Sample preparation issues for tissue imaging by imaging MS

Imaging MS is a powerful technique that combines the chemical and spatial analysis of surfaces. It allows spatial localization of multiple different compounds that are recorded in parallel without the need of a label. It is currently one of the rapidly developing techniques in the proteomics toolbox. Different complementary imaging MS methods, i.e. MALDI and secondary ion MS imaging for direct tissue analysis, can be applied on exactly the same tissue sample. This allows the identification of small molecules, peptides and proteins present on the same sample surface. Sample preparation is crucial to obtain high quality, reliable and reproducible complementary molecular images. It is essential to optimize the conditions for each step in the sample preparation protocol, ranging from sample collection and storage to surface modification. In this article, we review and discuss the importance of correct sample treatment in case of MALDI and secondary ion MS imaging experiments and describe the experimental requirements for optimal sample preparation.     

MALDI mass spectrometry in prostate cancer biomarker discovery

Matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry (MS) is a highly versatile and sensitive analytical technique, which is known for its soft ionisation of biomolecules such as peptides and proteins. Generally, MALDI MS analysis requires little sample preparation, and in some cases like MS profiling it can be automated through the use of robotic liquid-handling systems. For more than a decade now, MALDI MS has been extensively utilised in the search for biomarkers that could aid clinicians in diagnosis, prognosis, and treatment decision making. This review examines the various MALDI-based MS techniques like MS imaging, MS profiling and proteomics in-depth analysis where MALDI MS follows fractionation and separation methods such as gel electrophoresis, and how these have contributed to prostate cancer biomarker research. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.     

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 for direct tissue analysis: technological advancements and recent applications

Matrix assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is a method that allows the investigation of the molecular content of tissues within its morphological context. Since it is able to measure the distribution of hundreds of analytes at once, while being label free, this method has great potential which has been increasingly recognized in the field of tissue-based research. In the last few years, MALDI-IMS has been successfully used for the molecular assessment of tissue samples mainly in biomedical research and also in other scientific fields. The present article will give an update on the application of MALDI-IMS in clinical and preclinical research. It will also give an overview of the multitude of technical advancements of this method in recent years. This includes developments in instrumentation, sample preparation, computational data analysis and protein identification. It will also highlight a number of emerging fields for application of MALDI-IMS like drug imaging where MALDI-IMS is used for studying the spatial distribution of drugs in tissues.     

Review of Matrix-Assisted Laser Desorption Ionization-Imaging Mass Spectrometry for Lipid Biochemical Histopathology

Matrix-Assisted Laser Desorption Ionization-Imaging Mass Spectrometry (MALDI-IMS) is a rapidly evolving method used for the in situ visualization and localization of molecules such as drugs, lipids, peptides, and proteins in tissue sections. Therefore, molecules such as lipids, for which antibodies and other convenient detection reagents do not exist, can be detected, quantified, and correlated with histopathology and disease mechanisms. Furthermore, MALDI-IMS has the potential to enhance our understanding of disease pathogenesis through the use of “biochemical histopathology”. Herein, we review the underlying concepts, basic methods, and practical applications of MALDI-IMS, including post-processing steps such as data analysis and identification of molecules. The potential utility of MALDI-IMS as a companion diagnostic aid for lipid-related pathological states is discussed.     

Tissue imaging using MALDI-MS: a new frontier of histopathology proteomics

Modern pathology is an amalgam of many disciplines, such as microbiology, biochemistry and immunology, which historically have been intermingled with the practice of clinical medicine. For centuries, the pre-eminent pathological tool, at least in the context of patients, was a post-mortem examination. With the advent of optical microscopes, morphology became a predominant means of developing tissue classification. A further paradigm shift occurred in the attempt to understand the nature and origin of disease; the recognition that, ultimately, it is the derangement in the structure and function of genes and proteins that causes human disease. More recent progress in pathology has led to the use of genomics and molecular technologies, including DNA sequencing, microarray analysis, PCR, in situ hybridization and proteomics. Today, the newest frontier appears to be histopathology proteomics, which adds the mass spectrometer to the arsenal of tools for the direct analysis of tissue biopsies and molecular diagnosis. Typically called MALDI imaging, this technique takes mass spectral snapshots of intact tissue slices, revealing how proteins and peptides are spatially distributed within a given sample. In this review, MALDI imaging technology is presented as well as applications of such technology in cancer or neurodegenerative diseases.     

Imaging mass spectrometry to visualize biomolecule distributions in mouse brain tissue following hemispheric cortical spreading depression

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.     

Matrix-assisted laser desorption/ionization imaging mass spectrometry: a promising technique for reproductive research

Matrix-assisted laser desorption/ionization (MALDI) tissue imaging mass spectrometry is particularly promising among the numerous applications of mass spectrometry. It is used for probing and analyzing the spatial arrangement of a wide range of molecules, including proteins, peptides, lipids, drugs, and metabolites, directly in thin slices of tissue. In the field of proteomics, the technology avoids tedious and time-consuming extraction and fractionation steps classically required for sample analysis. MALDI imaging mass spectrometry is increasingly recognized as a powerful method for clinical proteomics, particularly in cancer research. The technology has particular potential for the discovery of new tissue biomarker candidates, classification of tumors, early diagnosis or prognosis, elucidating pathogenesis pathways, and therapy monitoring. Over recent years, MALDI imaging mass spectrometry has been used for molecular profiling and imaging directly in male and female reproductive tissues. This review will consider some of the recent publications in the field, addressing a range of issues covering embryo development, gene expression product profiling during gametogenesis, and seeking and identifying biomarkers of reproductive cancers. The wealth of advances in mass spectrometry imaging will inevitably attract biologists and clinicians as the advantages and power of this technology become more widely known. This review will also discuss bottlenecks and the many technical issues that remain to be resolved before laboratories in the field can adopt the technology. We foresee that MALDI imaging mass spectrometry will have a major impact in reproductive research by opening new avenues to the understanding of various molecular mechanisms and the diagnosis of reproductive pathologies.     

Imaging mass spectrometry: principle and application

Imaging mass spectrometry (IMS) is two-dimensional mass spectrometry to visualize the spatial distribution of biomolecules, which does not need either separation or purification of target molecules, and enables us to monitor not only the identification of unknown molecules but also the localization of numerous molecules simultaneously. Among the ionization techniques, matrix assisted laser desorption/ionization (MALDI) is one of the most generally used for IMS, which allows the analysis of numerous biomolecules ranging over wide molecular weights. Proper selection and preparation of matrix is essential for successful imaging using IMS. Tandem mass spectrometry, which is referred to MSⁿ, enables the structural analysis of a molecule detected by the first step of IMS. Applications of IMS were initially developed for studying proteins or peptides. At present, however, targets of IMS research have expanded to the imaging of small endogenous metabolites such as lipids, exogenous drug pharmacokinetics, exploring new disease markers, and other new scientific fields. We hope that this new technology will open a new era for biophysics.     

膨胀显微成像技术的原理及应用

膨胀显微成像技术（expansion microscopy,ExM）是一种新型超分辨成像技术。该技术借助可膨胀水凝胶均匀地物理放大生物样本,在常规光学成像条件下实现超分辨成像。ExM适用于细胞、组织切片等多种类型生物样本。蛋白质、核酸、脂质等生物大分子均可借助ExM进行超分辨成像。ExM可与共聚焦显微镜、光片显微镜、超高分辨显微镜联合使用,进一步提高成像分辨率。近年来,多种从基础ExM拓展而来的衍生技术进一步促进了该技术的实际应用。本文综述了ExM及其衍生技术的基本原理、ExM与不同成像技术联用的研究进展及ExM在不同类型生物样本中的应用进展,并对ExM技术的发展前景做出展望。     

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.     

Preparation and characterization of nitrilotriacetic-acid-terminated self-assembled monolayers on gold surfaces for matrix-assisted laser desorption ionization-time of flight-mass spectrometry analysis of proteins and peptides

On-target affinity capture, enrichment and purification of biomolecules improve detection of specific analytes from complex biological samples in matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis. In this paper, we report a simple method for preparation of a self-assembled nitrilotriacetic acid (NTA) monolayer on gold surface which can be used as a MALDI-TOF-MS sample target specifically for recombinant oligohistidine-tagged proteins/peptides and phosphorylated peptides. The NTA functional groups are immobilized to the gold surface via the linkage of 1,8-octanedithiol which forms a self-assembled monolayer on gold. Characterization by X-ray photoelectron spectroscopy and MALDI analysis of the modified surface are described. The chemically modified surface shows strong affinity toward the analytes of interest, which allows effective removal of the common interferences, e.g. salts and detergents, and therefore leads to improved signal/noise ratio and detection limit. The use of the modified surface simplifies the sample preparation for MALDI analysis of these targeted analytes.     

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.     

Tissue profiling by mass spectrometry: a review of methodology and applications

Matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) has become a valuable tool to address a broad range of questions in many areas of biomedical research. One such application allows spectra to be obtained directly from intact tissues, termed "profiling" (low resolution) and "imaging" (high resolution). In light of the fact that MALDI tissue profiling allows over a thousand peptides and proteins to be rapidly detected from a variety of tissues, its application to disease processes is of special interest. For example, protein profiles from tumors may allow accurate prediction of tumor behavior, diagnosis, and prognosis and uncover etiologies underlying idiopathic diseases. MALDI MS, in conjunction with laser capture microdissection, is able to produce protein expression profiles from a relatively small number of cells from specific regions of heterogeneous tissue architectures. Imaging mass spectrometry enables the investigator to assess the spatial distribution of proteins, drugs, and their metabolites in intact tissues. This article provides an overview of several tissue profiling and imaging applications performed by MALDI MS, including sample preparation, matrix selection and application, histological staining prior to MALDI analysis, tissue profiling, imaging, and data analysis. Several applications represent direct translation of this technology to clinically relevant problems.     

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the homogenization of the tissue of interest that could potentially cause changes in the metabolic pathways being studied¹. Mass spectrometric imaging (MSI) is a powerful analytical tool that can provide spatial information of analytes within intact slices of biological tissue samples^1-5. This technique has been used extensively to study various types of compounds including proteins, peptides, lipids, and small molecules such as endogenous metabolites. With matrix-assisted laser desorption/ionization (MALDI)-MSI, spatial distributions of multiple metabolites can be simultaneously detected. Herein, a method developed specifically for conducting untargeted metabolomics MSI experiments on legume roots and root nodules is presented which could reveal insights into the biological processes taking place. The method presented here shows a typical MSI workflow, from sample preparation to image acquisition, and focuses on the matrix application step, demonstrating several matrix application techniques that are useful for detecting small molecules. Once the MS images are generated, the analysis and identification of metabolites of interest is discussed and demonstrated. The standard workflow presented here can be easily modified for different tissue types, molecular species, and instrumentation.     

Detergent addition to tryptic digests and ion mobility separation prior to MS/MS improves peptide yield and protein identification for in situ proteomic investigation of frozen and formalin‐fixed paraffin‐embedded adenocarcinoma tissue sections

The identification of proteins involved in tumour progression or which permit enhanced or novel therapeutic targeting is essential for cancer research. Direct MALDI analysis of tissue sections is rapidly demonstrating its potential for protein imaging and profiling in the investigation of a range of disease states including cancer. MALDI‐mass spectrometry imaging (MALDI‐MSI) has been used here for direct visualisation and in situ characterisation of proteins in breast tumour tissue section samples. Frozen MCF7 breast tumour xenograft and human formalin‐fixed paraffin‐embedded breast cancer tissue sections were used. An improved protocol for on‐tissue trypsin digestion is described incorporating the use of a detergent, which increases the yield of tryptic peptides for both fresh frozen and formalin‐fixed paraffin‐embedded tumour tissue sections. A novel approach combining MALDI‐MSI and ion mobility separation MALDI‐tandem mass spectrometry imaging for improving the detection of low‐abundance proteins that are difficult to detect by direct MALDI‐MSI analysis is described. In situ protein identification was carried out directly from the tissue section by MALDI‐MSI. Numerous protein signals were detected and some proteins including histone H3, H4 and Grp75 that were abundant in the tumour region were identified.     

MALDI imaging mass spectrometry: molecular snapshots of biochemical systems

Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is emerging as a powerful tool for investigating the distribution of molecules within biological systems through the direct analysis of thin tissue sections. Unique among imaging methods, MALDI-IMS can determine the distribution of hundreds of unknown compounds in a single measurement. We discuss the current state of the art of MALDI-IMS along with some recent applications and technological developments that illustrate not only its current capabilities but also the future potential of the technique to provide a better understanding of the underlying molecular mechanisms of biological processes.     

MALDI imaging mass spectrometry for in situ proteomic analysis of preneoplastic lesions in pancreatic cancer

The identification of new biomarkers for preneoplastic pancreatic lesions (PanINs, IPMNs) and early pancreatic ductal adenocarcinoma (PDAC) is crucial due to the diseases high mortality rate upon late detection. To address this task we used the novel technique of matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) on genetically engineered mouse models (GEM) of pancreatic cancer. Various GEM were analyzed with MALDI IMS to investigate the peptide/protein-expression pattern of precursor lesions in comparison to normal pancreas and PDAC with cellular resolution. Statistical analysis revealed several discriminative m/z-species between normal and diseased tissue. Intraepithelial neoplasia (PanIN) and intraductal papillary mucinous neoplasm (IPMN) could be distinguished from normal pancreatic tissue and PDAC by 26 significant m/z-species. Among these m/z-species, we identified Albumin and Thymosin-beta 4 by liquid chromatography and tandem mass spectrometry (LC-MS/MS), which were further validated by immunohistochemistry, western blot, quantitative RT-PCR and ELISA in both murine and human tissue. Thymosin-beta 4 was found significantly increased in sera of mice with PanIN lesions. Upregulated PanIN expression of Albumin was accompanied by increased expression of liver-restricted genes suggesting a hepatic transdifferentiation program of preneoplastic cells. In conclusion we show that GEM of endogenous PDAC are a suitable model system for MALDI-IMS and subsequent LC-MS/MS analysis, allowing in situ analysis of small precursor lesions and identification of differentially expressed peptides and proteins.     

Identification of Protein Markers Specific for Papillary Renal Cell Carcinoma Using Imaging Mass Spectrometry

Since the emergence of proteomics methods, many proteins specific for renal cell carcinoma (RCC) have been identified. Despite their usefulness for the specific diagnosis of RCC, such proteins do not provide spatial information on the diseased tissue. Therefore, the identification of cancer-specific proteins that include information on their specific location is needed. Recently, matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) based imaging mass spectrometry (IMS) has emerged as a new tool for the analysis of spatial distribution as well as identification of either proteins or small molecules in tissues. In this report, surgical tissue sections of papillary RCC were analyzed using MALDI-IMS. Statistical analysis revealed several discriminative cancer-specific m/z-species between normal and diseased tissues. Among these m/z-species, two particular proteins, S100A11 and ferritin light chain, which are specific for papillary RCC cancer regions, were successfully identified using LC-MS/MS following protein extraction from independent RCC samples. The expressions of S100A11 and ferritin light chain were further validated by immunohistochemistry of human tissues and tissue microarrays (TMAs) of RCC. In conclusion, MALDI-IMS followed by LC-MS/MS analysis in human tissue identified that S100A11 and ferritin light chain are differentially expressed proteins in papillary RCC cancer regions.     

Tissue imaging using MALDI-MS: a new frontier of histopathology proteomics

Modern pathology is an amalgam of many disciplines, such as microbiology, biochemistry and immunology, which historically have been intermingled with the practice of clinical medicine. For centuries, the pre-eminent pathological tool, at least in the context of patients, was a post-mortem examination. With the advent of optical microscopes, morphology became a predominant means of developing tissue classification. A further paradigm shift occurred in the attempt to understand the nature and origin of disease; the recognition that, ultimately, it is the derangement in the structure and function of genes and proteins that causes human disease. More recent progress in pathology has led to the use of genomics and molecular technologies, including DNA sequencing, microarray analysis, PCR, in situ hybridization and proteomics. Today, the newest frontier appears to be histopathology proteomics, which adds the mass spectrometer to the arsenal of tools for the direct analysis of tissue biopsies and molecular diagnosis. Typically called MALDI imaging, this technique takes mass spectral snapshots of intact tissue slices, revealing how proteins and peptides are spatially distributed within a given sample. In this review, MALDI imaging technology is presented as well as applications of such technology in cancer or neurodegenerative diseases.