Analytical and Bioanalytical Chemistry (v.407, #8)

Current trends in mass spectrometry imaging by Andreas Römpp; Uwe Karst (2023-2025).
is a group leader and lecturer (“Privatdozent”) at the Institute of Inorganic and Analytical Chemistry of Justus Liebig University in Giessen, Germany. He worked as a research assistant at the Max Planck Institute for Chemistry and obtained his PhD from the University of Mainz in 2003. In 2004 he worked as a postdoctoral researcher at the FOM-AMOLF Institute in Amsterdam. His work has always been focused on method development for high resolution mass spectrometry. In recent years mass spectrometry imaging has become his main area of interest with a particular focus on increasing spatial resolution and obtaining reliable chemical information. Andreas Römpp is coordinator of the common data format for mass spectrometry imaging—imzML. He is also actively involved in the MS imaging COST Action BM1104 as management committee member and work group co-chair. obtained his Ph.D. in analytical chemistry from the University of Münster, Germany, in 1993, and was a Postdoctoral Research Associate at the University of Colorado in Boulder, CO, USA. After his return to the University of Münster, he finished his Habilitation in 1998. In 2001, he took over a position as Full Professor of Chemical Analysis at the University of Twente, the Netherlands. He accepted his current position as Chair of Analytical Chemistry at the University of Münster in 2005. His research interests focus on hyphenated analytical techniques and their (bio)medical and pharmaceutical applications, including elemental speciation analysis, metallomics, mass spectrometric imaging, and electrochemistry/MS.

A public repository for mass spectrometry imaging data by Andreas Römpp; Rui Wang; Juan Pablo Albar; Andrea Urbani; Henning Hermjakob; Bernhard Spengler; Juan Antonio Vizcaíno (2027-2033).
is a group leader and lecturer (Privatdozent) at the Institute of Inorganic and Analytical Chemistry of Justus Liebig University in Giessen, Germany. His work has always been focused on method development for high-resolution mass spectrometry. In recent years, mass spectrometry imaging has become his main area of interest. He is the coordinator of the common data format for mass spectrometry imaging—imzML. He is also actively involved in COST Action BM1104 as a management committee member and workgroup co-chair. works as a senior software engineer in the Proteomics Services Team at the European Bioinformatics Institute of the European Molecular Biology Laboratory. He has special interests in big data, data visualization, data analysis, enterprise application architecture, and bioinformatics. Previously, he has worked as a software engineer at a pharmaceutical start-up and at IBM. obtained a degree in chemistry from Complutense University of Madrid, where he obtained his Ph.D. degree in 1981. After several years working in the private sector, he started his scientific activity at the National Centre for Biotechnology (CSIC), becoming one of the pioneers of proteomics in Spain. He was Head of the CSIC Proteomics Facility, Biomolecular and Bioinformatics Resources Platform Coordinator, a member of the Human Proteome Organization Council, and a member of the Chromosome-Based Human Proteome Project Executive Committee. His work has been published in more than 160 scientific articles and was a source of inspiration for many scientists in the field. He passed away in July 2014 at the age of 61. is Head of the Proteomics and Metabonomics Laboratory at IRCCS – Fondazione Santa Lucia, Rome. He is Associate Professor in Clinical Biochemistry and Molecular Biology, and Faculty Chair at the University of Rome “Tor Vergata.” Since 2009, he has been organizing the National Congress of the Italian Proteomics Association ( http://www.itpa.it ) as President of the Italian Proteomics Association for 2009–2015, and he will be European Proteomics Association President for 2015–2017. Since he was awarded his Ph.D. degree in 1998, he has authored over 100 articles in the field of proteomics and metabonomics. leads the Proteomics Services Team at the European Bioinformatics Institute, providing a broad portfolio of resources for systems biology, ranging from protein expression (PRIDE) via molecular interactions (IntAct) and curated pathways (Reactome) to systems biology models (BioModels) at the highest level of abstraction. As a founding member and co-chair of the Human Proteome Organization Proteomics Standards Initiative, and as a senior editor of the journal Proteomics, he contributes to the standardization of data representation in proteomics. is Full Professor of Analytical Chemistry at Justus Liebig University in Giessen, Germany. He contributed to the development of matrix-assisted laser desorption ionization mass spectrometry in the 1980s, developed the postsource decay technique, and in 1994 introduced the matrix-assisted laser desorption ionization imaging method. He and his group have worked in the field of mass spectrometry imaging for many years, in several fields of atmospheric pressure in situ mass spectrometry techniques, and in aerosol analysis. is the project leader of the PRIDE database of mass spectrometry proteomics data at the European Bioinformatics Institute of the European Molecular Biology Laboratory (Cambridge, UK). He has also managed the ProteomeXchange consortium of proteomics resources since its inception in 2011. He has degrees in pharmacy and biochemistry and a Ph.D. degree in molecular biology from the University of Salamanca (Spain). In the last few years, he has been involved in the development of PRIDE and its related tools and several data standards (e.g., mzIdentML and mzTab), as part of his contribution to the Human Proteome Organization Proteomics Standards Initiative.

Discussion point: reporting guidelines for mass spectrometry imaging by Liam A. McDonnell; Andreas Römpp; Benjamin Balluff; Ron M. A. Heeren; Juan Pablo Albar; Per E. Andrén; Garry L. Corthals; Axel Walch; Markus Stoeckli (2035-2045).
is Director of Proteomics at the Fondazione Pisana per la Scienza ONLUS (Pisa, Italy) and Associate Professor at Leiden University Medical Center (Leiden, the Netherlands). He is chair of the European Imaging MS network COST Action BM1104 and former chair of the Mass Spectrometry Imaging interest group of the American Society for Mass Spectrometry. His research focus is the interface of fundamental mass spectrometry with clinical research, for which interdisciplinarity is key. is group leader and lecturer (“Privatdozent”) at the Institute of Inorganic and Analytical Chemistry of Justus Liebig University in Giessen, Germany. His work has always been focused on method development for high resolution mass spectrometry. In recent years mass spectrometry imaging has become his main area of interest. He is coordinator of the common data format for mass spectrometry imaging—imzML. He is also actively involved in the COST action BM1104 as management committee member and work group co-chair. received training in bioinformatics at the Weihenstephan University of Applies Sciences, Germany. During his PhD at the Institute of Pathology of the Helmholtz Zentrum München, Germany, he obtained experience in the application of mass spectrometry imaging (MSI) in pathology research of gastric cancer. As a post-doc at the Center of Proteomics and Metabolomics, Leiden University Medical Center, he focused on the computational analysis of MSI data sets from large patient cohorts by establishing novel algorithms for image segmentation, coregistration, and correlation of the results with clinical data. is a distinguished Professor of molecular imaging and Limburg chair at the University of Maastricht. He is the Director of the Maastricht MultiModal Molecular Imaging institute M4I. Previously, he headed the biomolecular imaging mass spectrometry group at FOM AMOLF that has now been incorporated into M4I. His research interests encompass fundamental, instrumentation, and applied research in multimodal imaging mass spectrometry relevant to industry and society. He is an active MS community member, evidenced by his positions as treasurer of the International MS Foundation, Dutch national representative in COST action BM1104 as well as advisory board memberships of several companies and academic institutions. (1953–2014) was one of the leading functional proteomics researchers in Spain and Europe. His research at the Spanish National Biotechnology Centre (CNB) focused on drawing a complete map of protein dynamics, interactions, and posttranslational modifications that take place in the cell and he was one of the driving forces behind the Human Proteome Project. Since 2005 he was the coordinator of ProteoRed, the Spain Proteomics Network, that formed the model on which the MSI European network COST Action BM1104 was based. is an Associate Professor at the Department of Pharmaceutical Biosciences, Biomolecular Imaging & Proteomics, Uppsala University, Sweden. He was recently awarded a special research position from the Swedish Research Council–Medicine in Functional Protein Chemistry. Dr. Andrén’s research is focused on the development and application of integrated approaches to neurodegenerative diseases such as Parkinson’s disease and drug discovery/development utilizing mass spectrometry imaging. research focuses on solving critical questions in science from a molecular perspective. The research of his group focuses on the development of technologies allied to mass spectrometry and applied to a range of biomedical applications. Innovative research is pursued in four key areas: 1. mass spectrometric tools and allied technologies aimed at sensitive and quantitative analysis of proteins and their posttranslational modifications; 2. separation sciences that can efficiently resolve complex molecular mixtures so their analysis is within the capacity of our analytical technology; 3. apply new chemistries (in methods and molecules) to biomedical applications to reveal molecular functional information of protein networks and protein complexes; and 4. computational methods to assist in the efficient validation and understanding of proteome-scale analysis of biomolecular systems. Most applications for these technologies are in health, disease, food, and forensics. Recently Garry has also established a new area of activity for HIMS in ‘Science for Art’. MD (1997) is a board-certified pathologist (2004) and lecturer at the Albert-Ludwigs-Universität Freiburg and Technische Universität München. He is the head of the Research Unit Analytical Pathology at Helmholtz Zentrum München and has (co)authored over 180 original scientific articles, including several reviews, h-index 33.Axel Walch’s applied clinical research is related to tissue pathology and can be defined as “reverse translational research” by obtaining new knowledge directly in human pathology and translating this back to basic researcher. His general research interests lie in the discovery of temporal and spatial processes in tissues using microscopic in vitro and in vivo imaging approaches such as imaging mass spectrometry. Applications have focused on qualitative and quantitative detection of endogenous compounds in intact tissues of patients and in animal systems. is a leading MSI scientist with a strong background in mass spectrometric techniques applied to biomedical research. He is pioneering label-free molecular imaging by MALDI mass spectrometry and currently applying it to study compound and metabolite distributions in laboratory animals at Novartis. He is known for writing the software “BioMap”, building the iMatrixSpray, and disseminating information on www.ms-imaging.org .

The choice of colour scheme used to present data can have a dramatic effect on the perceived structure present within the data. This is of particular significance in mass spectrometry imaging (MSI), where ion images that provide 2D distributions of a wide range of analytes are used to draw conclusions about the observed system. Commonly employed colour schemes are generally suboptimal for providing an accurate representation of the maximum amount of data. Rainbow-based colour schemes are extremely popular within the community, but they introduce well-documented artefacts which can be actively misleading in the interpretation of the data. In this article, we consider the suitability of colour schemes and composite image formation found in MSI literature in the context of human colour perception. We also discuss recommendations of rules for colour scheme selection for ion composites and multivariate analysis techniques such as principal component analysis (PCA). Graphical Abstract at Visualisation of the same data (unnormalised m/z 826 from the cerebellum region of a mouse brain) using colour schemes found in the MSI literature. Intensity spans from 0 to 100 counts. a Grayscale, b red, c green, d blue, e green to white, f cyan to white, g blue to white, h red to white, i pink to white, j copper to white, k hot, l pink hot, m green to yellow, n cyan to magenta to yellow, o double scale (blue to green, red to yellow), p temperature-based, q–t rainbow-based
Keywords: Mass spectrometry imaging; Colour scheme; Data visualisation

A micropixelated ion-imaging detector for mass resolution enhancement of a QMS instrument by Sarfaraz U. A. H. Syed; Gert B. Eijkel; Simon Maher; Piet Kistemaker; Stephen Taylor; Ron M. A. Heeren (2055-2062).
An in-vacuum position-sensitive micropixelated detector (Timepix) is used to investigate the time-dependent spatial distribution of different charge state (and hence different mass-to-charge (m/z)) ions exiting an electrospray ionization (ESI)-based quadrupole mass spectrometer (QMS) instrument. Ion images obtained from the Timepix detector provide a detailed insight into the positions of stable and unstable ions of the mass peak as they exit the QMS. With the help of image processing algorithms and by selecting areas on the ion images where more stable ions impact the detector, an improvement in mass resolution by a factor of 5 was obtained for certain operating conditions. Moreover, our experimental approach of mass resolution enhancement was confirmed by in-house-developed novel QMS instrument simulation software. Utilizing the imaging-based mass resolution enhancement approach, the software predicts instrument mass resolution of ∼1,0000 for a single-filter QMS instrument with a 210-mm long mass filter and a low operating frequency (880 kHz) of the radio frequency (RF) voltage.
Keywords: Mass spectrometry; Imaging; Spectroscopy/instrumentation

Three-dimensional imaging of lipids and metabolites in tissues by nanospray desorption electrospray ionization mass spectrometry by Ingela Lanekoff; Kristin Burnum-Johnson; Mathew Thomas; Jeeyeon Cha; Sudhansu K. Dey; Pengxiang Yang; Maria C. Prieto Conaway; Julia Laskin (2063-2071).
Three-dimensional (3D) imaging of tissue sections is a new frontier in mass spectrometry imaging (MSI). Here, we report on fast 3D imaging of lipids and metabolites associated with mouse uterine decidual cells and embryo at the implantation site on day 6 of pregnancy. 2D imaging of 16–20 serial tissue sections deposited on the same glass slide was performed using nanospray desorption electrospray ionization (nano-DESI)—an ambient ionization technique that enables sensitive localized analysis of analytes on surfaces without special sample pretreatment. In this proof-of-principle study, nano-DESI was coupled to a high-resolution Q-Exactive instrument operated at high repetition rate of >5 Hz with moderate mass resolution of 35,000 (mm at m/z 200), which enabled acquisition of the entire 3D image with a spatial resolution of ∼150 μm in less than 4.5 h. The results demonstrate localization of acetylcholine in the primary decidual zone (PDZ) of the implantation site throughout the depth of the tissue examined, indicating an important role of this signaling molecule in decidualization. Choline and phosphocholine—metabolites associated with cell growth—are enhanced in the PDZ and abundant in other cellular regions of the implantation site. Very different 3D distributions were obtained for fatty acids (FA), oleic acid and linoleic acid (FA 18:1 and FA 18:2), differing only by one double bond. Localization of FA 18:2 in the PDZ indicates its important role in decidualization while FA 18:1 is distributed more evenly throughout the tissue. In contrast, several lysophosphatidylcholines (LPC) observed in this study show donut-like distributions with localization around the PDZ. Complementary distributions with minimal overlap were observed for LPC 18:0 and FA 18:2 while the 3D image of the potential precursor phosphatidylcholine 36:2 (PC 36:2) showed a significant overlap with both LPC 18:0 and FA 18:2.
Keywords: Three-dimensional (3D) imaging mass spectrometry; Nanospray desorption electrospray ionization (nano-DESI); Mouse embryo; Phospholipids; Metabolites

Quantitative mass spectrometry imaging of emtricitabine in cervical tissue model using infrared matrix-assisted laser desorption electrospray ionization by Mark T. Bokhart; Elias Rosen; Corbin Thompson; Craig Sykes; Angela D. M. Kashuba; David C. Muddiman (2073-2084).
A quantitative mass spectrometry imaging (QMSI) technique using infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is demonstrated for the antiretroviral (ARV) drug emtricitabine in incubated human cervical tissue. Method development of the QMSI technique leads to a gain in sensitivity and removal of interferences for several ARV drugs. Analyte response was significantly improved by a detailed evaluation of several cationization agents. Increased sensitivity and removal of an isobaric interference was demonstrated with sodium chloride in the electrospray solvent. Voxel-to-voxel variability was improved for the MSI experiments by normalizing analyte abundance to a uniformly applied compound with similar characteristics to the drug of interest. Finally, emtricitabine was quantified in tissue with a calibration curve generated from the stable isotope-labeled analog of emtricitabine followed by cross-validation using liquid chromatography tandem mass spectrometry (LC-MS/MS). The quantitative IR-MALDESI analysis proved to be reproducible with an emtricitabine concentration of 17.2 ± 1.8 μg/gtissue. This amount corresponds to the detection of 7 fmol/voxel in the IR-MALDESI QMSI experiment. Adjacent tissue slices were analyzed using LC-MS/MS which resulted in an emtricitabine concentration of 28.4 ± 2.8 μg/gtissue.
Keywords: Mass spectrometry imaging; IR-MALDESI; Absolute quantification; Drug distribution; HIV; Selected reaction monitoring

The use of hydrazine-based derivatization reagents for improved sensitivity and detection of carbonyl containing compounds using MALDI-MSI by Bryn Flinders; Josie Morrell; Peter S. Marshall; Lisa E. Ranshaw; Malcolm R. Clench (2085-2094).
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.
Keywords: Derivatization; MALDI-MSI; Reactive matrices; Glucocorticoids

In the last few years, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) has been successfully used to study the distribution of lipids within tissue sections. However, few efforts have been made to acquire reliable quantitative data regarding the localized concentrations of these molecules. Here we propose an approach based on brain homogenates for the quantification of phosphatidylcholines (PCs) in brain section by MALDI MSI. Homogenates were spiked with a range of PC(16:0 d31/18:1) concentrations. Sections from homogenates and intact brain were simultaneously prepared before being analyzed by MALDI MSI using a Fourier transform ion cyclotron resonance (FT-ICR) analyzer. Standard curves were generated from the signal intensity of the different PC(16:0 d31/18:1) ionic species ([M+H]+, [M+Na]+ and [M+K]+) detected from the homogenate sections. Localized quantitative data were finally extracted by correlating the standard curves with the signal intensities of endogenous PC (especially PC(16:0/18:1)) ionic species detected on different areas of the brain section. They were consistent with quantitative values found in the literature. This work introduces a new method to take directly into account biological matrix effects for the quantification of lipids as well as other endogenous compounds, in tissue sections by MALDI MSI. Graphical abstract A spiked tissue-based method for lipids quantification by MALDI mass spectrometry imaging.
Keywords: MALDI; Imaging; Lipids; Quantification

Distribution and quantification of irinotecan and its active metabolite SN-38 in colon cancer murine model systems using MALDI MSI by Achim Buck; Susanne Halbritter; Christoph Späth; Annette Feuchtinger; Michaela Aichler; Horst Zitzelsberger; Klaus-Peter Janssen; Axel Walch (2107-2116).
Tissue distribution and quantitative analysis of small molecules is a key to assess the mechanism of drug action and evaluate treatment efficacy. The prodrug irinotecan (CPT-11) is widely used for chemotherapeutic treatment of colorectal cancer. CPT-11 requires conversion into its active metabolite SN-38 to exert the desired pharmacological effect. MALDI-Fourier transform ion cyclotron resonance (FT-ICR) and MALDI-time-of-flight (TOF) mass spectrometry imaging (MSI) were performed for detection of CPT-11 and SN-38 in tissue sections from mice post CPT-11 injection. In-depth information was gained about the distribution and quantity of drug compounds in normal and tumor tissue. The prodrug was metabolized, as proven by the detection of SN-38 in liver, kidney and digestive tract. In tumors from genetic mouse models for colorectal cancer (Apc 1638N/wt x pvillin-Kras V12G ), CPT-11 was detected but not the active metabolite. In order to correlate drug distribution relative to vascularization, MALDI data were superimposed with CD31 (PECAM-1) immunohistochemistry. This analysis indicated that intratumoral access of CPT-11 mainly occurred by extravasation from microvessels. The present study exploits the power of MALDI MSI in drug analysis, and presents a novel approach to monitor drug distribution in relation to vessel functionality in preclinical and clinical research.
Keywords: Mass spectrometry; MALDI imaging; Drug monitoring/drug screening; Irinotecan; SN-38

Droplet-based liquid microjunction surface sampling coupled with high-performance liquid chromatography (HPLC)-electrospray ionization (ESI)-tandem mass spectrometry (MS/MS) for spatially resolved analysis provides the possibility of effective analysis of complex matrix samples and can provide a greater degree of chemical information from a single spot sample than is typically possible with a direct analysis of an extract. Described here is the setup and enhanced capabilities of a discrete droplet liquid microjunction surface sampling system employing a commercially available CTC PAL autosampler. The system enhancements include incorporation of a laser distance sensor enabling unattended analysis of samples and sample locations of dramatically disparate height as well as reliably dispensing just 0.5 μL of extraction solvent to make the liquid junction to the surface, wherein the extraction spot size was confined to an area about 0.7 mm in diameter; software modifications improving the spatial resolution of sampling spot selection from 1.0 to 0.1 mm; use of an open bed tray system to accommodate samples as large as whole-body rat thin tissue sections; and custom sample/solvent holders that shorten sampling time to approximately 1 min per sample. The merit of these new features was demonstrated by spatially resolved sampling, HPLC separation, and mass spectral detection of pharmaceuticals and metabolites from whole-body rat thin tissue sections and razor blade (“crude”) cut mouse tissue. Graphical abstract Workflow of the droplet based liquid microjunction surface sampling process
Keywords: Liquid microjunction; Droplet-based liquid extraction; Autosampler; Spatial distribution; Laser distance sensor

MALDI imaging mass spectrometry of N-linked glycans on formalin-fixed paraffin-embedded murine kidney by Ove J. R. Gustafsson; Matthew T. Briggs; Mark R. Condina; Lyron J. Winderbaum; Matthias Pelzing; Shaun R. McColl; Arun V. Everest-Dass; Nicolle H. Packer; Peter Hoffmann (2127-2139).
Recent developments in spatial proteomics have paved the way for retrospective in situ mass spectrometry (MS) analyses of formalin-fixed paraffin-embedded clinical tissue samples. This type of analysis is commonly referred to as matrix-assisted laser desorption/ionization (MALDI) imaging. Recently, formalin-fixed paraffin-embedded MALDI imaging analyses were augmented to allow in situ analyses of tissue-specific N-glycosylation profiles. In the present study, we outline an improved automated sample preparation method for N-glycan MALDI imaging, which uses in situ PNGase F-mediated release and measurement of N-linked glycans from sections of formalin-fixed murine kidney. The sum of the presented data indicated that N-glycans can be cleaved from proteins within formalin-fixed tissue and characterized using three strategies: (i) extraction and composition analysis through on-target MALDI MS and liquid chromatography coupled to electrospray ionization ion trap MS; (ii) MALDI profiling, where N-glycans are released and measured from large droplet arrays in situ; and (iii) MALDI imaging, which maps the tissue specificity of N-glycans at a higher resolution. Thus, we present a complete, straightforward method that combines MALDI imaging and characterization of tissue-specific N-glycans and complements existing strategies. Graphical Abstract MALDI imaging MS of N-linked glycans released from formalin-fixed paraffin-embedded murine kidney sections. Ion intensity maps for (Hex)2(HexNAc)3(Deoxyhexose)3+(Man)3(GlcNAc)2 (m/z 2304.932, red), (Hex)6+(Man)3(GlcNAc)2 (m/z 1905.742, green) and (Hex)2(HexNAc)2+(Man)3(GlcNAc)2 (m/z 1663.756, blue)
Keywords: MALDI imaging; MALDI; Mass spectrometry; Glycans; N-linked

Lateral resolution in NALDI MSI: back to the future by Lukas Krasny; Oldřich Benada; Marcela Strnadova; Karel Lemr; Vladimir Havlicek (2141-2147).
Nanostructure-assisted laser desorption/ionization (NALDI) has been recognized as a powerful matrix-free mass spectrometry tool ideal for imaging of small molecules. In this report, the NALDI approach was compared with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry in terms of sensitivity, reproducibility, and lateral resolution, which can be achieved in mass spectrometry imaging (MSI) experiments using a Nd:YAG laser. Scanning electron microscopy was used for surface topology analysis and evaluation of a putative surface-enhanced sensitivity effect, which was observed upon reduction of the laser focus. NALDI was identified as a more reproducible technique lacking MSI artifacts arising from distant tissue removal known from MALDI oversampling. Graphical Abstract Scanning electron microscopy photographs of a NALDI surface
Keywords: Mass spectrometry; Imaging; NALDI; Nanostructure; Silicon nanowires; Lateral resolution

Desorption electrospray ionization (DESI) mass spectrometry (MS) imaging was used to image locusts dosed with the antihistamine drug terfenadine. The study was conducted in order to elucidate a relatively high elimination rate of terfenadine from the locust hemolymph. In this one of the few MS imaging studies on insects, a method for cryosectioning of whole locusts was developed, and the distributions of a number of endogenous compounds are reported, including betaine and a number of amino acids and phospholipids. Terfenadine was detected in the stomach region and the intestine walls, whereas three different metabolites—terfenadine acid (fexofenadine), terfenadine glucoside, and terfenadine phosphate—were detected in significantly smaller amounts and only in the unexcreted feces in the lower part of the intestine. The use of MS/MS imaging was necessary in order to detect the metabolites. With use of DESI-MS imaging, no colocalization of the drug and the metabolites was observed, suggesting a very rapid excretion of metabolites into the feces. Additional liquid chromatography–MS investigations were performed on hemolymph and feces and showed some abundance of terfenadine and the three metabolites, although at low levels, in both the hemolymph and the feces. Graphical Abstract ᅟ
Keywords: Drug metabolitsm; Insects; Mass spectrometry imaging; Desorption electrospray ionization mass spectrometry

Imaging mass spectrometry (IMS) is a technique in full expansion used in many clinical and biological applications. A common limitation of the technology, particularly true for protein analysis, is that only the most abundant and/or more easily ionizable molecules are typically detected. One approach to overcome this limitation is to transfer proteins contained within tissue sections onto functionalized surfaces with high spatial fidelity for IMS analysis. In this case, only proteins with an affinity for the surface will be retained whereas others will be removed. The chemical nature of the surface is therefore critical. The research work presented herein proposes a high spatial fidelity transfer method for proteins from thin tissue sections onto a nitrocellulose surface. The method employs a home-built apparatus that allows the transfer process to be performed without any direct physical contact between the section and the transfer surface while maintaining physical pressure between the surfaces to help protein migration. The performance of this system was demonstrated using mouse liver and kidney sections. Serials sections were also collected either to be stained with hematoxylin and eosin (H&E) to assess the spatial fidelity of the transfer process or to be directly analyzed as a control sample to differentiate the signals detected after transfer. IMS results showed a high spatial fidelity transfer of a subset of proteins. Some of the detected proteins were poorly observed or not observed with conventional direct tissue analysis, demonstrating an increase in detection sensitivity and specificity with the newly developed method. Graphical Abstract Imaging MS of proteins transferred from tissue sections to a capture membrane
Keywords: Imaging mass spectrometry; MALDI; Proteins; Nitrocellulose; Transfer

Towards imaging metabolic pathways in tissues by Tim J. A. Dekker; Emrys A. Jones; Willem E. Corver; René J. M. van Zeijl; André M. Deelder; Rob A. E. M. Tollenaar; Wilma E. Mesker; Hans Morreau; Liam A. McDonnell (2167-2176).
Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging using 9-aminoacridine as the matrix leads to the detection of low mass metabolites and lipids directly from cancer tissues. These included lactate and pyruvate for studying the Warburg effect, as well as succinate and fumarate, metabolites whose accumulation is associated with specific syndromes. By using the pathway information present in the human metabolome database, it was possible to identify regions within tumor tissue samples with distinct metabolic signatures that were consistent with known tumor biology. We present a data analysis workflow for assessing metabolic pathways in their histopathological context.
Keywords: Metabolites; Warburg effect; Imaging mass spectrometry; Metabolic pathways

Quantification in MALDI-MS imaging: what can we learn from MALDI-selected reaction monitoring and what can we expect for imaging? by Tiffany Porta; Antoine Lesur; Emmanuel Varesio; Gérard Hopfgartner (2177-2187).
Quantification by mass spectrometry imaging (Q-MSI) is one of the hottest topics of the current discussions among the experts of the MS imaging community. If MSI is established as a powerful qualitative tool in drug and biomarker discovery, its reliability for absolute and accurate quantification (QUAN) is still controversial. Indeed, Q-MSI has to deal with several fundamental aspects that are difficult to control, and to account for absolute quantification. The first objective of this manuscript is to review the state-of-the-art of Q-MSI and the current strategies developed for absolute quantification by direct surface sampling from tissue sections. This includes comments on the quest for the perfect matrix-matched standards and signal normalization approaches. Furthermore, this work investigates quantification at a pixel level to determine how many pixels must be considered for accurate quantification by ultraviolet matrix-assisted laser desorption/ionization (MALDI), the most widely used technique for MSI. Particularly, this study focuses on the MALDI-selected reaction monitoring (SRM) in rastering mode, previously demonstrated as a quantitative and robust approach for small analyte and peptide-targeted analyses. The importance of designing experiments of good quality and the use of a labeled compound for signal normalization is emphasized to minimize the signal variability. This is exemplified by measuring the signal for cocaine and a tryptic peptide (i.e., obtained after digestion of a monoclonal antibody) upon different experimental conditions, such as sample stage velocity, laser power and frequency, or distance between two raster lines. Our findings show that accurate quantification cannot be performed on a single pixel but requires averaging of at least 4–5 pixels. The present work demonstrates that MALDI-SRM/MSI is quantitative with precision better than 10–15 %, which meets the requirements of most guidelines (i.e., in bioanalysis or toxicology) for quantification of drugs or peptides from tissue homogenates. Graphical Abstract MALDI-SRM/MSI is quantitative with precision better than 10–15 % when instrumental parameters are correctly set and after pixel-by-pixel normalization
Keywords: MALDI; Mass spectrometry imaging; Quantification; Selected reaction monitoring; Signal normalization

Mass spectrometry imaging provides for non-targeted, label-free chemical imaging. In this study, atmospheric pressure high-resolution scanning microprobe matrix-assisted laser desorption/ionization mass spectrometry imaging (AP-SMALDI MSI) was used for the first time to describe the chemical distribution of the defensive compounds pederin, pseudopederin, and pederon in tissue sections (16 μm thick) of the rove beetle Paederus riparius. The whole-insect tissue section was scanned with a 20-μm step size. Mass resolution of the orbital trapping mass spectrometer was set to 100,000 at m/z 200. Additionally, organ-specific compounds were identified for brain, nerve cord, eggs, gut, ovaries, and malpighian tubules. To confirm the distribution of the specific compounds, individual organs from the insect were dissected, and MSI experiments were performed on the dissected organs. Three ganglia of the nerve cord, with a dimension of 250–500 μm, were measured with 10-μm spatial resolution. High-quality m/z images, based on high spatial resolution and high mass accuracy were generated. These features helped to assign mass spectral peaks with high confidence. Mass accuracy of the imaging experiments was <3 ppm root mean square error, and mapping of different compound classes from a single experiment was possible. This approach improved the understanding of the biochemistry of P. riparius. Concentration differences and distributions of pederin and its analogues could be visualized in the whole-insect section. Without any labeling, we assigned key lipids for specific organs to describe their location in the body and to identify morphological structures with a specificity higher than with staining or immunohistology methods.
Keywords: Paederus riparius ; Insects; Pederin; High-resolution mass spectrometry imaging; MALDI Imaging

Characterization of freeze-fractured epithelial plasma membranes on nanometer scale with ToF-SIMS by Felix Draude; Martin Körsgen; Andreas Pelster; Tanja Schwerdtle; Johannes Müthing; Heinrich F. Arlinghaus (2203-2211).
Time-of-flight secondary ion mass spectrometry (ToF-SIMS) was used to characterize the freeze-fracturing process of human epithelial PANC-1 and UROtsa cells. For this purpose, phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, and phosphatidylserine standard samples were investigated to find specific signals with both high specificity and signal intensity. The results were used to investigate single cells of subconfluent cell layers prepared with a special silicon wafer sandwich preparation technique. This freeze-fracturing technique strips cell membranes off the cells, isolating them on opposing silicon wafer substrates. Criteria were found for defining regions with stripped off cell membranes and, on the opposing wafer, complementary regions with the remaining cells. Measured ethanolamine/choline and serine/choline ratios in these regions clearly showed that in the freeze-fracturing process, the lipid bilayer of the plasma membrane is split along its central zone. Accordingly, only the outer lipid monolayer is stripped off the cell, while the inner lipid monolayer remains attached to the cell on the opposing wafer, thus allowing detailed analysis of a single lipid monolayer. Furthermore, it could be shown that using different washing procedures did not influence the transmembrane lipid distribution. Under optimized preparation conditions, it became feasible to detect lipids with a lateral resolution of approximately 100 nm. The data indicate that ToF-SIMS would be a very useful technique to study with very high lateral resolution changes in lipid composition caused, for example, by lipid storage diseases or pharmaceuticals that interfere with the lipid metabolism.
Keywords: ToF-SIMS imaging; Life science; Lipid; Freeze-fracturing; Membrane; Transmembrane asymmetry

The remodeling of the synovial membrane, which normally lubricates the joints by producing synovial fluid, is one of the most characteristic events in the pathology of osteoarthritis (OA). The heterogeneity and spatial distribution of proteins in the synovial membrane are poorly studied and we hypothesized that they constitute excellent molecular disease classifiers for the accurate diagnosis of the disease. Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) allows for the study of the localization and identification of hundreds of different molecules with high sensitivity in very thin tissue sections. In this work, we employed MALDI-MSI in combination with principal component analysis and discriminant analysis to reveal the specific profile and distribution of digested proteins in human normal and OA synovial membranes. Proteins such as hemoglobin subunit alpha 2, hemoglobin subunit beta, actin aortic smooth muscle, biglycan, and fibronectin have been directly identified from human synovial biopsies. In addition, we have determined the location of disease-specific OA markers. Some of them which are located in areas of low inflammation provide valuable information on tissue heterogeneity. Finally, we described the OA molecular protein signatures common to synovial and other articular tissues such as cartilage. For the first time, normal and OA human synovial membranes have been classified by MALDI-MSI, thus offering a new sensitive tool for the study of rheumatic pathologies.
Keywords: MALDI-MSI; OA; Peptides; Digestion

The challenge of on-tissue digestion for MALDI MSI— a comparison of different protocols to improve imaging experiments by Hanna C. Diehl; Birte Beine; Julian Elm; Dennis Trede; Maike Ahrens; Martin Eisenacher; Katrin Marcus; Helmut E. Meyer; Corinna Henkel (2223-2243).
Mass spectrometry imaging (MSI) has become a powerful and successful tool in the context of biomarker detection especially in recent years. This emerging technique is based on the combination of histological information of a tissue and its corresponding spatial resolved mass spectrometric information. The identification of differentially expressed protein peaks between samples is still the method’s bottleneck. Therefore, peptide MSI compared to protein MSI is closer to the final goal of identification since peptides are easier to measure than proteins. Nevertheless, the processing of peptide imaging samples is challenging due to experimental complexity. To address this issue, a method development study for peptide MSI using cryoconserved and formalin-fixed paraffin-embedded (FFPE) rat brain tissue is provided. Different digestion times, matrices, and proteases were tested to define an optimal workflow for peptide MSI. All practical experiments were done in triplicates and analyzed by the SCiLS Lab software, using structures derived from myelin basic protein (MBP) peaks, principal component analysis (PCA) and probabilistic latent semantic analysis (pLSA) to rate the experiments’ quality. Blinded experimental evaluation in case of defining countable structures in the datasets was performed by three individuals. Such an extensive method development for peptide matrix-assisted laser desorption/ionization (MALDI) imaging experiments has not been performed so far, and the resulting problems and consequences were analyzed and discussed. Graphical abstract Example of experimental setup: Comparison of matrices DHB vs. HCCA (II) using FFPE tissue digested for 2 h. Overview of the statistic and structure analysis. (a) pLSA, only components with at least two clearly visible structures are displayed. (b) Mean of counted structures for all visible m/z values of theoretically digested MBP. The three numbers for each experimental condition are derived from counts of three different researchers (R1, R2, and R3). Color coding for (c) and (d): HCCA (II) in red and DHB in blue. (c) PCA of the mean spectra and (d) PCA of the spectra group
Keywords: Matrix-assisted laser desorption/ionization (MALDI); Mass spectrometry imaging (MSI); Sample preparation; Peptide imaging; Formalin-fixed paraffin-embedded (FFPE); Tryptic digestion; LysC-mix

Localization of sunitinib in in vivo animal and in vitro experimental models by MALDI mass spectrometry imaging by James J. Connell; Yutaka Sugihara; Szilvia Török; Balázs Döme; József Tóvári; Thomas E. Fehniger; György Marko-Varga; Ákos Végvári (2245-2253).
The spatial distribution of an anticancer drug and its intended target within a tumor plays a major role on determining how effective the drug can be at tackling the tumor. This study provides data regarding the lateral distribution of sunitinib, an oral antiangiogenic receptor tyrosine kinase inhibitor using an in vitro animal model as well as an in vitro experimental model that involved deposition of a solution of sunitinib onto tissue sections. All tumor sections were analyzed by matrix-assisted laser desorption/ionization mass spectrometry imaging and compared with subsequent histology staining. Six tumors at four different time points after commencement of in vivo sunitinib treatment were examined to observe the patterns of drug uptake. The levels of sunitinib present in in vivo treated tumor sections increased continuously until day 7, but a decrease was observed at day 10. Furthermore, the in vitro experimental model was adjustable to produce a drug level similar to that obtained in the in vivo model experiments. The distribution of sunitinib in tissue sections treated in vitro appeared to agree with the histological structure of tumors, suggesting that this approach may be useful for testing drug update.
Keywords: Mass spectrometry; MALDI; MALDI-MSI; In vivo models; Drug; Sunitinib; Colorectal adenocarcinoma

Conventional mass spectrometry image preprocessing methods used for denoising, such as the Savitzky-Golay smoothing or discrete wavelet transformation, typically do not only remove noise but also weak signals. Recently, memory-efficient principal component analysis (PCA) in conjunction with random projections (RP) has been proposed for reversible compression and analysis of large mass spectrometry imaging datasets. It considers single-pixel spectra in their local context and consequently offers the prospect of using information from the spectra of adjacent pixels for denoising or signal enhancement. However, little systematic analysis of key RP-PCA parameters has been reported so far, and the utility and validity of this method for context-dependent enhancement of known medically or pharmacologically relevant weak analyte signals in linear-mode matrix-assisted laser desorption/ionization (MALDI) mass spectra has not been explored yet. Here, we investigate MALDI imaging datasets from mouse models of Alzheimer’s disease and gastric cancer to systematically assess the importance of selecting the right number of random projections k and of principal components (PCs) L for reconstructing reproducibly denoised images after compression. We provide detailed quantitative data for comparison of RP-PCA-denoising with the Savitzky-Golay and wavelet-based denoising in these mouse models as a resource for the mass spectrometry imaging community. Most importantly, we demonstrate that RP-PCA preprocessing can enhance signals of low-intensity amyloid-β peptide isoforms such as Aβ1-26 even in sparsely distributed Alzheimer’s β-amyloid plaques and that it enables enhanced imaging of multiply acetylated histone H4 isoforms in response to pharmacological histone deacetylase inhibition in vivo. We conclude that RP-PCA denoising may be a useful preprocessing step in biomarker discovery workflows.
Keywords: Random projections; PCA; Principal component analysis; Denoising; MALDI imaging; Amyloid-β peptide; Histone deacetylase

Mass spectrometry imaging (MSI) allows for the direct and simultaneous analysis of the spatial distribution of molecular species from sample surfaces such as tissue sections. One of the goals of MSI is monitoring the distribution of compounds at the cellular resolution in order to gain insights about the biology that occurs at this spatial level. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) imaging of cervical tissue sections was performed using a spot-to-spot distance of 10 μm by utilizing the method of oversampling, where the target plate is moved by a distance that is less than the desorption radius of the laser. In addition to high spatial resolution, high mass accuracy (±1 ppm) and high mass resolving power (140,000 at m/z = 200) were achieved by coupling the IR-MALDESI imaging source to a hybrid quadrupole Orbitrap mass spectrometer. Ion maps of cholesterol in tissues were generated from voxels containing <1 cell, on average. Additionally, the challenges of imaging at the cellular level in terms of loss of sensitivity and longer analysis time are discussed. Graphical abstract Cellular-level mass spectrometry imaging using IR-MALDESI and oversampling
Keywords: Mass spectrometry imaging; Infrared; MALDESI; Cellular analysis; Oversampling; Tissue analysis; Lipids

Monitoring metabolites from Schizophyllum commune interacting with Hypholoma fasciculare combining LESA–HR mass spectrometry and Raman microscopy by Riya C. Menezes; Marco Kai; Katrin Krause; Christian Matthäus; Aleš Svatoš; Jürgen Popp; Erika Kothe (2273-2282).
Microbial competition for territory and resources is inevitable in habitats with overlap between niches of different species or strains. In fungi, competition is brought about by antagonistic mycelial interactions which alter mycelial morphology, metabolic processes, secondary metabolite release, and extracellular enzyme patterns. Until now, we were not able study in vivo chemical interactions of different colonies growing on the same plate. In this report, we developed a fast and least invasive approach to identify, quantify, and visualize co culture-induced metabolites and their location of release within Schizophyllum commune. The pigments indigo, indirubin, and isatin were used as examples to show secondary metabolite production in the interaction zone with Hypholoma fasciculare. Using a combinatory approach of Raman spectroscopy imaging, liquid extraction surface analysis (LESA), and high-resolution mass spectrometry, we identified, quantified, and visualized the presence of indigo and indirubin in the interaction zone. This approach allows the investigation of metabolite patterns between wood degrading species in competition to gain insight in community interactions, but could also be applied to other microorganisms. This method advances analysis of living, still developing colonies and are in part not destructive as Raman spectroscopy imaging is implemented.
Keywords: Raman spectroscopy; LESA–HRMS; Mass spectrometry pigment production; Indigo; Wood-decaying fungi; Basidiomycetes

Fabry disease: renal sphingolipid distribution in the α-Gal A knockout mouse model by mass spectrometric and immunohistochemical imaging by Ladislav Kuchar; Helena Faltyskova; Lukas Krasny; Robert Dobrovolny; Helena Hulkova; Jana Ledvinova; Michael Volny; Martin Strohalm; Karel Lemr; Lenka Kryspinova; Befekadu Asfaw; Jitka Rybová; Robert J. Desnick; Vladimir Havlicek (2283-2291).
Fabry disease is an X-linked lysosomal storage disease due to deficient α-galactosidase A (α-Gal A) activity and the resultant lysosomal accumulation of globotriaosylceramide (Gb3) and related lipids primarily in blood vessels, kidney, heart, and other organs. The renal distribution of stored glycolipid species in the α-Gal A knockout mouse model was compared to that in mice to assess relative distribution and absolute amounts of accumulated sphingolipid isoforms. Twenty isoforms of five sphingolipid groups were visualized by mass spectrometry imaging (MSI), and their distribution was compared with immunohistochemical (IHC) staining of Gb3, the major stored glycosphingolipid in consecutive tissue sections. Quantitative bulk lipid analysis of tissue sections was assessed by electrospray ionization with tandem mass spectrometry (ESI-MS/MS). In contrast to the findings in wild-type mice, all three analytical techniques (MSI, IHC, and ESI-MS/MS) revealed increases in Gb3 isoforms and ceramide dihexosides (composed mostly of galabiosylceramides), respectively. To our knowledge, this is the first report of the distribution of individual molecular species of Gb3 and galabiosylceramides in kidney sections in Fabry disease mouse. In addition, the spatial distribution of ceramides, ceramide monohexosides, and sphingomyelin forms in renal tissue is presented and discussed in the context of their biosynthesis. Graphical Abstract Immunohistochemical images of a wild type (left) and Fabry mouse kidney (right)
Keywords: Fabry disease; Kidney; Glycosphingolipids; Mass spectrometry imaging; Quantitation

High-resolution time and spatial imaging of tobacco and its pyrolysis products during a cigarette puff by microprobe sampling photoionisation mass spectrometry by R. Hertz-Schünemann; S. Ehlert; T. Streibel; C. Liu; K. McAdam; R. R. Baker; R. Zimmermann (2293-2299).
The time- and space-resolved chemical signatures of gases and vapours formed in solid-state combustion processes are difficult to examine using recent analytical techniques. A machine-smoked cigarette represents a very reproducible model system for dynamic solid-state combustion. By using a special sampling system (microprobe unit) that extracts the formed gases from inside of the burning cigarette, which is coupled to a photoionisation mass spectrometer, it was possible to study the evolution of organic gases during a 2-s cigarette puff. The concentrations of various pyrolysis and combustion products such as 1,3-butadiene, toluene, acetaldehyde and phenol were monitored on-line at different sampling points within cigarettes. A near-microscopic-scale spatial resolution and a 200-ms time resolution were achieved. Finally, the recorded information was combined to generate time-resolved concentration maps, showing the formation and destruction zones of the investigated compounds in the burning cigarette. The combustion zone at the tip of cigarette, where e.g. 1,3-butadiene is predominately formed, was clearly separable from the pyrolysis zones. Depending on the stability of the precursor (e.g. lignin or cellulose), the position of pyrolytic formation varies. In conclusion, it was demonstrated that soft photoionisation mass spectrometry in conjunction with a microprobe sampling device can be used for time- and space-resolved analysis of combustion and pyrolysis reactions. In addition to studies on the model cigarette, further model systems may be studied with this approach. This may include further studies on the combustion of biomass or coal chunks, on heterogeneously catalysed reactions or on spray, dust and gas combustion processes.
Keywords: Solid-state combustion; Imaging; Pyrolysis; Combustion product mapping; Microprobe sampling; Photoionisation mass spectrometry

Subcellular-level resolution MALDI-MS imaging of maize leaf metabolites by MALDI-linear ion trap-Orbitrap mass spectrometer by Andrew R. Korte; Marna D. Yandeau-Nelson; Basil J. Nikolau; Young Jin Lee (2301-2309).
A significant limiting factor in achieving high spatial resolution for matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) imaging is the size of the laser spot at the sample surface. Here, we present modifications to the beam-delivery optics of a commercial MALDI-linear ion trap-Orbitrap instrument, incorporating an external Nd:YAG laser, beam-shaping optics, and an aspheric focusing lens, to reduce the minimum laser spot size from ~50 μm for the commercial configuration down to ~9 μm for the modified configuration. This improved system was applied for MALDI-MS imaging of cross sections of juvenile maize leaves at 5-μm spatial resolution using an oversampling method. A variety of different metabolites including amino acids, glycerolipids, and defense-related compounds were imaged at a spatial resolution well below the size of a single cell. Such images provide unprecedented insights into the metabolism associated with the different tissue types of the maize leaf, which is known to asymmetrically distribute the reactions of C4 photosynthesis among the mesophyll and bundle sheath cell types. The metabolite ion images correlate with the optical images that reveal the structures of the different tissues, and previously known and newly revealed asymmetric metabolic features are observed. Figure Laser optics modification for subcellular-level MS imaging of maize leaf
Keywords: Mass spectrometry imaging; Metabolite; Maize

A method to prevent protein delocalization in imaging mass spectrometry of non-adherent tissues: application to small vertebrate lens imaging by David M. G. Anderson; Kyle A. Floyd; Stephen Barnes; Judy M. Clark; John I. Clark; Hassane Mchaourab; Kevin L. Schey (2311-2320).
MALDI imaging requires careful sample preparation to obtain reliable, high-quality images of small molecules, peptides, lipids, and proteins across tissue sections. Poor crystal formation, delocalization of analytes, and inadequate tissue adherence can affect the quality, reliability, and spatial resolution of MALDI images. We report a comparison of tissue mounting and washing methods that resulted in an optimized method using conductive carbon substrates that avoids thaw mounting or washing steps, minimizes protein delocalization, and prevents tissue detachment from the target surface. Application of this method to image ocular lens proteins of small vertebrate eyes demonstrates the improved methodology for imaging abundant crystallin protein products. This method was demonstrated for tissue sections from rat, mouse, and zebrafish lenses resulting in good-quality MALDI images with little to no delocalization. The images indicate, for the first time in mouse and zebrafish, discrete localization of crystallin protein degradation products resulting in concentric rings of distinct protein contents that may be responsible for the refractive index gradient of vertebrate lenses.
Keywords: Imaging mass spectrometry; Matrix-assisted laser desorption ionization (MALDI); Lens; Crystallins

Distributed computing strategies for processing of FT-ICR MS imaging datasets for continuous mode data visualization by Donald F. Smith; Carl Schulz; Marco Konijnenburg; Mehmet Kilic; Ron M. A. Heeren (2321-2327).
High-resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry imaging enables the spatial mapping and identification of biomolecules from complex surfaces. The need for long time-domain transients, and thus large raw file sizes, results in a large amount of raw data (“big data”) that must be processed efficiently and rapidly. This can be compounded by large-area imaging and/or high spatial resolution imaging. For FT-ICR, data processing and data reduction must not compromise the high mass resolution afforded by the mass spectrometer. The continuous mode “Mosaic Datacube” approach allows high mass resolution visualization (0.001 Da) of mass spectrometry imaging data, but requires additional processing as compared to feature-based processing. We describe the use of distributed computing for processing of FT-ICR MS imaging datasets with generation of continuous mode Mosaic Datacubes for high mass resolution visualization. An eight-fold improvement in processing time is demonstrated using a Dutch nationally available cloud service. Graphical abstract ᅟ
Keywords: Imaging mass spectrometry; FTMS; Supercomputing; Cloud computing; Parallel processing; MALDI

Mass spectrometry imaging of biological tissue: an approach for multicenter studies by Andreas Römpp; Jean-Pierre Both; Alain Brunelle; Ron M. A. Heeren; Olivier Laprévote; Brendan Prideaux; Alexandre Seyer; Bernhard Spengler; Markus Stoeckli; Donald F. Smith (2329-2335).
Mass spectrometry imaging has become a popular tool for probing the chemical complexity of biological surfaces. This led to the development of a wide range of instrumentation and preparation protocols. It is thus desirable to evaluate and compare the data output from different methodologies and mass spectrometers. Here, we present an approach for the comparison of mass spectrometry imaging data from different laboratories (often referred to as multicenter studies). This is exemplified by the analysis of mouse brain sections in five laboratories in Europe and the USA. The instrumentation includes matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF), MALDI-QTOF, MALDI-Fourier transform ion cyclotron resonance (FTICR), atmospheric-pressure (AP)-MALDI-Orbitrap, and cluster TOF-secondary ion mass spectrometry (SIMS). Experimental parameters such as measurement speed, imaging bin width, and mass spectrometric parameters are discussed. All datasets were converted to the standard data format imzML and displayed in a common open-source software with identical parameters for visualization, which facilitates direct comparison of MS images. The imzML conversion also allowed exchange of fully functional MS imaging datasets between the different laboratories. The experiments ranged from overview measurements of the full mouse brain to detailed analysis of smaller features (depending on spatial resolution settings), but common histological features such as the corpus callosum were visible in all measurements. High spatial resolution measurements of AP-MALDI-Orbitrap and TOF-SIMS showed comparable structures in the low-micrometer range. We discuss general considerations for planning and performing multicenter studies in mass spectrometry imaging. This includes details on the selection, distribution, and preparation of tissue samples as well as on data handling. Such multicenter studies in combination with ongoing activities for reporting guidelines, a common data format (imzML) and a public data repository can contribute to more reliability and transparency of MS imaging studies. Comparison of MS imaging platforms in international multicenter study
Keywords: Mass spectrometry imaging; Multicenter studies; Multimodal imaging; Data format imzML; Data handling and processing

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.
Keywords: MALDI; Transmission geometry; High spatial resolution; MS imaging; Protein imaging