

Matrix pre‐coated targets for high throughput MALDI imaging of proteins

We have developed matrix pre‐coated targets for imaging proteins in thin tissue sections by matrix‐assisted laser desorption/ionization mass spectrometry. Gold covered microscope slides were coated with sinapinic acid (SA) in batches in advance and were shown to be stable for over 6 months when kept in the dark. The sample preparation protocol using these SA pre‐coated targets involves treatment with diisopropylethylamine (DIEA)‐H₂O vapor, transforming the matrix layer to a viscous ionic liquid. This SA‐DIEA ionic liquid layer extracts proteins and other analytes from tissue sections that are thaw mounted to this target. DIEA is removed by the immersion of the target into diluted acetic acid, allowing SA to co‐crystallize with extracted analytes directly on the target. Ion images (3–70 kDa) of sections of mouse brain and rat kidney at spatial resolution down to 10 µm were obtained. Use of pre‐coated slides greatly reduces sample preparation time for matrix‐assisted laser desorption/ionization imaging while providing high throughput, low cost and high spatial resolution images. Copyright © 2014 John Wiley & Sons, Ltd.     

Matrix optimization for matrix-assisted laser desorption/ionization mass spectrometry of oligosaccharides from human milk

Neutral and acidic oligosaccharides from human milk were analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS). These experiments require suitable matrices; their selection and particularly their preparation protocols must be optimized. Important criteria are sensitivity, reproducibility, tolerance against impurities and resolution over a wide mass range. For analytical investigations of these oligosaccharides, containing labile fucosylated and sialylated components, another property of a matrix becomes a significant factor, namely the influence on ion stability and the extent of (metastable) fragmentation. The experience gained with the MALDI/MS of neutral and acidic oligosaccharides is summarized taking into account different intentions of measurement and typical problems, such as impurities after enzymatic treatment. For a rapid screening of an oligosaccharide sample, superior results were obtained with a new preparation technique using 5-chloro-2-mercaptobenzothiazole (CMBT) as the first layer for 2,5-dihydroxybenzoic acid. For structural analysis by post-source decay, CMBT as the first layer for 3-aminoquinoline is a favoured preparation protocol, because extensive fragmentation is achieved. For acidic oligosaccharides, a special preparation protocol makes it possible to determine the number of sialic acids by inducing highly effective cationization. Matrix-assisted laser desorption/ionization mass spectrometry; matrices; oligosaccharides; post-source decay.     

Applications of laser microprobe mass spectrometry in organic analysis

The ability to focus the laser accurately onto the sample with a small beam diameter (2.0–3.0 μm) enables laser mass spectrometry to be used as a microprobe. Results from a fully automated ion-mapping system for laser mass spectrometry are described. These results show that the spatial resolution of the laser microprobe is primarily limited by the diameter of the laser beam. Factors such as laser power density, laser focus, sample preparation, and chemical environment influence the reproducibility of laser mass spectra significantly. Calibration curves obtained in the analysis of mixtures of phenanthrolines demonstrate that laser mass spectrometry can be used to quantify organic components. Preliminary results on the detection of neutral molecules resulting from metastable decomposition in the flight tube are also presented.