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A multimodal mass spectrometry imaging (MSI) approach was used to investigate the chemotherapy drug-induced response of a Multicellular Tumour Spheroid (MCTS) 3D cell culture model of osteosarcoma (OS). The work addresses the critical demand for enhanced translatable early drug discovery approaches by demonstrating a robust spatially resolved molecular distribution analysis in tumour models following chemotherapeutic intervention. Advanced high-resolution techniques were employed, including desorption electrospray ionisation (DESI) mass spectrometry imaging (MSI), to assess the interplay between metabolic and cellular pathways in response to chemotherapeutic intervention. Endogenous metabolite distributions of the human OS tumour models were complemented with subcellularly resolved protein localisation by the detection of metal-tagged antibodies using Imaging Mass Cytometry (IMC). The first application of matrix-assisted laser desorption ionization-immunohistochemistry (MALDI-IHC) of 3D cell culture models is reported here. Protein localisation and expression following an acute dosage of the chemotherapy drug doxorubicin demonstrated novel indications for mechanisms of region-specific tumour survival and cell-cycle-specific drug-induced responses. Previously unknown doxorubicin-induced metabolite upregulation was revealed by DESI-MSI of MCTSs, which may be used to inform mechanisms of chemotherapeutic resistance. The demonstration of specific tumour survival mechanisms that are characteristic of those reported for in vivo tumours has underscored the increasing value of this approach as a tool to investigate drug resistance.

In order to achieve even more widespread adoption over the next 5 years, a number of issues in mass spectrometry imaging need to be addressed. These are non-observation of compounds (due to ionization suppression), sample throughput, imaging of low-abundant species, and how to extract information from the large volumes of data generated. In this article, how current research indicates that these issues will be resolved along with potential application areas that MSI could look to exploit is discussed., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Careful formulation of pharmaceuticals for oral delivery is essential to ensure that the optimal amount of the active ingredient reaches its intended site of action. This chapter demonstrates how mass spectrometry can be used in conjunction with ex vivo tissue and an adapted milli-fluidics system to carry out a drug absorption study. MALDI MSI is used to visualize the drug within the small intestine tissue from the absorption experimentation. LC-MS/MS is used to complete a mass balance of the experiment and quantify the amount of drug that has permeated through the tissue., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) has seen a growing interest as a leading technique in the pharmaceutical industry for mapping label-free exogenous and endogenous species in biological tissues. However, the use of MALDI-MSI to perform spatially resolved absolute quantitation of species directly in tissues is still challenging, and robust quantitative mass spectrometry imaging (QMSI) methods need to be developed. In this study, we describe the microspotting technique for analytical and internal standard deposition, matrix sublimation, powerful QMSI software, and mass spectrometry imaging setup to obtain absolute quantitation of drug distribution in 3D skin models., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

This is the first report of the use of laser ablation-inductively coupled plasma time-of-flight mass spectrometry (LA-ICP-TOFMS) to analyze human malignant pleural mesothelioma (MPM) samples at the cellular level. MPM is an aggressive, incurable cancer associated with asbestos exposure, with a long latency and poor overall survival. Following careful optimization of the laser fluence, the simultaneous ablation of soft biological tissue and hard mineral fibers was possible, allowing the spatial detection of elements such as Si, Mg, Ca, and Fe, which are also present in the glass substrate. A low-dispersion LA setup was employed, which provided the high spatial resolution necessary to identify the asbestos fibers and fiber fragments in the tissue and to characterize the metallome at the cellular level (a pixel size of 2 μm), with a high speed (at 250 Hz). The multielement LA-ICP-TOFMS imaging approach enabled (i) the detection of asbestos fibers/mineral impurities within the MPM tissue samples of patients, (ii) the visualization of the tissue structure with the endogenous elemental pattern at high spatial resolution, and (iii) obtaining insights into the metallome of MPM patients with different pathologies in a single analysis run. Asbestos and other mineral fibers were detected in the lung and pleura tissue of MPM patients, respectively, based on their multielement pattern (Si, Mg, Ca, Fe, and Sr). Interestingly, strontium was detected in asbestos fibers, suggesting a link between this potential toxic element and MPM pathogenesis. Furthermore, monitoring the metallome around the talc deposit regions (characterized by elevated levels of Al, Mg, and Si) revealed significant tissue damage and inflammation caused by talc pleurodesis. LA-ICP-TOFMS results correlated to Perls' Prussian blue and histological staining of the corresponding serial sections. Ultimately, the ultra-high-speed and high-spatial-resolution capabilities of this novel LA-ICP-TOFMS setup may become an important clinical tool for simultaneous asbestos detection, metallome monitoring, and biomarker identification.

Absorption studies on oral drugs can be difficult due to the challenge of replicating the complex structure and environment of the GI tract. Drug absorption studies can be conducted using in vivo and ex vivo animal tissue or animal-free techniques. These studies typically involve the use of Caco-2 cells. However, Caco-2 cells do not incorporate all the cell types found in intestinal tissue and lack P450 metabolizing enzymes. The QV600 LLI system is a microfluidics system designed for use with cell culture. Here, it has been adapted to house appropriate sections of ex vivo porcine tissue to act as a system that models the duodenum section of the small intestine. A pH regulated solution of Atorvastatin was flowed over the apical layer of the GI tissue and a nutrient solution flowed over the basal layer of the tissue to maintain tissue viability. The tissue samples were snap-frozen, cryosectioned, and imaged using MALDI Mass Spectrometry Imaging (MSI). A proof-of-concept study on the effect of excipients on absorption was conducted. Different concentrations of the solubilizing agent were added to the donor circuit of the QV600 LLI. The amount of Atorvastatin in the acceptor circuit was determined to study the effect of the excipient on the amount of drug that had permeated through the tissue. Using these data, Papp, pig values were calculated and compared with the literature.

Matrix assisted laser desorption ionisation mass spectrometry imaging (MALDI-MSI), was used to obtain images of lipids and metabolite distribution in formalin fixed and embedded in paraffin (FFPE) whole eye sections containing primary uveal melanomas (UM). Using this technique, it was possible to obtain images of lysophosphatidylcholine (LPC) type lipid distribution that highlighted the tumour regions. Laser ablation inductively coupled plasma mass spectrometry images (LA-ICP-MS) performed on UM sections showed increases in copper within the tumour periphery and intratumoural zinc in tissue from patients with poor prognosis. These preliminary data indicate that multi-modal MSI has the potential to provide insights into the role of trace metals and cancer metastasis.

Osteosarcoma (OS) is the most common primary bone malignancy and largely effects adolescents and young adults, with 60% of patients under the age of 25. There are multiple cell models of OS described in vitro that express the specific genetic alterations of the sarcoma. In the work reported here, multiple mass spectrometry imaging (MSI) modalities were employed to characterise two aggregated cellular models of OS models formed using the MG63 and SAOS-2 cell lines. Phenotyping of the metabolite activity within the two OS aggregoid models was achieved and a comparison of the metabolite data with OS human tissue samples revealed relevant fatty acid and phospholipid markers. Although, annotations of these species require MS/MS analysis for confident identification of the metabolites. From the putative assignments however, it was suggested that the MG63 aggregoids are an aggressive tumour model that exhibited metastatic-like potential. Alternatively, the SAOS-2 aggregoids are more mature osteoblast-like phenotype that expressed characteristics of cellular differentiation and bone development. It was determined the two OS aggregoid models shared similarities of metabolic behaviour with different regions of OS human tissues, specifically of the higher metastatic grade.

Despite being a critical molecule in the brain, mass spectrometry imaging (MSI) of cholesterol has been under-reported compared to other lipids due to the difficulty in ionizing the sterol molecule. In the present work, we have employed an on-tissue enzyme-assisted derivatization strategy to improve detection of cholesterol in brain tissue sections. We report distribution and levels of cholesterol across specific structures of the mouse brain, in a model of Niemann-Pick type C1 disease, and during brain development. MSI revealed that in the adult mouse, cholesterol is the highest in the pons and medulla and how its distribution changes during development. Cholesterol was significantly reduced in the corpus callosum and other brain regions in the Npc1 null mouse, confirming hypomyelination at the molecular level. Our study demonstrates the potential of MSI to the study of sterols in neuroscience.

In skin penetration studies, HPLC-MS/MS analysis on extracts of heat-separated epidermis and dermis provides an estimate of the amount of drug penetrated. In this study, MALDI-MSI enabled qualitative skin distribution analysis of endogenous molecules and the drug molecule, tofacitinib and quantitative analysis of the amount of tofacitinib in the epidermis. The delivery of tofacitinib to the skin was investigated in a Franz diffusion cell using three different formulations (two oil-in-water creams, C1 and C2 and an aqueous gel). Further, in vitro release testing (IVRT) was performed and resulted in the fastest release of tofacitinib from the aqueous gel and the lowest from C2. In the ex vivo skin penetration and permeation study, C1 showed the largest skin retention of tofacitinib, whereas, lower retention and higher permeation were observed for the gel and C2. The quantitative MALDI-MSI analysis showed that the content of tofacitinib in the epidermis for the C1 treated samples was comparable to HPLC-MS/MS analysis, whereas, the samples treated with C2 and the aqueous gel were below LOQ. The study demonstrates that MALDI-MSI can be used for the quantitative determination of drug penetration in epidermis, as well as, to provide valuable information on qualitative skin distribution of tofacitinib., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Methodology combining mass spectrometry imaging (MSI) with ion mobility separation (IMS) has emerged as a biological imaging technique due to its versatility, sensitivity and label-free approach. This technique has been shown to separate isomeric compounds such as lipids, amino acids, carboxylic acids and carbohydrates. This report describes mass spectrometry imaging in combination with traveling-wave ion mobility separation and matrix-assisted laser desorption/ionization (MALDI). Positive ionization mode was used to locate fructans on tissue printed sections of Agave rhizome and stem tissue and distinguished fructan isoforms. Here we show the location of fructans ranging from DP3 to DP17 to be differentially abundant across the stem tissue and for the first time, experimental collision cross sections of endogenous fructan structures have been collected, revealing at least two isoforms for fructans of DP4, DP5, DP6, DP7, DP8, DP10, and DP11. This demonstrates that complex fructans such as agavins can be located and their isoforms resolved using a combination of MALDI, IMS, and MSI, without the need for extraction or derivatization. Use of this methodology uncovered patterns of fructan localization consistent with functional differences where higher DP fructans are found toward the central section of the stem supporting a role in long term carbohydrate storage whereas lower DP fructans are concentrated in the highly vascularized central core of rhizomes supporting a role in mobilization of carbohydrates from the mother plant to developing offsets. Tissue specific patterns of expression of genes encoding enzymes involved in fructan metabolism are consistent with fructan structures and localization., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Pérez-López, Simpson, Clench, Gomez-Vargas and Ordaz-Ortiz.)

Rationale: Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is routinely employed to monitor the distribution of compounds in tissue sections and generate two-dimensional (2D) images. Whilst informative the images do not represent the distribution of the analyte of interest through the entire organ. The generation of 3D images is an exciting field that can provide a deeper view of the analyte of interest throughout an entire organ., Methods: Serial sections of mouse and rat lung tissue were obtained at 120 μm depth intervals and imaged individually. Homogenate registration markers were incorporated in order to aid the final 3D image construction. Using freely available software packages, the images were stacked together to generate a 3D image that showed the distribution of endogenous species throughout the lungs., Results: Preliminary tests were performed on 16 serial tissue sections of mouse lungs. A 3D model showing the distribution of phosphocholine at m/z 184.09 was constructed, which defined the external structure of the lungs and trachea. Later, a second experiment was performed using 24 serial tissue sections of the left lung of a rat. Two molecular markers, identified as [PC (32:1) + K] + at m/z 770.51 and [PC (36:4) + K] + at m/z 820.52, were used to generate 3D models of the parenchyma and airways, respectively., Conclusions: A straightforward method to generate 3D MALDI-MS images of selected molecules in lung tissue has been presented. Using freely available imaging software, the 3D distributions of molecules related to different anatomical features were determined., (© 2020 John Wiley & Sons, Ltd.)

We report for the first time label-free quantification of xenobiotic metabolizing enzymes (XME), transporters, redox enzymes, proteases, and nucleases in six human skin explants and a three-dimensional living skin equivalent model from LabSkin. We aimed to evaluate the suitability of LabSkin as an alternative to animal testing for the development of topical formulations. More than 2000 proteins were identified and quantified from total cellular protein. Alcohol dehydrogenase 1C, the most abundant phase I XME in human skin, and glutathione S-transferase pi 1, the most abundant phase II XME in human skin, were present in similar abundance in LabSkin. Several esterases were quantified and esterase activity was confirmed in LabSkin using substrate-based mass spectrometry imaging. No cytochrome P450 (P450) activity was observed for the substrates tested, in agreement with the proteomics data, where the cognate P450s were absent in both human skin and LabSkin. Label-free protein quantification allowed insights into other related processes such as redox homeostasis and proteolysis. For example, the most abundant antioxidant enzymes were thioredoxin and peroxiredoxin-1. This systematic determination of functional equivalence between human skin and LabSkin is a key step toward the construction of a representative human in vitro skin model, which can be used as an alternative to current animal-based tests for chemical safety and for predicting dosage of topically administered drugs. SIGNIFICANCE STATEMENT: The use of label-free quantitative mass spectrometry to elucidate the abundance of xenobiotic metabolizing enzymes, transporters, redox enzymes, proteases, and nucleases in human skin enhance our understanding of the skin physiology and biotransformation of topical drugs and cosmetics. This will help to develop mathematical models to predict drug metabolism in human skin and to develop more robust in vitro engineered human skin tissue as alternatives to animal testing., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

Rationale: Malignant pleural mesothelioma is an extremely aggressive and incurable malignancy associated with prior exposure to asbestos fibres. Difficulties remain in relation to early diagnosis, notably due to impeded identification of asbestos in lung tissue. This study describes a novel laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) imaging approach to identify asbestos within mesothelioma models with clinical significance., Methods: Human mesothelioma cells were exposed to different types of asbestos fibres and prepared on plastic slides for LA-ICP-MS analysis. No further sample preparation was required prior to analysis, which was performed using an NWR Image 266 nm laser ablation system coupled to an Element XR sector-field ICP mass spectrometer, with a lateral resolution of 2 μm. Data was processed using LA-ICP-MS ImageTool v1.7 with the final graphic production made using DPlot software., Results: Four different mineral fibres were successfully identified within the mesothelioma samples based on some of the most abundant elements that make up these fibres (Si, Mg and Fe). Using LA-ICP-MS as an imaging tool provided information on the spatial distribution of the fibres at cellular level, which is essential in asbestos detection within tissue samples. Based on the metal counts generated by the different types of asbestos, different fibres can be identified based on shape, size, and elemental composition. Detection of Ca was attempted but requires further optimisation., Conclusions: Detection of asbestos fibres in lung tissues is very useful, if not necessary, to complete the pathological dt9iagnosis of asbestos-related malignancies in the medicolegal field. For the first time, this study demonstrates the successful application of LA-ICP-MS imaging to identify asbestos fibres and other mineral fibres within mesothelioma samples. Ultimately, high-resolution, fast-speed LA-ICP-MS analysis has the potential to be integrated into clinical workflow to aid earlier detection and stratification of mesothelioma patient samples., (© 2020 The Authors. Rapid Communications in Mass Spectrometry published by John Wiley & Sons Ltd.)

Introduction : Three-dimensional (3D) cell cultures have become increasingly important materials to investigate biological processes and drug efficacy and toxicity. The ability of 3D cultures to mimic the physiology of primary tissues and organs in the human body enables further insight into cellular behavior and is hence highly desirable in early-stage drug development. Analyzing the spatial distribution of drug compounds and endogenous molecules provides an insight into the efficacy of a drug whilst simultaneously giving information on biological responses. Areas Covered : In this review we will examine the main 3D cell culture systems employed and applications, which describe their integration with mass spectrometry imaging (MSI). Expert Opinion : MSI is a powerful technique that can map a vast range of molecules simultaneously in tissues without the addition of labels that can provide insights into the efficacy and safety of a new drug. The combination of MSI and 3D cell cultures has emerged as a promising tool in early-stage drug analysis. However, the most common administration route for pharmaceutical drugs is via oral delivery. The use of MSI in combination with models of the GI tract is an area that has been little explored to date, the reasons for this are discussed.

The reliable identification of blood, as well as the determination of its origin (human or animal) is of great importance in a forensic investigation. Whilst presumptive tests are rapid and deployed in situ, their very nature requires confirmatory tests to be performed remotely. However, only serological tests can determine blood provenance. The present study improves on a previously devised Matrix Assisted Laser Desorption Ionisation Mass Spectrometry (MALDI MS)-proteomics based method for the reliable detection of blood by enabling the determination of blood provenance. The overall protocol was developed to be more specific than presumptive tests and faster/easier than the gold standard liquid chromatography (LC) MS/MS analysis. This is considered a pre-validation study that has investigated stains and fingermarks made in blood, other biofluids and substances that can elicit a false-positive response to colorimetric or presumptive tests, in a blind fashion. Stains and marks were either untreated or enhanced with a range of presumptive tests. Human and animal blood were correctly discriminated from other biofluids and non-biofluid related matrices; animal species determination was also possible within the system investigated. The procedure is compatible with the prior application of presumptive tests. The refined strategy resulting from iterative improvements through a trial and error study of 56 samples was applied to a final set of 13 blind samples. This final study yielded 12/13 correct identifications with the 13th sample being correctly identified as animal blood but with no species attribution. This body of work will contribute towards the validation of MALDI MS based methods and deployment in violent crimes involving bloodshed.

Mass spectrometry imaging (MSI) is an established analytical tool capable of defining and understanding complex tissues by determining the spatial distribution of biological molecules. Three-dimensional (3D) cell culture models mimic the pathophysiological environment of in vivo tumors and are rapidly emerging as a valuable research tool. Here, multimodal MSI techniques were employed to characterize a novel aggregated 3D lung adenocarcinoma model, developed by the group to mimic the in vivo tissue. Regions of tumor heterogeneity and the hypoxic microenvironment were observed based on the spatial distribution of a variety of endogenous molecules. Desorption electrospray ionization (DESI)-MSI defined regions of a hypoxic core and a proliferative outer layer from metabolite distribution. Targeted metabolites (e.g., lactate, glutamine, and citrate) were mapped to pathways of glycolysis and the TCA cycle demonstrating tumor metabolic behavior. The first application of imaging mass cytometry (IMC) with 3D cell culture enabled single-cell phenotyping at 1 μm spatial resolution. Protein markers of proliferation ( K i -67) and hypoxia (glucose transporter 1) defined metabolic signaling in the aggregoid model, which complemented the metabolite data. Laser ablation inductively coupled plasma (LA-ICP)-MSI analysis localized endogenous elements including magnesium and copper, further differentiating the hypoxia gradient and validating the protein expression. Obtaining a large amount of molecular information on a complementary nature enabled an in-depth understanding of the biological processes within the novel tumor model. Combining powerful imaging techniques to characterize the aggregated 3D culture highlighted a future methodology with potential applications in cancer research and drug development.

Three-dimensional (3D) cell culture is a rapidly emerging field, which mimics some of the physiological conditions of human tissues. In cancer biology, it is considered a useful tool in predicting in vivo chemotherapy responses, compared with conventional two-dimensional (2D) cell culture. We have developed a novel 3D cell culture model of osteosarcoma composed of aggregated proliferative tumour spheroids, which shows regions of tumour heterogeneity formed by aggregated spheroids of polyclonal tumour cells. Aggregated spheroids show local necrotic and apoptotic regions and have sizes suitable for the study of spatial distribution of metabolites by mass spectrometry imaging (MSI). We have used this model to perform a proof-of-principle study showing a heterogeneous distribution of endogenous metabolites that colocalise with the necrotic core and apoptotic regions in this model. Cytotoxic chemotherapy (doxorubicin) responses were significantly attenuated in our 3D cell culture model compared with those of standard cell culture, as determined by resazurin assay, despite sufficient doxorubicin diffusion demonstrated by localisation throughout the 3D constructs. Finally, changes to the distribution of endogenous metabolites in response to doxorubicin were readily detected by MSI. Principal component analysis identified 50 metabolites which differed most in their abundance between treatment groups, and of these, 10 were identified by both in-software t test and mixed-effects analysis of variance (ANOVA). Subsequent independent MSIs of identified species were consistent with principle component analysis findings. This proof-of-principle study shows for the first time that chemotherapy-induced changes in metabolite abundance and distribution may be determined in 3D cell culture by MSI, highlighting this method as a potentially useful tool in the elucidation of chemotherapy responses as an alternative to in vivo testing., (© 2019 John Wiley & Sons, Ltd.)

Dysregulated cholesterol metabolism is implicated in a number of neurological disorders. Many sterols, including cholesterol and its precursors and metabolites, are biologically active and important for proper brain function. However, spatial cholesterol metabolism in brain and the resulting sterol distributions are poorly defined. To better understand cholesterol metabolism in situ across the complex functional regions of brain, we have developed on-tissue enzyme-assisted derivatization in combination with microliquid extraction for surface analysis and liquid chromatography-mass spectrometry to locate sterols in tissue slices (10 µm) of mouse brain. The method provides sterolomic analysis at 400-µm spot diameter with a limit of quantification of 0.01 ng/mm 2 It overcomes the limitations of previous mass spectrometry imaging techniques in analysis of low-abundance and difficult-to-ionize sterol molecules, allowing isomer differentiation and structure identification. Here we demonstrate the spatial distribution and quantification of multiple sterols involved in cholesterol metabolic pathways in wild-type and cholesterol 24S-hydroxylase knockout mouse brain. The technology described provides a powerful tool for future studies of spatial cholesterol metabolism in healthy and diseased tissues., Competing Interests: Competing interest statement: Y.W. and W.J.G. are listed as inventors on the patent “Kit and method for quantitative detection of steroids” US9851368B2., (Copyright © 2020 the Author(s). Published by PNAS.)

Since the birth of proteomics science in the 1990, the number of applications and of sample preparation methods has grown exponentially, making a huge contribution to the knowledge in life science disciplines. Continuous improvements in the sample treatment strategies unlock and reveal the fine details of disease mechanisms, drug potency, and toxicity as well as enable new disciplines to be investigated such as forensic science.This chapter will cover the most recent developments in sample preparation strategies for tissue proteomics in three areas, namely, cancer, toxicology, and forensics, thus also demonstrating breath of application within the domain of health and well-being, pharmaceuticals, and secure societies.In particular, in the area of cancer (human tumor biomarkers), the most efficient and multi-informative proteomic strategies will be covered in relation to the subsequent application of matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) and liquid extraction surface analysis (LESA), due to their ability to provide molecular localization of tumor biomarkers albeit with different spatial resolution.With respect to toxicology, methodologies applied in toxicoproteomics will be illustrated with examples from its use in two important areas: the study of drug-induced liver injury (DILI) and studies of effects of chemical and environmental insults on skin, i.e., the effects of irritants, sensitizers, and ionizing radiation. Within this chapter, mainly tissue proteomics sample preparation methods for LC-MS/MS analysis will be discussed as (i) the use of LC-MS/MS is majorly represented in the research efforts of the bioanalytical community in this area and (ii) LC-MS/MS still is the gold standard for quantification studies.Finally, the use of proteomics will also be discussed in forensic science with respect to the information that can be recovered from blood and fingerprint evidence which are commonly encountered at the scene of the crime. The application of proteomic strategies for the analysis of blood and fingerprints is novel and proteomic preparation methods will be reported in relation to the subsequent use of mass spectrometry without any hyphenation. While generally yielding more information, hyphenated methods are often more laborious and time-consuming; since forensic investigations need quick turnaround, without compromising validity of the information, the prospect to develop methods for the application of quick forensic mass spectrometry techniques such as MALDI-MS (in imaging or profiling mode) is of great interest.

The determination of the type of deposition mechanism of blood within fingermarks at the scene of violent crimes is of great importance for the reconstruction of the bloodshed dynamics. However, to date, evaluation still relies on the subjective visual examination of experts. Practitioners encounter three types of scenarios in which blood may be found in fingermarks and they refer to the following three deposition mechanisms: (i) blood marks, originating from a bloodied fingertip; (ii) marks in blood, originating from a clean fingertip contacting a blood contaminated surface; (iii) coincidental deposition mechanisms, originating from a clean fingertip contacting a clean surface, leaving a latent fingermark, and subsequent contamination with blood. The authors hypothesised that, due to differences in distribution of blood in the furrows and on the ridges, the height of blood depositions on the ridges and furrows (and their relative proportions), will differ significantly across the three depositions mechanisms. A second hypothesis was made that the differences would be significant and consistent enough to exploit their measurement as a quantitative and objective way to differentiate the deposition mechanisms. In recent years, infinite focus microscopy (IFM) has been developed, allowing for the computational generation of a 3D image of the topology of a sample via acquisition of images on multiple focal planes. On these bases, it was finally hypothesised that the application of this technique would allow the distinction of deposition mechanisms (i) to (iii). A set of preliminary experiments were designed to test whether IFM was "fit for purpose" and, subsequently, to test if any of the three deposition mechanisms scenarios could be differentiated. Though IFM enabled the analysis of tape lifted samples with some success, for samples produced and analysed directly on the surface of deposition, the results show that the measurements from any scenario will be highly dependent on the original surface of deposition (both in terms of its nature and of the variable exposure to environment); as crime scenes exhibit a wide range of possible relevant surfaces of deposition, the technique showed to not have the desired wide appeal for inclusion into a standardised set of protocols within a routine crime scene workflow., (Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.)

The combination of microspotting of analytical and internal standards, matrix sublimation, and recently developed software for quantitative mass spectrometry imaging has been used to develop a high-resolution method for the determination of terbinafine hydrochloride in the epidermal region of a full thickness living skin equivalent model. A quantitative assessment of the effect of the addition of the penetration enhancer (dimethyl isosorbide (DMI)) to the delivery vehicle has also been performed, and data have been compared to those obtained from LC-MS/MS measurements of homogenates of isolated epidermal tissue. At 10% DMI, the levels of signal detected for the drug in the epidermis were 0.20 ± 0.072 mg/g tissue for QMSI and 0.28 ± 0.040 mg/g tissue for LC-MS/MS at 50% DMI 0.69 ± 0.23 mg/g tissue for QMSI and 0.66 ± 0.057 mg/g tissue for LC-MS/MS. Comparison of means and standard deviations indicates no significant difference between the values obtained by the two methods.

A 3D cell culture is an artificially created environment in which cells are permitted to grow/interact with their surroundings in all three dimensions. Derived from 3D cell culture, organoids are generally small-scale constructs of cells that are fabricated in the laboratory to serve as 3D representations of in vivo tissues and organs. Due to regulatory, economic and societal issues concerning the use of animals in scientific research, it seems clear that the use of 3D cell culture and organoids in for example early stage studies of drug efficacy and toxicity will increase. The combination of such 3D tissue models with mass spectrometry imaging provides a label-free methodology for the study of drug absorption/penetration, drug efficacy/toxicity, and drug biotransformation. In this article, some of the successes achieved to date and challenges to be overcome before this methodology is more widely adopted are discussed., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Described is a quantitative-mass-spectrometry-imaging (qMSI) methodology for the analysis of lactate and glutamate distributions in order to delineate heterogeneity among mouse tumor models used to support drug-discovery efficacy testing. We evaluate and report on preanalysis-stabilization methods aimed at improving the reproducibility and efficiency of quantitative assessments of endogenous molecules in tissues. Stability experiments demonstrate that optimum stabilization protocols consist of frozen-tissue embedding, post-tissue-sectioning desiccation, and storage at -80 °C of tissue sections sealed in vacuum-tight containers. Optimized stabilization protocols are used in combination with qMSI methodology for the absolute quantitation of lactate and glutamate in tumors, incorporating the use of two different stable-isotope-labeled versions of each analyte and spectral-clustering performed on each tissue section using k-means clustering to allow region-specific, pixel-by-pixel quantitation. Region-specific qMSI was used to screen different tumor models and identify a phenotype that has low lactate heterogeneity, which will enable accurate measurements of lactate modulation in future drug-discovery studies. We conclude that using optimized qMSI protocols, it is possible to quantify endogenous metabolites within tumors, and region-specific quantitation can provide valuable insight into tissue heterogeneity and the tumor microenvironment.

Objective: Examination of the skin barrier repair/wound healing process using a living skin equivalent (LSE) model and matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) to identify lipids directly involved as potential biomarkers. These biomarkers may be used to determine whether an in vivo wound is going to heal for example if infected., Methods: An in vitro LSE model was wounded with a scalpel blade and assessed at day 4 post-wounding by histology and MALDI-MSI. Samples were sectioned at wound site and were either formalin-fixed paraffin-embedded (FFPE) for histology or snapped frozen (FF) for MSI analysis., Results: The combination of using an in vitro wounded skin model with MSI allowed the identification of lipids involved in the skin barrier repair/wound healing process. The technique was able to highlight lipids directly in the wound site and distinguish differences in lipid distribution between the epidermis and wound site., Conclusion: This novel method of coupling an in vitro LSE with MSI allowed in-depth molecular analysis of the skin barrier repair/wound healing process. The technique allowed the identification of lipids directly involved in the skin barrier repair/wound healing process, indicating these biomarkers may be potentially be used within the clinic. These biomarkers will help to determine, which stage of the skin barrier repair/wound healing process the wound is in to provide the best treatment., (© 2018 Society of Cosmetic Scientists and the Société Française de Cosmétologie.)

Numerous studies have indicated that yellow semen syndrome (YSS) of turkey is associated with the production of low semen quality, resulting in reduced fertility and hatchability. It is unknown at present if the etiology of YSS also could be linked to low-molecular weight metabolites. The aim of this study was to examine the metabolome of white and yellow seminal plasma of turkeys. Two different metabolomics approaches, shotgun (direct infusion) and liquid chromatography-mass spectrometry (LC-MS), were employed to identify metabolites differentially abundant in yellow seminal plasma. Significant changes in the levels of 1549 and 2093 metabolites were detected in yellow vs. white seminal plasma using shotgun and LC-MS, respectively. Of these, 354 metabolites (189 increased and 165 decreased) after shotgun and 936 metabolites (363 increased and 573 decreased) after LC-MS were putatively identified using the Human Metabolome Database. Significantly differentiated metabolites were subjected to Ingenuity Pathway Analysis. Lipid metabolism, molecular transport, and nucleic acid metabolism were the top pathways that differentiated white and yellow seminal plasma. These data strongly suggest that disturbance of carbohydrate and lipid metabolism is characteristic for YSS. The abnormal metabolism of lipids may contribute to the numerous lipid vacuoles previously observed in the reproductive tracts of YSS males. An increased level of riboflavin in YSS may be responsible for yellow turkey semen pigmentation. A disturbance in thyroid hormone metabolism visible at protein and metabolic levels may be involved in YSS in turkey. The low quality of YSS may be linked with the presence of drug residues in the reproductive tract.

Introduction: The distinction of papillary thyroid carcinomas from benign thyroid lesions has important implication for clinical man-agement. Classification based on histopathological features can be supported by molecular biomarkers, including lipidomic signatures, identified with the use of high-throughput mass spectrometry techniques. Formalin fixation is a standard procedure for stabilization and preservation of tissue samples, therefore this type of samples constitute highly valuable source of clinical material for retrospective molecular studies. In this study we used mass spectrometry imaging to detect lipids discriminating papillary cancer from not cancerous thyroid directly in formalin-fixed tissue sections., Material and Methods: For this purpose imaging and profiling of lipids present in non-malignant and cancerous thyroid tissue specimens were conducted. High resolution MALDI-Q-Ion Mobility-TOF-MS technique was used for lipidomic analysis of formalin fixed thyroid tissue samples. Lipids were identified by the comparison of the exact molecular masses and fragmentation pathways of the protonated molecule ions, recorded during the MS/MS experiments, with LIPID MAPS database., Results: Several phosphatidylcholines (32:0, 32:1, 34:1 and 36:3), sphingomyelins (34:1 and 36:1) and phosphatidic acids (36:2 and 36:3) were detected and their abundances were significantly higher in cancerous tissue compared to non-cancerous tissue. The same lipid species were detected in formalin-fixed as in fresh-frozen tissue, but [M + Na]+ ions were the most abundant in formalin fixed whereas [M + K]+ ions were predominant in fresh tissue., Conclusions: Our results prove the viability of MALDI-MSI for analysis of lipid distribution directly in formalin-fixed tissue, and the potential for their use in the classification of thyroid diseases.

Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) has been employed to rapidly screen longitudinally sectioned drug user hair samples for cocaine and its metabolites using continuous raster imaging. Optimization of the spatial resolution and raster speed were performed on intact cocaine contaminated hair samples. The optimized settings (100 × 150 μm at 0.24 mm/s) were subsequently used to examine longitudinally sectioned drug user hair samples. The MALDI-MS/MS images showed the distribution of the most abundant cocaine product ion at m/z 182. Using the optimized settings, multiple hair samples obtained from two users were analyzed in approximately 3 h: six times faster than the standard spot-to-spot acquisition method. Quantitation was achieved using longitudinally sectioned control hair samples sprayed with a cocaine dilution series. A multiple reaction monitoring (MRM) experiment was also performed using the 'dynamic pixel' imaging method to screen for cocaine and a range of its metabolites, in order to differentiate between contaminated hairs and drug users. Cocaine, benzoylecgonine, and cocaethylene were detectable, in agreement with analyses carried out using the standard LC-MS/MS method. Graphical Abstract ᅟ.

MALDI-mass spectrometry imaging (MALDI-MSI) has been shown to allow the study of protein distribution and identification directly within formalin-fixed paraffin-embedded (FFPE) tissue sections. However, direct protein identification from tissue sections remains challenging due to signal interferences and/or existing post-translational or other chemical modifications. The use of antigen retrieval (AR) has been demonstrated for unlocking proteins prior to in situ enzymatic digestion and MALDI-MSI analysis of FFPE tissue sections. In the work reported here, the identification of proline oxidation, which may occur when performing the AR protocol, is described. This facilitated and considerably increased the number of identified peptides when adding proline oxidation as a variable modification to the MASCOT search criteria. This article is part of a Special Issue entitled: MALDI Imaging, edited by Dr. Corinna Henkel and Prof. Peter Hoffmann., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Rationale: The incubation of CPAQOP (1-[(2R)-2-[[4-[3-chloro-4-(2-pyridyloxy)anilino]quinazolin-5-yl]oxymethyl]-1-piperidyl]-2-hydroxy) with human liver microsomes generated several metabolites that highlighted the hydroxyacetamide side chain was a major site of metabolism for the molecule. The metabolites were derived predominantly from oxidative biotransformations; however, two unexpected products were detected by liquid chromatography/ultraviolet/mass spectrometry (LC/UV/MS) and identified as methanol adducts. This observation prompted further LC/MS investigations into their formation., Methods: Three separate incubations of CPAQOP were conducted in human liver microsomes; Naïve, fortified with methoxyamine and fortified with glutathione. Separation was achieved via ultra-high-performance liquid chromatography with either methanol or acetonitrile gradients containing formic acid. MS analysis was conducted by electrospray ionisation LTQ Orbitrap mass spectrometry acquiring accurate mass full scan, data-dependent MS 2 and all ion fragmentation., Results: No methanol adducts were detected by MS when acetonitrile was used in the mobile phase instead of methanol, verifying that a metabolite was reacting with methanol on column. Although this reactive metabolite could not be isolated or structurally characterised by LC/MS directly, product ion spectra of the methanol adducts confirmed addition of methanol on the hydroxyacetamide side chain. Additional experiments using methoxyamine showed the disappearance of the two methanol adducts and appearance of a methoxyamine adduct, confirming the presence of an aldhyde. Product ion spectra of the methoxyamine adduct confirmed addition of methoxyamine to the hydroxyacetamide side chain., Conclusions: The proposed bioactivation of CPAQOP occurred via the reactive aldehyde intermediate, which readily reacted with methanol in the mobile phase to form a pair of isomeric hemiacetal methanol adducts. In acidified methanol the equilibrium favoured the methanol adduct and in acidified acetonitrile it favoured the hydrate; therefore, the reactive aldehyde metabolite was not detected and could not be structurally characterised directly. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) is now a well-established technique for imaging analysis of sectioned biological tissues. One of the growing areas of interest is in the analysis of skin. MALDI-MSI can provide a wealth of information from within sections of skin. This includes information on the distribution of pharmaceuticals following topical treatments, through to the examination of the composition of different skin layers and studies of proteomic, lipidomic, and metabolomic responses to disease, wounds, and external stimuli. Here, we describe the handling procedures, preparatory treatment, and mass spectrometry setup required for the MALDI MSI analysis of lipids within human skin samples.

Liquid extraction surface analysis mass spectrometry imaging (LESA-MSI) has been shown to be an effective tissue profiling and imaging technique, producing robust and reliable qualitative distribution images of an analyte or analytes in tissue sections. Here, we expand the use of LESA-MSI beyond qualitative analysis to a quantitative analytical technique by employing a mimetic tissue model previously shown to be applicable for MALDI-MSI quantitation. Liver homogenate was used to generate a viable and molecularly relevant control matrix for spiked drug standards which can be frozen, sectioned and subsequently analyzed for the generation of calibration curves to quantify unknown tissue section samples. The effects of extraction solvent composition, tissue thickness and solvent/tissue contact time were explored prior to any quantitative studies in order to optimize the LESA-MSI method across several different chemical entities. The use of a internal standard to normalize regional differences in ionization response across tissue sections was also investigated. Data are presented comparing quantitative results generated by LESA-MSI to LC-MS/MS. Subsequent analysis of adjacent tissue sections using DESI-MSI is also reported.

Mass spectrometry imaging (MSI) is a powerful tool for the study of intact tissue sections. The use of matrix-assisted laser desorption/ionisation (MALDI) MSI for the study of the distribution and effect of emollient treatment on sections of reconstructed living skin equivalents during their development and maturation is described. Living skin equivalent (LSE) samples were obtained at 14days development, re-suspended in maintenance medium and incubated for 24h after delivery. The medium was changed, the LSE treated with either Physiogel A.I.® or Oilatum Junior® emollients and then re-incubated and samples taken at 4, 6 and 24h time points. Mass spectra and mass spectral images were recorded from 12μm sections of the LSE taken at each time point for comparison using MALDI mass spectrometry (MS). It was possible to detect ions characteristic of each emollient in the LSE. In addition a number of lipid species previously reported as being significant in the maturation of the LSE were observable. At the 24h time point, the images revealed what appeared to be differences in the organisation of the skin cells observed across the Physiogel A.I.® treatment group tissue sections when directly compared to the untreated tissue group., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Arginine is an important amino acid but has been barely studied in plants. The little research that has been done indicates that the pathways of synthesis are similar to those found in animals and procaryotes. However little is known about the cellular and tissue localization of the amino acid in plants. The research reported in this paper was designed to examine whether MALDI-MSI was sufficiently sensitive to examine the distribution of this amino acid in plant material, and whether the synthetic pathways were co-located. In wheat and orchid roots, the amount of arginine in tissues varies greatly and the pathways for its synthesis were not always detected with the amino acid., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A bottom up in situ proteomic method has been developed enabling the mapping of multiple blood signatures on the intact ridges of blood fingermarks by Matrix Assisted Laser Desorption Mass Spectrometry Imaging (MALDI-MSI). This method, at a proof of concept stage, builds upon recently published work demonstrating the opportunity to profile and identify multiple blood signatures in bloodstains via a bottom up proteomic approach. The present protocol addresses the limitation of the previously developed profiling method with respect to destructivity; destructivity should be avoided for evidence such as blood fingermarks, where the ridge detail must be preserved in order to provide the associative link between the biometric information and the events of bloodshed. Using a blood mark reference model, trypsin concentration and spraying conditions have been optimised within the technical constraints of the depositor eventually employed; the application of MALDI-MSI and Ion Mobility MS have enabled the detection, confirmation and visualisation of blood signatures directly onto the ridge pattern. These results are to be considered a first insight into a method eventually informing investigations (and judicial debates) of violent crimes in which the reliable and non-destructive detection and mapping of blood in fingermarks is paramount to reconstruct the events of bloodshed., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

MALDI-MS Imaging is a novel label-free technique that can be used to visualize the changes in multiple mass responses following treatment. Following treatment with proinflammatory cytokine interleukin-22 (IL-22), the epidermal differentiation of Labskin, a living skin equivalent (LSE), successfully modeled psoriasis in vitro. Masson's trichrome staining enabled visualization and quantification of epidermal differentiation between the untreated and IL-22 treated psoriatic LSEs. Matrix-assisted laser desorption ionization mass spectrometry imaging was used to observe the spatial location of the psoriatic therapy drug acetretin following 48 h treatments within both psoriatic and normal LSEs. After 24 h, the drug was primarily located in the epidermal regions of both the psoriatic and nonpsoriatic LSE models whereas after 48 h it was detectible in the dermis., (© 2016 The Authors. Proteomics Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Blood evidence is frequently encountered at the scene of violent crimes and can provide valuable intelligence in the forensic investigation of serious offences. Because many of the current enhancement methods used by crime scene investigators are presumptive, the visualisation of blood is not always reliable nor does it bear additional information. In the work presented here, two methods employing a shotgun bottom up proteomic approach for the detection of blood are reported; the developed protocols employ both an in solution digestion method and a recently proposed procedure involving immobilization of trypsin on hydrophobin Vmh2 coated MALDI sample plate. The methods are complementary as whilst one yields more identifiable proteins (as biomolecular signatures), the other is extremely rapid (5 minutes). Additionally, data demonstrate the opportunity to discriminate blood provenance even when two different blood sources are present in a mixture. This approach is also suitable for old bloodstains which had been previously chemically enhanced, as experiments conducted on a 9-year-old bloodstain deposited on a ceramic tile demonstrate.

Liquid extraction surface analysis mass spectrometry (LESA-MS) is a surface sampling technique that incorporates liquid extraction from the surface of tissue sections with nanoelectrospray mass spectrometry. Traditional tissue analysis techniques usually require homogenization of the sample prior to analysis via high-performance liquid chromatography mass spectrometry (HPLC-MS), but an intrinsic weakness of this is a loss of all spatial information and the inability of the technique to distinguish between actual tissue penetration and response caused by residual blood contamination. LESA-MS, in contrast, has the ability to spatially resolve drug distributions and has historically been used to profile discrete spots on the surface of tissue sections. Here, we use the technique as a mass spectrometry imaging (MSI) tool, extracting points at 1 mm spatial resolution across tissue sections to build an image of xenobiotic and endogenous compound distribution to assess drug blood-brain barrier penetration into brain tissue. A selection of penetrant and "nonpenetrant" drugs were dosed to rats via oral and intravenous administration. Whole brains were snap-frozen at necropsy and were subsequently sectioned prior to analysis by matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) and LESA-MSI. MALDI-MSI, as expected, was shown to effectively map the distribution of brain penetrative compounds but lacked sufficient sensitivity when compounds were marginally penetrative. LESA-MSI was used to effectively map the distribution of these poorly penetrative compounds, highlighting its value as a complementary technique to MALDI-MSI. The technique also showed benefits when compared to traditional homogenization, particularly for drugs that were considered nonpenetrant by homogenization but were shown to have a measurable penetration using LESA-MSI.

Background: Mass spectrometry imaging (MSI) is a powerful tool for the study of intact tissue sections. Here, its application to the study of the distribution of lipids in sections of reconstructed living skin equivalents during their development and maturation is described., Methods: Living skin equivalent (LSE) samples were obtained at 14 days development, re-suspended in maintenance medium and incubated for 24 h after delivery. The medium was then changed, the LSE re-incubated and samples taken at 4, 6 and 24 h time points. Mass spectra and mass spectral images were recorded from 12 μm sections of the LSE taken at each time point for comparison using matrix assisted laser desorption ionisation mass spectrometry., Results: A large number of lipid species were identified in the LSE via accurate mass-measurement MS and MSMS experiments carried out directly on the tissue sections. MS images acquired at a spatial resolution of 50 μm × 50 μm showed the distribution of identified lipids within the developing LSE and changes in their distribution with time. In particular development of an epidermal layer was observable as a compaction of the distribution of phosphatidylcholine species., Conclusions: MSI can be used to study changes in lipid composition in LSE. Determination of the changes in lipid distribution during the maturation of the LSE will assist in the identification of treatment responses in future investigations.

Rationale: 5,6-Dimethylxanthenone-4-acetic acid (DMXAA) is a low molecular weight drug of the flavonoid group, which has an anti-vascular effect in tumours causing endothelial cell apoptosis and activation of cytokines. Flavonoid-based compounds have been reported to lead to an upregulation in the expression of lysophosphatidylcholines (LPC)-type lipids in solid tumours. A study employing TLC/MALDI-MS and MALDI-MS imaging to examine LS174T colorectal adenocarcinoma xenografts following administration of DMXAA has been conducted into this effect., Methods: LS174T colorectal adenocarcinoma xenografts grown in male immune-deficient mice were treated with 27.5 mg/kg DMXAA. The control (before treatment) and 4 h and 24 h post-treatment tumours were excised and divided into two. MALDI-MS imaging experiments were carried out on 12 µm cryosections sections taken from one half of the tumours and from the other half the lipids were extracted and analysed by TLC/MALDI-MS. These experiments were carried out in triplicate., Results: Statistical analysis of the MALDI-MS imaging data set indicated an increased amount of LPC in the 24 h post-treated sample and a decreased amount of PC in the 24 h post-treated sample, compared with the 4 h post-treated sample and the control. These effects were confirmed by the TLC/MALDI-MS data. The lipid extracts were separated into six spots on the TLC plate. These were identified as arising from different lipids classes, i.e. LPC, sphingomyelins (SM), phosphatidylcholines (PC) and phosphatidylethanolamines (PE). The TLC/MALDI-MS data indicated that LPC were highly expressed in the 4 h and 24 h post-treated tumour samples compared with the control. Examination of the mass spectrometric images confirms this increase and demonstrates additionally that the increase in the signals arising from LPC appears to be localised primarily within the central areas of the xenograft., Conclusions: An increase in expression of LPC lipids in solid tumours treated with DMXAA has been demonstrated and shown to be localised in the central area of the tumour., (Copyright © 2015 John Wiley & Sons, Ltd.)

Despite recent improvements to in situ proteolysis strategies, a higher efficiency is still needed to increase both the number of peptides detected and the associated ion intensity, leading to a complete and reliable set of biomarkers for diagnostic or prognostic purposes. In the study presented here, an extract of a systematic study is illustrated investigating a range of surfactants assisting trypsin proteolytic activity. Method development was trialled on fingermarks; this specimen results from a transfer of sweat from an individual's fingertip to a surface upon contact. As sweat carries a plethora of biomolecules, including peptides and proteins, fingermarks are, potentially, a very valuable specimen for non-invasive prognostic or diagnostic screening. A recent study has demonstrated the opportunity to quickly detect peptides and small proteins in fingermarks using Matrix Assisted Laser Desorption Ionization Mass Spectrometry Profiling (MALDI MSP). However, intact detection bears low sensitivity and does not allow species identification; therefore, a shotgun proteomic approach was employed involving in situ proteolysis. Data demonstrate that in fingermarks, further improvements to the existing method can be achieved using MEGA-8 as surfactant in higher percentages as well as combinations of different detergents. Also, for the first time, Rapigest SF, normally used in solution digestions, has been shown to successfully work also for in situ proteolysis.

Over the last decade, MALDI-MS imaging has been used by researchers to explore areas of proteomics, lipidomics and metabolomics in samples of clinical origin for both targeted and global biomarker analysis. Numerous technological advancements in MS and clinical tissue MS imaging have been accomplished; hence, in this article we aim to critically discuss whether MS imaging has now in fact become a true champion of the 'Omics Era'. In order to assess the potential for it to be routinely used in the clinical setting, it is pertinent to discuss some of its limitations, and to examine how these have been addressed by researchers. The key limitations of the technique we will discuss in this viewpoint article are as follows: sample throughput; relevance to patients, the availability of validated/standardised techniques; and integration with conventional pathology and other medical imaging techniques. Good progress has been made over the last 5 years in overcoming these limitations that had previously restricted the use of this technology in the clinical setting., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Hydrazine-based derivatization reagents have been used to detect the presence of the carbonyl containing glucocorticoid fluticasone proprionate in rat lung tissue by MALDI-MSI. Such reagents also act as a matrix for analysis by MALDI-MS and have been termed "reactive matrices". Cryosections of rat lung tissue (12 μm), spotted with a range of concentrations of fluticasone proprionate, were derivatized in situ with 2,4-dinitrophenylhydrazine (DNPH) and 4-dimethylamino-6-(4-methoxy-1-naphthyl)-1,3,5-triazine-2-hydrazine (DMNTH) by the use of an acoustic reagent spotter. It has been demonstrated that DMNTH gave superior results compared to DNPH and that analysis of samples immediately after application of DMNTH resulted in the detection of the protonated hydrazone derivative ([MD + H](+)) of fluticasone propionate at a concentration of 500 ng/μL. It has been further shown that a prolonged reaction time (~48 h) improves the detection limit of the protonated hydrazone derivative to 50 ng/μL and that improvements in sensitivity and limits of detection are obtained when a conventional MALDI matrix CHCA is employed in conjunction with the DNPH/DMNTH reactive matrix.

Pharmacodynamics and toxicodynamics are the study of the biochemical and physiological effects of therapeutic agents and toxicants and their mechanisms of action. MALDI-MS imaging offers great potential for the study of pharmaco/toxicodynamic responses in tissue owing is its ability to study multiple biomarkers simultaneously in a label-free manner. Here, existing examples of such studies examining anticancer drugs and topically applied treatments are described. Examination of the literature shows that the use of MS imaging in pharmaco/toxicodynamic studies is in fact quite low. The reasons for this are discussed and potential developments in the methodology that might lead to its further use are described.

MS imaging allows profiling and imaging of compounds directly from tumor tissue, tissue microarrays and tissue-engineered models of tumors. Methodologies for the quantitative analysis of localized/colocalized ion signals from a single cancer cell would be a major advance. Alternative methods of generating ions to matrix-assisted laser desorption ionization are increasingly employed. Desorption electrospray ionization has been used for the intraoperative diagnosis of human brain tumors and secondary ion MS imaging with cluster primary ion sources has been used for high spatial resolution imaging tumor sections. Extensive validation of the technique for the analysis of disease biomarkers is required, if imaging MS is to have a future role in the clinic.