Evaluation of Quantitative Platforms for Single Target Mass Spectrometry Imaging.

(1) Imaging of pharmaceutical compounds in tissue is an increasingly important subsection of Mass Spectrometry Imaging (MSI). Identifying proper target engagement requires MS platforms with high sensitivity and spatial resolution. Three prominent categories of drugs are small molecule drugs, antibody-drug conjugate payloads, and protein degraders. (2) We tested six common MSI platforms for their limit of detection (LoD) on a representative compound for each category: a Matrix-Assisted Laser Desorption/Ionization (MALDI) Fourier Transform Ion Cyclotron, a MALDI-2 Time-of-Flight (ToF), a MALDI-2 Trapped Ion Mobility Spectrometry ToF, a Desorption Electrospray Ionization Orbitrap, and 2 Atmospheric Pressure-MALDI Triple Quadrupoles. Samples were homogenized tissue mimetic models of rat liver spiked with known concentrations of analytes. (3) We found that the AP-MALDI-QQQ platform outperformed all 4 competing platforms by a minimum of 2- to 52-fold increase in LoD for representative compounds from each category of pharmaceutical. (4) AP-MALDI-QQQ platforms are effective, cost-efficient mass spectrometers for the identification of targeted analytes of interest.

Optimization of Zebrafish Larvae Sectioning for Mass Spectrometry Imaging.

The utility of zebrafish is becoming more frequent due to lower costs and high similarities to humans. Zebrafish larvae are attractive subjects for drug screening and drug metabolism research. However, obtaining good quality zebrafish larvae sections for batch samples at designated planes, angles, and locations for comparison purposes is a challenging task. We report here the optimization of fresh frozen zebrafish larvae sectioning for mass spectrometry imaging. We utilized the gelatin solutions that were created at two different temperatures (50 and 85 °C) as embedding media. Gelatin-50 (gelatin created under 50 °C, solid gel under room temperature) was used to make a larvae-shaped mold and gelatin-85 (gelatin created under 85 °C, liquid under room temperature) was used to embed the larvae. H&E staining of sections shows well-preserved morphology and minimal histological interference. More importantly, the position of the larvae was well controlled resulting in more consistent sectioning of the larvae.

Catalase and superoxide dismutase response and the underlying molecular mechanism for naphthalene.

Naphthalene, a naturally-occurring polyaromatic hydrocarbon, pose potential threats to health for its wide exposures in environment. Naphthalene could disrupt the redox equilibrium resulting in oxidative damage. Antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD) are considered to be the efficient defense barriers to protect organisms from negative impacts of toxicants. Limited information is available regarding the underlying molecular mechanism between antioxidant enzymes and naphthalene. In this paper, structural and functional alterations of CAT and SOD for low dose (1.6-25.6 mg/L) naphthalene exposure have been investigated at the molecular and cellular levels. The enzyme activity responses of CAT and SOD in hepatocytes for naphthalene were consistent with the molecular, in which the activity of CAT increased and the activity of SOD slightly inhibited. Spectroscopy methods and molecular docking were carried out to investigate the underlying binding mechanisms. Naphthalene exposure significantly changed the conformation of CAT with secondary structure alteration (α-helix increase) but only changed the skeleton structure of SOD without secondary structure alteration. Naphthalene could bind to CAT and SOD primarily via H-binding force accompanied with the particle size of CAT/SOD agglomerates decreasing. Naphthalene preferentially bound to the surface of CAT and SOD. Besides, naphthalene could also bind directly to the active center of CAT with the key residues Arg364 and Tyr 357 for activity. This paper provides a combined cellular and molecular strategy to research biomarker responses for toxicants exposure. Besides, this study offers detailed basic data for the comprehensive understanding of naphthalene toxicity.

A recommended and verified procedure for in situ tryptic digestion of formalin-fixed paraffin-embedded tissues for analysis by matrix-assisted laser desorption/ionization imaging mass spectrometry.

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) is a molecular imaging technology uniquely capable of untargeted measurement of proteins, lipids, and metabolites while retaining spatial information about their location in situ. This powerful combination of capabilities has the potential to bring a wealth of knowledge to the field of molecular histology. Translation of this innovative research tool into clinical laboratories requires the development of reliable sample preparation protocols for the analysis of proteins from formalin-fixed paraffin-embedded (FFPE) tissues, the standard preservation process in clinical pathology. Although ideal for stained tissue analysis by microscopy, the FFPE process cross-links, disrupts, or can remove proteins from the tissue, making analysis of the protein content challenging. To date, reported approaches differ widely in process and efficacy. This tutorial presents a strategy derived from systematic testing and optimization of key parameters, for reproducible in situ tryptic digestion of proteins in FFPE tissue and subsequent MALDI IMS analysis. The approach describes a generalized method for FFPE tissues originating from virtually any source.

The Ca2+ transient as a feedback sensor controlling cardiomyocyte ionic conductances in mouse populations.

Conductances of ion channels and transporters controlling cardiac excitation may vary in a population of subjects with different cardiac gene expression patterns. However, the amount of variability and its origin are not quantitatively known. We propose a new conceptual approach to predict this variability that consists of finding combinations of conductances generating a normal intracellular Ca2+ transient without any constraint on the action potential. Furthermore, we validate experimentally its predictions using the Hybrid Mouse Diversity Panel, a model system of genetically diverse mouse strains that allows us to quantify inter-subject versus intra-subject variability. The method predicts that conductances of inward Ca2+ and outward K+ currents compensate each other to generate a normal Ca2+ transient in good quantitative agreement with current measurements in ventricular myocytes from hearts of different isogenic strains. Our results suggest that a feedback mechanism sensing the aggregate Ca2+ transient of the heart suffices to regulate ionic conductances.

Novel vacuum stable ketone-based matrices for high spatial resolution MALDI imaging mass spectrometry.

We describe the use of aromatic ketones and cinnamyl ketones that have high vacuum stability for analyzing tissue sections using matrix-assisted laser desorption/ionization imaging mass spectrometry. Specifically, the matrix, (E)-4-(2,5-dihydroxyphenyl)but-3-en-2-one (2,5-cDHA) provides high sensitivity and high vacuum stability while producing small size crystals (1-2 µm). A high throughput and highly reproducible sample preparation method was developed for these matrices that first involves using an organic spray solution for small matrix crystal seeding followed by spraying of the matrix in a 30% acetonitrile/70% water solution on the tissue surface to obtain a homogeneous coating of small crystals, suitable for high spatial resolution imaging.

Trypsin and MALDI matrix pre-coated targets simplify sample preparation for mapping proteomic distributions within biological tissues by imaging mass spectrometry.

Prefabricated surfaces containing α-cyano-4-hydroxycinnamic acid and trypsin have been developed to facilitate enzymatic digestion of endogenous tissue proteins prior to matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS). Tissue sections are placed onto slides that were previously coated with α-cyano-4-hydroxycinnamic acid and trypsin. After incubation to promote enzymatic digestion, the tissue is analyzed by MALDI IMS to determine the spatial distribution of the tryptic fragments. The peptides detected in the MALDI IMS dataset were identified by Liquid chromatography-tandem mass spectrometry/mass spectrometry. Protein identification was further confirmed by correlating the localization of unique tryptic fragments originating from common parent proteins. Using this procedure, proteins with molecular weights as large as 300 kDa were identified and their distributions were imaged in sections of rat brain. In particular, large proteins such as myristoylated alanine-rich C-kinase substrate (29.8 kDa) and spectrin alpha chain, non-erythrocytic 1 (284 kDa) were detected that are not observed without trypsin. The pre-coated targets simplify workflow and increase sample throughput by decreasing the sample preparation time. Further, the approach allows imaging at higher spatial resolution compared with robotic spotters that apply one drop at a time. Copyright © 2016 John Wiley & Sons, Ltd.

Image fusion of mass spectrometry and microscopy: a multimodality paradigm for molecular tissue mapping.

We describe a predictive imaging modality created by 'fusing' two distinct technologies: imaging mass spectrometry (IMS) and microscopy. IMS-generated molecular maps, rich in chemical information but having coarse spatial resolution, are combined with optical microscopy maps, which have relatively low chemical specificity but high spatial information. The resulting images combine the advantages of both technologies, enabling prediction of a molecular distribution both at high spatial resolution and with high chemical specificity. Multivariate regression is used to model variables in one technology, using variables from the other technology. We demonstrate the potential of image fusion through several applications: (i) 'sharpening' of IMS images, which uses microscopy measurements to predict ion distributions at a spatial resolution that exceeds that of measured ion images by ten times or more; (ii) prediction of ion distributions in tissue areas that were not measured by IMS; and (iii) enrichment of biological signals and attenuation of instrumental artifacts, revealing insights not easily extracted from either microscopy or IMS individually.

Tissue protein imaging at 1 µm laser spot diameter for high spatial resolution and high imaging speed using transmission geometry MALDI TOF MS.

We have achieved protein imaging mass spectrometry capabilities at sub-cellular spatial resolution and at high acquisition speed by integrating a transmission geometry ion source with time of flight mass spectrometry. The transmission geometry principle allowed us to achieve a 1-µm laser spot diameter on target. A minimal raster step size of the instrument was 2.5 µm. Use of 2,5-dihydroxyacetophenone robotically sprayed on top of a tissue sample as a matrix together with additional sample preparation steps resulted in single pixel mass spectra from mouse cerebellum tissue sections having more than 20 peaks in a range 3-22 kDa. Mass spectrometry images were acquired in a standard step raster microprobe mode at 5 pixels/s and in a continuous raster mode at 40 pixels/s.

Automated anatomical interpretation of ion distributions in tissue: linking imaging mass spectrometry to curated atlases.

Imaging mass spectrometry (IMS) has become a prime tool for studying the distribution of biomolecules in tissue. Although IMS data sets can become very large, computational methods have made it practically feasible to search these experiments for relevant findings. However, these methods lack access to an important source of information that many human interpretations rely upon: anatomical insight. In this work, we address this need by (1) integrating a curated anatomical data source with an empirically acquired IMS data source, establishing an algorithm-accessible link between them and (2) demonstrating the potential of such an IMS-anatomical atlas link by applying it toward automated anatomical interpretation of ion distributions in tissue. The concept is demonstrated in mouse brain tissue, using the Allen Mouse Brain Atlas as the curated anatomical data source that is linked to MALDI-based IMS experiments. We first develop a method to spatially map the anatomical atlas to the IMS data sets using nonrigid registration techniques. Once a mapping is established, a second computational method, called correlation-based querying, gives an elementary demonstration of the link by delivering basic insight into relationships between ion images and anatomical structures. Finally, a third algorithm moves further beyond both registration and correlation by providing automated anatomical interpretation of ion images. This task is approached as an optimization problem that deconstructs ion distributions as combinations of known anatomical structures. We demonstrate that establishing a link between an IMS experiment and an anatomical atlas enables automated anatomical annotation, which can serve as an important accelerator both for human and machine-guided exploration of IMS experiments.

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)-H2 O 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.

Implementation of a Gaussian beam laser and aspheric optics for high spatial resolution MALDI imaging MS.

We have investigated the use of a Gaussian beam laser for MALDI Imaging Mass Spectrometry to provide a precisely defined laser spot of 5 µm diameter on target using a commercial MALDI TOF instrument originally designed to produce a 20 µm diameter laser beam spot at its smallest setting. A Gaussian beam laser was installed in the instrument in combination with an aspheric focusing lens. This ion source produced sharp ion images at 5 µm spatial resolution with signals of high intensity as shown for images from thin tissue sections of mouse brain.

Laser beam filtration for high spatial resolution MALDI imaging mass spectrometry.

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.

Matrix precoated targets for direct lipid analysis and imaging of tissue.

We have developed targets precoated with matrix for imaging lipids in tissues using matrix-assisted laser desorption ionization mass spectrometry (MALDI MS). Thin tissue sections (rat kidney and mouse or rat brains) were placed onto 1,5-diaminonaphthalene precoated targets (prepared beforehand by a protocol utilizing sublimation) and were washed with ammonium formate solution. After a brief drying period, the target slides were imaged by MALDI MS. The resulting images from these sections were of equivalent quality to those obtained using the usual postcoating approach, such as sublimation and spraying, in terms of the sharpness of substructures in the images demonstrated by imaging at spatial resolutions of 100, 10, and 5 µm. Matrix precoated targets have a shelf life of more than 6 months when kept in a dark, nonhumid environment such as a nontransparent desiccator.

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.

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

Discussions about MALDI imaging frequently turn to the topic of spatial resolution and the eff orts of some researchers in the field to push towards routine imaging of tissue sections at a cellular scale. Some factors that limit resolution are, the size of the focused desorption laser beam and analyte delocalization from the solution-based sample preparation. With solvent-free matrix application techniques analyte delocalization is less of a concern and the size of the focused laser is the major limiter of spatial resolution. In the Special Feature, Professor Caprioli and co-workers at Vanderbilt University demonstrate a new instrumental approach for improving spatial resolution. They have modifi ed a MALDI-TOF system to use transmission-mode geometry, in which the desorption laser is focused onto the matrix crystals from behind and through the target and sample rather than conventional front-side illumination where the laser is focused onto the crystals directly. They show that by moving the laser source behind the sample target, they can optimize the laser focus to achieve cellular resolution for MALDI imaging.

Targeted multiplex imaging mass spectrometry with single chain fragment variable (scfv) recombinant antibodies.

Recombinant scfv antibodies specific for CYP1A1 and CYP1B1 P450 enzymes were combined with targeted imaging mass spectrometry to simultaneously detect the P450 enzymes present in archived, paraffin-embedded, human breast cancer tissue sections. By using CYP1A1 and CYP1B1 specific scfv, each coupled to a unique reporter molecule (i.e., a mass tag) it was possible to simultaneously detect multiple antigens within a single tissue sample with high sensitivity and specificity using mass spectrometry. The capability of imaging multiple antigens at the same time is a significant advance that overcomes technical barriers encountered when using present day approaches to develop assays that can simultaneously detect more than a single antigen in the same tissue sample.

Activity-based probes linked with laser-cleavable mass tags for signal amplification in imaging mass spectrometry: analysis of serine hydrolase enzymes in mammalian tissue.

A novel functional imaging mass spectrometry technology is described that utilizes activity-based probes for imaging enzyme active sites in tissue sections. We demonstrate this technology using an activity-based probe (fluorophosphate) that is specific for serine hydrolases. A dendrimer containing multiple mass tags that is attached to the activity-based probe is used to analyze the binding sites of the probe through release and measurement of the mass tags on laser irradiation. A generation 8 poly(amido amine) dendrimer with 1024 amino groups was labeled with an azide group, and then, more than 900 mass tags were attached in order to achieve signal amplification of nearly 3 orders of magnitude. The experimental protocol first involves binding of the activity-based probe containing an alkyne group to serine hydrolases in the tissue section followed by attachment of the dendrimer labeled with mass tags to the bound probe by Click chemistry. On irradiation of the labeled tissue by the laser beam in a raster pattern, the mass tags are liberated and recorded by the mass analyzer; consequently, the ion image of the mass tag reveals the distribution of serine hydrolases in the tissue. This process was shown using rat brain and mouse embryo sections. Targeted imaging has the advantage of providing high spatial resolution and high sensitivity through the use of signal amplification chemistry with high target specificity through the use of an enzyme activity probe.

Genetic variants in MARCO are associated with the susceptibility to pulmonary tuberculosis in Chinese Han population.

BACKGROUND: Susceptibility to tuberculosis is not only determined by Mycobacterium tuberculosis infection, but also by the genetic component of the host. Macrophage receptor with a collagenous structure (MARCO) is essential components required for toll like receptor-signaling in macrophage response to Mycobacterium tuberculosis, which may contribute to tuberculosis risk. PRINCIPAL FINDINGS: To specifically investigated whether single nucleotide polymorphisms (SNPs) in MARCO gene are associated with pulmonary tuberculosis in Chinese Han population. By selecting tagging SNPs in MARCO gene, 17 tag SNPs were identified and genotyped in 923 pulmonary tuberculosis patients and 1033 healthy control subjects using a hospital based case-control association study. Single-point and haplotype analysis revealed an association in intron and exon region of MARCO gene. One SNP (rs17009726) was associated with susceptibility to pulmonary tuberculosis, where the carriers of the G allele had a 1.65 fold (95% CI = 1.32-2.05, p(corrected) = 9.27E-5) increased risk of pulmonary tuberculosis. Haplotype analysis revealed that haplotype GC containing G allele of 17009726 and haplotype TGCC (rs17795618T/A, rs1371562G/T, rs6761637T/C, rs2011839C/T) were also associated with susceptibility to pulmonary tuberculosis (p(corrected) = 0.0001 and 0.029, respectively). CONCLUSIONS: Our study suggested that genetic variants in MARCO gene were associated with pulmonary tuberculosis susceptibility in Chinese Han population, and the findings emphasize the importance of MARCO mediated immune responses in the pathogenesis of tuberculosis.

Protective role of transient pore openings in calcium handling by cardiac mitochondria.

Long-lasting mitochondrial permeability transition pore (mPTP) openings damage mitochondria, but transient mPTP openings protect against chronic cardiac stress. To probe the mechanism, we subjected isolated cardiac mitochondria to gradual Ca(2+) loading, which, in the absence of BSA, induced long-lasting mPTP opening, causing matrix depolarization. However, with BSA present to mimic cytoplasmic fatty acid-binding proteins, the mitochondrial population remained polarized and functional, even after matrix Ca(2+) release caused an extramitochondrial free [Ca(2+)] increase to >10 µM, unless mPTP openings were inhibited. These findings could be explained by asynchronous transient mPTP openings allowing individual mitochondria to depolarize long enough to flush accumulated matrix Ca(2+) and then to repolarize rapidly after pore closure. Because subsequent matrix Ca(2+) reuptake via the Ca(2+) uniporter is estimated to be >100-fold slower than matrix Ca(2+) release via mPTP, only a tiny fraction of mitochondria (<1%) are depolarized at any given time. Our results show that transient mPTP openings allow cardiac mitochondria to defend themselves collectively against elevated cytoplasmic Ca(2+) levels as long as respiratory chain activity is able to balance proton influx with proton pumping. We found that transient mPTP openings also stimulated reactive oxygen species production, which may engage reactive oxygen species-dependent cardioprotective signaling.