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Matrix assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) of intact proteins is mostly performed using time-of-flight (TOF) based mass spectrometers, operated in linear mode. Linear MALDI-TOF systems provide limited mass resolving power and mass accuracy, which complicates assigning identities to the peaks in the MSI datasets. In this work we report ultra-high mass resolution MALDI-MSI based on 15T Fourier transform ion cyclotron resonance (FTICR) mass spectrometry for the analysis of intact proteins directly from non-embedded and OCT-embedded mouse and human (control and type 2 diabetes) pancreas so that small endocrine compartments (islets of Langerhans) may be analyzed in control and pathological tissues. Sample preparation methods, in terms of increased sensitivity while limiting lateral diffusion of analytes, have been investigated. By combining protein localization, high mass accuracy, and the clearly resolved isotope patterns we were able to assign protein identities with additional confidence, including proteins of similar average mass and with interspersed isotopomers. These capabilities allowed us to ascertain the presence of many protein adducts that, with a low resolving power instrument, could be misinterpreted as distinct protein ions. (C) 2017 Published by Elsevier B.V

Neuronal hyperexcitability often results from an unbalance between excitatory and inhibitory neurotransmission, but the synaptic alterations leading to enhanced seizure propensity are only partly understood. Here we assess changes in functional synaptic pools in mouse visual cortex made epileptic by tetanus neurotoxin (TeNT) injection. Using an ultrastructural measure of synaptic activity, we quantified functional differences at excitatory and inhibitory synapses. We found homeostatic changes in epileptic networks, expressed as an early onset lengthening of active zones at inhibitory synapses, followed by spatial reorganization of recycled vesicles at excitatory synapses. A proteomic analysis of synaptic content revealed an upregulation of Carboxypeptidase E (CPE) following TeNT injection; remarkably, its inhibition rapidly decreased network discharges in vivo. Altogether, our analyses reveal a complex landscape of changes affecting the epileptic synaptic release machinery, unveiling presynaptic mechanisms which may impact vesicles’ release timing rather than synaptic strength. Our results indicate i) altered positioning of release-competent vesicles as a novel signature of epileptic networks and ii) inhibiting CPE a potential therapeutic strategy to counteract epileptic discharges.

Subclinical atherosclerosis cannot be predicted and novel therapeutic targets are needed. The molecular anatomy of healthy and atherosclerotic tissue is pursued to identify ongoing molecular changes in atherosclerosis development. Mass Spectrometry Imaging (MSI) accounts with the unique advantage of analyzing proteins and metabolites (lipids) while preserving their original localization; thus two dimensional maps can be obtained. Main molecular alterations were investigated in a rabbit model in response to early development of atherosclerosis. Aortic arterial layers (intima and media) and calcified regions were investigated in detail by MALDI-MSI and proteins and lipids specifically defining those areas of interest were identified. These data further complement main findings previously published in J Proteomics (M. Martin-Lorenzo et al., J. Proteomics. (In press); M. Martin-Lorenzo et al., J. Proteomics 108 (2014) 465–468.) [1,2].

The molecular anatomy of healthy and atherosclerotic tissue is pursued here to identify ongoing molecular changes in atherosclerosis development. Subclinical atherosclerosis cannot be predicted and novel therapeutic targets are needed. Mass spectrometry imaging (MSI) is a novel unexplored ex vivo imaging approach in CVD able to provide in-tissue molecular maps. A rabbit model of early atherosclerosis was developed and high-spatial-resolution MALDI-MSI was applied to comparatively analyze histologically-based arterial regions of interest from control and early atherosclerotic aortas. Specific protocols were applied to identify lipids and proteins significantly altered in response to atherosclerosis. Observed protein alterations were confirmed by immunohistochemistry in rabbit tissue, and additionally in human aortas. Molecular features specifically defining different arterial regions were identified. Localized in the intima, increased expression of SFA and lysolipids and intimal spatial organization showing accumulation of PI, PG and SM point to endothelial dysfunction and triggered inflammatory response. TG, PA, SM and PE-Cer were identified specifically located in calcified regions. Thymosin β4 (TMSB4X) protein was upregulated in intima versus media layer and also in response to atherosclerosis. This overexpression and localization was confirmed in human aortas. In conclusion, molecular histology by MS Imaging identifies spatial organization of arterial tissue in response to atherosclerosis.

MALDI mass spectrometry can simultaneously measure hundreds of biomolecules directly from tissue. Using essentially the same technique but different sample preparation strategies, metabolites, lipids, peptides and proteins can be analyzed. Spatially correlated analysis, imaging MS, enables the distributions of these biomolecular ions to be simultaneously measured in tissues. A key advantage of imaging MS is that it can annotate tissues based on their MS profiles and thereby distinguish biomolecularly distinct regions even if they were unexpected or are not distinct using established histological and histochemical methods e.g. neuropeptide and metabolite changes following transient electrophysiological events such as cortical spreading depression (CSD), which are spreading events of massive neuronal and glial depolarisations that occur in one hemisphere of the brain and do not pass to the other hemisphere , enabling the contralateral hemisphere to act as an internal control. A proof-of-principle imaging MS study, including 2D and 3D datasets, revealed substantial metabolite and neuropeptide changes immediately following CSD events which were absent in the protein imaging datasets. The large high dimensionality 3D datasets make even rudimentary contralateral comparisons difficult to visualize. Instead non-negative matrix factorization (NNMF), a multivariate factorization tool that is adept at highlighting latent features, such as MS signatures associated with CSD events, was applied to the 3D datasets. NNMF confirmed that the protein dataset did not contain substantial contralateral differences, while these were present in the neuropeptide dataset. This article is part of a Special Issue entitled: Imaging Mass Spectrometry: A User’s Guide to a New Technique for Biological and Biomedical Research.

Since the introduction of matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS), numerous instrumental developments have been presented. The introduction of microscope MALDI IMS was a major breakthrough, making micron-range resolution MALDI imaging of kiloDalton mass species possible. We discuss new developments that makes large (cm range) field of view, high resolution (μm range) microscope mode imaging mass spectrometry (IMS) possible in a single experiment, using three different data acquisition approaches simultaneously. We demonstrate how a combination of these acquisition approaches is used to correlate mass spectral and high resolution imaging data.

Imaging mass spectrometry technology is rapidly developing. New desorption and ionization methods allow access to an increasingly large number of biomolecular systems. Sensitivity, speed and spatial resolution are continuously improving with new technologies such as cluster ion sources and microscope mode imaging approaches. MALDI imaging and SIMS are providing complementary molecular insights into biomolecular distributions on the surfaces of tissue sections and cells and demonstrate the great promise of biomolecular mass spectrometric imaging. There is one element, all MS imaging researchers agree upon. Sample preparation is crucial to the success of the method. Unfortunately, each application and each MS imaging technology requires a different type of sample preparation. Matrix coating, metal coating, sample morphology, sample history, and local chemical environment all influence the desorption and ionization mechanisms that lie at the basis of all imaging techniques. As images by themselves are a semi-quantitative representation of molecular distributions this obviously raises questions on their reliability. In this paper, a discussion will be devoted to the basic sample preparation requirements for biomedical imaging mass spectrometry. The differences and similarities for the two major imaging MS methods, SIMS and MALDI, will be addressed. Examples of extreme compound suppression as well as different matrix preparation methods for ME-SIMS and MALDI will be discussed.

Matrix assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) has been used to determine peptide distributions directly from rat, mouse and human pituitary tissue sections. Since these organs are small (10 2 –10 3 m) the spatial resolution of IMS is a key issue in molecular imaging of pituitary tissue sections. Here we show that high-resolution IMS allows localization of neuropeptide distributions within different cell clusters of a single organ of a pituitary tissue section. The sample preparation protocol does not result in analyte redistribution and is therefore applicable to IMS experiments at cellular length scales. The stigmatic imaging mass spectrometer used in this study produces selected-ion-count images with pixel sizes of 500 nm and a resolving power of 4m, yielding superior spatial detail compared to images obtained in microprobe imaging experiments. Furthermore, we show that with imaging mass spectrometry a distinction can be made between different mammalian tissue sections based on differences in the amino acid sequence of neuropeptides with the same function. This example demonstrates the power of IMS for label-free molecular imaging at relevant biological length scales.

In producing a method for selective protein nitration, we previously demonstrated the electrochemical nitration of hen egg white lysozyme to be at Tyr23 initially, followed by bisnitration at Tyr20, but with no trisnitration at Tyr53. The nitration site was determined by sequencing a tryptic peptide that included Tyr23 and Tyr20, but possible effects on other regions of the protein were not determined. Moreover, the electrooxidation conditions were harsh, involving an oxidation potential of +1.2 V (vs. saturated calomel electrode [SCE]), no added nitrogen source except the lysozyme itself, and long reaction periods with copper flag electrodes. Here we report a gentler procedure using much shorter reaction times with nitrite as the nitration source, a lower potential (+0.85 V vs. SCE), and a platinum basket electrode. Intact protein analysis by electrospray Fourier transform ion cyclotron resonance mass spectrometry identified mono- and bisnitration products with mass increases of +45 and +90 Da, respectively, consistent with the substitution of NO2 for H. In addition, the results revealed that no other covalent change in the protein occurred following electrooxidation. Nozzle skimmer dissociation of the intact mononitrated species localized the modification site to Tyr20 or Tyr23. Matrix-assisted laser desorption/ ionization time-of-flight and electrospray ionization time-of-flight analysis of the tryptic peptides of mononitrated lysozyme identified the site of nitration as Tyr23. (c) 2006 Elsevier Inc. All rights reserved.

Secondary ion mass spectrometry is commonly used to study many different types of complex surfaces. Yet, compared with MALDI and ESI– MS, SIMS has not made a significant impact in biological or biomedical research. The key features of the technique, namely high spatial resolution, high detection efficiency of ions spanning a wide m/z range, surface sensitivity and the high scan rates seem to match ideally with several questions posed at the cellular level. To this date, SIMS has had only limited success in the biological arena. Why is this and what is needed to change this? This discussion paper will critically review the advances and the usefulness of SIMS in biomedical research and compare it to other approaches that offer spatially resolved molecular information available to a researcher with a biological interest. We will demonstrate that the type of information generated by the various incarnations of SIMS is strongly dependent on sample preparation and surface condition and these phenomena are only poorly understood. Modern approaches such as the cluster gun developments, ME-SIMS, gold coating and MALDI stigmatic imaging on a SIMS instrument might change the perception of SIMS being a tool for semiconductor manufacturers and physicists, and might persuade biologists to use these innovative mass spectrometric imaging tools. # 2006 Published by Elsevier B.V.

Here, we present the results of extensive SIMION 7.0 modelling of a new linear octopole ion trap. The octopole was designed to increase the efficiency of an electrospray ion source coupled to a Fourier Transform Ion Cyclotron Resonance (FTICR) mass spectrometer. This improvement was achieved by applying a pulsed axial field to the octopole to eject the ion packet with a time and energy distribution that better match the acceptance criteria of the FTICR cell, thus increasing the trapping efficiency and sensitivity. The axial field was produced by applying a pulsed dc potential to the custom-designed ejection electrodes located between the octopole rods. The time and energy profiles of the ejected ion packets for several electrode shapes were calculated and are discussed in terms of their compatibility with efficient trapping of the ion packet in the FTICR cell. Preliminary experimental results show increased signal using the dc ejection electrodes of approximately 100%.

A new ion cell for Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), which can be operated within the temperature range 77–438 K, has been designed and constructed. It has an elongated open-ended cylindrical configuration with capacitively coupled trapping electrodes. Fast and accurate thermal control of the cell is realized by embedding a heating element and a cooling pipe into the ceramic jacket holding the cell electrode plates. To determine the geometry factor, β , of the cell a novel empirical methodology has been developed that is applicable to any ICR cell. This was achieved by comparing breakdown diagrams of protonated leucine enkephalin obtained using the new cell with those obtained using a well characterized cell. Energy-resolved collision-activated dissociation (CAD) of protonated leucine enkephalin, performed using the new cell, was applied to probe the internal energy content of ions at different ICR cell temperatures. These experiments demonstrate that the trapped ion population reaches the preset temperature of the cell through thermal equilibration with the cell walls by blackbody infrared radiation. This has permitted FT-ICR-MS studies (dissociation or ion–molecule reactions, etc.) to be performed at a wide temperature range, including low temperatures.

Imaging mass spectrometry : a user’s guide to a new technique for biological and biomedical research