MALDI imaging mass spectrometry for direct tissue analysis: a new frontier for molecular histology

Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is a powerful tool for investigating the distribution of proteins and small molecules within biological systems through the in situ analysis of tissue sections. MALDI-IMS can determine the distribution of hundreds of unknown compounds in a single measurement and enables the acquisition of cellular expression profiles while maintaining the cellular and molecular integrity. In recent years, a great many advances in the practice of imaging mass spectrometry have taken place, making the technique more sensitive, robust, and ultimately useful. In this review, we focus on the current state of the art of MALDI-IMS, describe basic technological developments for MALDI-IMS of animal and human tissues, and discuss some recent applications in basic research and in clinical settings.     

MALDI imaging mass spectrometry to investigate endogenous peptides in an animal model of Usher's disease

Imaging MS (MSI) has emerged as a valuable tool to study the spatial distribution of biomolecules in the brain. Herein, MALDI‐MSI was used to determine the distribution of endogenous peptides in a rat model of Usher's disease. This rare disease is considered as a leading cause of deaf‐blindness in humans worldwide. Cryosections of brain tissue were analyzed by MALDI‐MSI to differentiate between healthy and diseased rats. MSI results were highly reproducible. Tissue‐specific peptides were identified by MS/MS using LC‐Orbitrap and MALDI‐TOF/TOF analyses. These peptides were proposed for histological classification due to their particular spatial distribution in the brain, for example, substantia nigra, corpus callosum, and hippocampus. Several endogenous peptides showed significantly increased ion densities, particularly in the colliculi superiores and in the substantia nigra of diseased rats, including peptides derived from Fsd1, dystrobrevin‐β, and ProSAAS. Furthermore, several proteolytic degradation products of the myelin basic protein were identified, of which one peptide is most likely mediated by calpain‐2. Our findings contribute to the characterization of this animal model and include possible peptide markers of disease.     

MALDI imaging mass spectrometry as a novel tool for detecting histone modifications in clinical tissue samples

Histone post-translational modifications (PTMs), histone variants and enzymes responsible for the incorporation or the removal of the PTMs are being increasingly associated with human disease. Combinations of histone PTMs and the specific incorporation of variants contribute to the establishment of cellular identity and hence are potential markers that could be exploited in disease diagnostics and prognostics and therapy response prediction. Due to the scarcity of suitable antibodies and the pre-requirement of tissue homogenization for more advanced analytical techniques, comprehensive information regarding the spatial distribution of these factors at the tissue level has been lacking. MALDI imaging mass spectrometry provides an ideal platform to measure histone PTMs and variants from tissues while maintaining the information about their spatial distribution. Discussed in this review are the relevance of histones in the context of human disease and the contribution of MALDI imaging mass spectrometry in measuring histones in situ.     

Proteomics in atherothrombosis: a future perspective

Atherothrombosis is the primary cause of death in Western countries. The cellular and molecular mechanisms underlying atherosclerosis remain widely unknown. The complex nature of atherosclerotic cardiovascular diseases demands the development of novel technologies that enable discovery of new biomarkers for early disease detection and risk stratification, which may predict clinical outcome. In this review, we outline potential sources and recent proteomic approaches that could be applied in the search of novel biomarkers of cardiovascular risk. In addition, we describe some issues raised in relation to the application of proteomics to blood samples, as well as two novel emerging concepts, such as peptidomics and population proteomics. In the future, the use of high-throughput techniques (proteomic, genomics and metabolomics) will potentially identify novel patterns of biomarkers, which, along with traditional risk factors and imaging techniques, could help to target vulnerable patients and monitor the beneficial effects of pharmacological agents.     

Molecular histoproteomy by MALDI mass spectrometry imaging to uncover markers of the impact of Nosema on Apis mellifera

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) is a powerful technology used to investigate the spatio-temporal distribution of a huge number of molecules throughout a body/tissue section. In this paper, we report the use of MALDI IMS to follow the molecular impact of an experimental infection of Apis mellifera with the microsporidia Nosema ceranae. We performed representative molecular mass fingerprints of selected tissues obtained by dissection. This was followed by MALDI IMS workflows optimization including specimen embedding and positioning as well as washing and matrix application. We recorded the local distribution of peptides/proteins within different tissues from experimentally infected versus non infected honeybees. As expected, a distinction in these molecular profiles between the two conditions was recorded from different anatomical sections of the gut tissue. More importantly, we observed differences in the molecular profiles in the brain, thoracic ganglia, hypopharyngeal glands, and hemolymph. We introduced MALDI IMS as an effective approach to monitor the impact of N. ceranae infection on A. mellifera. This opens perspectives for the discovery of molecular changes in peptides/proteins markers that could contribute to a better understanding of the impact of stressors and toxicity on different tissues of a bee in a single experiment.     

Food allergen detection by mass spectrometry: From common to novel protein ingredients

Food allergens are molecules, mainly proteins, that trigger immune responses in susceptible individuals upon consumption even when they would otherwise be harmless. Symptoms of a food allergy can range from mild to acute; this last effect is a severe and potentially life-threatening reaction. The European Union (EU) has identified 14 common food allergens, but new allergens are likely to emerge with constantly changing food habits. Mass spectrometry (MS) is a promising alternative to traditional antibody-based assays for quantifying multiple allergenic proteins in complex matrices with high sensitivity and selectivity. Here, the main allergenic proteins and the advantages and drawbacks of some MS acquisition protocols, such as multiple reaction monitoring (MRM) and data-dependent analysis (DDA) for identifying and quantifying common allergenic proteins in processed foodstuffs are summarized. Sections dedicated to novel foods like microalgae and insects as new sources of allergenic proteins are included, emphasizing the significance of establishing stable marker peptides and validated methods using database searches. The discussion involves the in-silico digestion of allergenic proteins, providing insights into their potential impact on immunogenicity. Finally, case studies focussing on microalgae highlight the value of MS as an effective analytical tool for ensuring regulatory compliance throughout the food control chain.     

Recent advances and clinical insights into the use of proteomics in the study of atherosclerosis

Introduction: The application of new proteomics methods may help to identify new diagnostic/predictive molecular markers in an attempt to improve the clinical management of atherosclerosis.

Areas covered: Technological advances in proteomics have enhanced its sensitivity and multiplexing capacity, as well as the possibility of studying protein interactions and tissue structure. These advances will help us better understand the molecular mechanisms at play in atherosclerosis as a biological system. Moreover, this should help identify new predictive/diagnostic biomarkers and therapeutic targets that may facilitate effective risk stratification and early diagnosis, with the ensuing rapid implementation of treatment. This review provides a comprehensive overview of the novel methods in proteomics, including state-of-the-art techniques, novel biological samples and applications for the study of atherosclerosis.

Expert commentary: Collaboration between clinicians and researchers is crucial to further validate and introduce new molecular markers to manage atherosclerosis that are identified using the most up to date proteomic approaches.     

Integrating multiplex immunofluorescent and mass spectrometry imaging to map myeloid heterogeneity in its metabolic and cellular context

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Immunoaffinity techniques coupled to mass spectrometry for the analysis of human peptide hormones: advances and applications

Introduction: The accurate and comprehensive determination of peptide hormones from biological fluids has represented a considerable challenge to analytical chemists for decades. Besides long-established bioanalytical ligand binding assays (or ELISA, RIA, etc.), more and more mass spectrometry-based methods have been developed recently for purposes commonly referred to as targeted proteomics. Eventually the combination of both, analyte extraction by immunoaffinity and subsequent detection by mass spectrometry, has shown to synergistically enhance the test methods’ performance characteristics.

Areas covered: The review provides an overview about the actual state of existing methods and applications concerning the analysis of endogenous peptide hormones. Here, special focus is on recent developments considering the extraction procedures with immobilized antibodies, the subsequent separation of target analytes, and their detection by mass spectrometry.

Expert commentary: Key aspects of procedures aiming at the detection and/or quantification of peptidic analytes in biological matrices have experienced considerable improvements in the last decade, particularly in terms of the assays’ sensitivity, the option of multiplexing target compounds, automatization, and high throughput operation. Despite these advances and progress as expected to be seen in the near future, immunoaffinity purification coupled to mass spectrometry is not yet a standard procedure in routine analysis compared to ELISA/RIA.     

Electrospray-ionization mass spectrometry as a tool for fast screening of protein structural properties

Since the early 1990s, electrospray-ionization mass spectrometry (ESI-MS) has encountered growing interest as a complementary tool to established biochemical and biophysical methods for investigating protein structure and conformation. Nowadays, applications of ESI-MS to protein investigation span from the area of analytical biochemistry to that of structural biology. This review focuses on applications of this technique to the analysis of protein conformational properties and molecular interactions, underscoring their possible relevance for molecular biotechnology, although representing a still very young field. An introductive section presents the major issues related to theoretical and technical aspects of ESI-MS under non-denaturing conditions. Examples from our work and from the literature illustrate which kind of information can be obtained concerning key issues in biotechnology such as stability and aggregation of proteins under both near-native and challenging conditions, and interactions with other proteins, ligands and cofactors.     

Tracking fatty acid kinetics in distinct lipoprotein fractions in vivo: a novel high-throughput approach for studying dyslipidemia in rodent models

Isotopic tracers have been used to examine lipid trafficking for many years, and data from those studies have typically yielded novel insight regarding the pathophysiology of dyslipidemia. Previous experimental designs were suitable for studies in humans because relatively large volumes of plasma could be regularly sampled. We have expanded on the earlier logic by applying high-throughput analytical methods that require reduced sample volumes. Specifically, we have examined the possibility of coupling gel-based separations of lipoproteins (e.g., lipoprint) with LC-MS/MS analyses of complex lipid mixtures as a way to routinely measure the labeling profiles of distinct lipids in discrete lipoprotein subfractions. We demonstrate the ability to measure the incorporation of [U-¹³C]oleate into triglycerides (TG), PLs (PL), and cholesterol esters (CE) in VLDL, LDL, and HDL particles in mice. Although rodent models of dyslipidemia are inherently different from humans because of alterations in enzyme activities and underlying metabolism, rodent models can be used to screen novel compounds for efficacy in altering a given biochemical pathway and therein enable studies of target engagement in vivo. We expect that it is possible to translate our approach for application in other systems, including studies in humans.     

Going forward: Increasing the accessibility of imaging mass spectrometry

The driving force behind the high and increasing popularity of imaging mass spectrometry is its demonstrated potential for the determination of new diagnostic/prognostic biomarkers and its ability to simultaneously trace the distributions of pharmaceuticals and their metabolites in tissues without the need to develop expensive radioactively-labeled analogues. Both of these applications would benefit from standardized methods, for the development of novel MS-based molecular histology tests and governmental-approved MS-based assays for pharmaceutical development. In addition, the broader scientific community would benefit from the increased accessibility of the technique. Currently imaging MS studies are individual endeavors, utilizing the individual expertise and infrastructure of a single laboratory and their immediate collaborators. A wide array of tissue preparation, data acquisition and data analysis techniques has been developed but lacks an international collaborative structure and data sharing capabilities. Such a collaborative framework would enable methodological exchange and detailed comparisons of analytical capabilities, to explore synergies between the different methods and result in the development of robust standardized methods. Here we describe the activities of a new European imaging MS network that will explicitly compare and contrast existing methods to provide best practice guidelines for the entire healthcare research community.     

Spatial metabolomics using imaging mass spectrometry to identify the localization of asparaptine A in Asparagus officinalis

Spatial metabolomics uses imaging mass spectrometry (IMS) to localize metabolites within tissue section. Here, we performed matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance-IMS (MALDI-FTICR-IMS) to identify the localization of asparaptine A, a naturally occurring inhibitor of angiotensin-converting enzyme, in green spears of asparagus (Asparagus officinalis). Spatial metabolome data were acquired in an untargeted manner. Segmentation analysis using the data characterized tissue-type-dependent and independent distribution patterns in cross-sections of asparagus spears. Moreover, asparaptine A accumulated at high levels in developing lateral shoot tissues. Quantification of asparaptine A in lateral shoots using liquid chromatography-tandem mass spectrometry (LC-MS/MS) validated the IMS analysis. These results provide valuable information for understanding the function of asparaptine A in asparagus, and identify the lateral shoot as a potential region of interest for multiomics studies to examine gene-to-metabolite associations in the asparaptine A biosynthesis.     

Cross-linking and mass spectrometry as a tool for studying the structural biology of ribonucleoproteins

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The discovery of protein biomarkers in pre-eclampsia: the promising role of mass spectrometry

Abstract

Context: Pre-eclampsia (PE) is a common hypertensive disorder of pregnancy that substantially affects maternal and neonatal morbidity and mortality worldwide. The aetiology of the disease remains poorly understood with lack of reliable diagnostic tests. PE is a multisystem disorder so it is very unlikely that a single or a small group of biomarkers will accurately predict the disease. Mass spectrometry (MS) is indispensable analytical tool in protein analysis studies. MS-based proteomics have the ability to detect the entire protein complement to provide a useful window into a range of biological processes and allow the identification of differentially expressed proteins between samples.

Objective: The aim of this review is to summarise, discuss and evaluate the current predominant MS-based approaches applied for protein biomarker discovery. The paper also seeks to evaluate the current potential PE biomarkers described in the literature and identify issues that can guide future research.

Conclusion: MS-based proteomics studies are promising alternatives to classical hypothesis-driven approaches to discover novel biomarkers and provide new insights into the underlying phathophysiological mechanisms of PE. This should aid in the early diagnosis of PE and the understanding of the aetiology of the disease.     

Protein labeling by iTRAQ: a new tool for quantitative mass spectrometry in proteome research

A novel, MS-based approach for the relative quantification of proteins, relying on the derivatization of primary amino groups in intact proteins using isobaric tag for relative and absolute quantitation (iTRAQ) is presented. Due to the isobaric mass design of the iTRAQ reagents, differentially labeled proteins do not differ in mass; accordingly, their corresponding proteolytic peptides appear as single peaks in MS scans. Because quantitative information is provided by isotope-encoded reporter ions that can only be observed in MS/MS spectra, we analyzed the fragmentation behavior of ESI and MALDI ions of peptides generated from iTRAQ-labeled proteins using a TOF/TOF and/or a QTOF instrument. We observed efficient liberation of reporter ions for singly protonated peptides at low-energy collision conditions. In contrast, increased collision energies were required to liberate the iTRAQ label from lysine side chains of doubly charged peptides and, thus, to observe reporter ions suitable for relative quantification of proteins with high accuracy. We then developed a quantitative strategy that comprises labeling of intact proteins by iTRAQ followed by gel electrophoresis and peptide MS/MS analyses. As proof of principle, mixtures of five different proteins in various concentration ratios were quantified, demonstrating the general applicability of the approach presented here to quantitative MS-based proteomics.     

Antidepressant treatment effects on hippocampal protein turnover: Molecular and spatial insights from mass spectrometry

A major challenge in managing depression is that antidepressant drugs take a long time to exert their therapeutic effects. For the development of faster-acting treatments, it is crucial to get an improved understanding of the molecular mechanisms underlying antidepressant mode of action. Here, we used a novel mass spectrometry-based workflow to investigate how antidepressant treatment affects brain protein turnover at single-cell and subcellular resolution. We combined stable isotope metabolic labeling, quantitative Tandem Mass Spectrometry (qTMS) and Multi-isotope Imaging Mass Spectrometry (MIMS) to simultaneously quantify and image protein synthesis and turnover in hippocampi of mice treated with the antidepressant paroxetine. We identified changes in turnover of individual hippocampal proteins that reveal altered metabolism-mitochondrial processes and report on subregion-specific turnover patterns upon paroxetine treatment. This workflow can be used to investigate brain protein turnover changes in vivo upon pharmacological interventions at a resolution and precision that has not been possible with other methods to date. Our results reveal acute paroxetine effects on brain protein turnover and shed light on antidepressant mode of action.