Revisiting Rat Spermatogenesis with MALDI Imaging at 20-μm Resolution

Matrix-assisted laser desorption/ionization (MALDI) molecular imaging technology attracts increasing attention in the field of biomarker discovery. The unambiguous correlation between histopathology and MALDI images is a key feature for success. MALDI imaging mass spectrometry (MS) at high definition thus calls for technological developments that were established by a number of small steps. These included tissue and matrix preparation steps, dedicated lasers for MALDI imaging, an increase of the robustness against cell debris and matrix sublimation, software for precision matching of molecular and microscopic images, and the analysis of MALDI imaging data using multivariate statistical methods. The goal of these developments is to approach single cell resolution with imaging MS. Currently, a performance level of 20-μm image resolution was achieved with an unmodified and commercially available instrument for proteins detected in the 2–16-kDa range. The rat testis was used as a relevant model for validating and optimizing our technological developments. Indeed, testicular anatomy is among the most complex found in mammalian bodies. In the present study, we were able to visualize, at 20-μm image resolution level, different stages of germ cell development in testicular seminiferous tubules; to provide a molecular correlate for its well established stage-specific classification; and to identify proteins of interest using a top-down approach and superimpose molecular and immunohistochemistry images.MALDI imaging mass spectrometry (IMS)¹ is increasingly recognized as a powerful approach for the discovery of tissue biomarkers candidates and tissue leakage products in clinical proteomics (1–6). The technology holds an enormous potential for differentiation of disease states, classification of tumors, and early diagnosis or prognosis but also for elucidating pathogenesis pathways and as a follow-up for pharmacological treatments (1, 7–9).IMS offers some unique advantages that can support or complement most conventional techniques. It allows multiplex spatial localization of biomolecules on tissue sections without the need for time-consuming processing steps, specific probes, or labeling of analytes that can alter their native localization or function (10). Interestingly, in the field of biomarker detection, it is now recognized that in many cases a single protein cannot serve as a reliable disease-specific marker, whereas a panel of biomarkers candidates can potentially provide a clear diagnosis (8, 11). Although well established and extremely powerful, conventional differential proteomics approaches based on the analysis of tissue homogenates represent an oversimplification of tissue morphology in a “black versus white” manner (even if combined with tissue microdissection). In particular, cancer tissue consists of several different cell types at various developmental stages (12). An efficient, sensitive, and selective analysis of tumor tissue, therefore, ultimately requires the ability to perform molecular histological analyses on the level of individual cells.Many technical challenges remain to be faced in IMS among which the improvement of lateral resolution, which is currently in the range of 100–200 μm corresponding, at the best, to a few cells. Lateral resolution is mainly limited by the matrix application step and the laser beam diameter and shape, whereas reduction of the irradiated sample area naturally reduces the ion yield. Consequently, a compromise between high lateral resolution and spectral quality has generally to be found (13). However, the adequate evaluation of histological images after conventional hematoxylin and eosin (H&E) or immunohistological stainings routinely requires higher resolution down to the level of individual cellular nuclei. To beneficially integrate MALDI images into the histo(patho)logical evaluation, there are two key requirements. 1) MALDI images should have utmost resolution to correlate them with the tissue morphology at least on the level of single cells (i.e. 10 μm). 2) Overlapping of the transmission microscopic and MALDI images is mandatory to correlate molecular marker distributions with tissue morphology at typical microscopic image resolution (i.e. ∼100 nm). One of the driving forces behind improvement of lateral resolution is that the detection of biomarker candidates can thus be confined to very fine structures of the tissue and even single cells. Improvements in resolution, which is indeed a methodological challenge, must be demonstrated using a tissue sample that on the one hand has clear morphological features of appropriate size and that is well described by classical histology.In the present study, we combined readily available technologies to achieve 20-μm lateral image resolution and to match this information to high quality optical microscopy images as they are used in the histological practice. Because of its highly complex anatomical features, we used the mammalian testis as a model to establish the complete work flow from the acquisition of MALDI images with lateral resolution near the single cell level, the proper match with histological information derived from standard optical microscopy, to the identification of proteins of interest using a top-down approach.     

New access to Alzheimer’s and other neurodegenerative diseases

Single-cell analysis is gaining popularity in the field of mass spectrometry as a method for analyzing protein and peptide content in cells. The spatial resolution of MALDI mass spectrometry (MS) imaging is by a large extent limited by the laser focal diameter and the displacement of analytes during matrix deposition. Owing to recent advancements in both laser optics and matrix deposition methods, spatial resolution on the order of a single eukaryotic cell is now achievable by MALDI MS imaging. Provided adequate instrument sensitivity, a lateral resolution of ?10 µm is currently attainable with commercial instruments. As a result of these advances, MALDI MS imaging is poised to become a transformative clinical technology. In this article, the crucial steps needed to obtain single-cell resolution are discussed, as well as potential applications to disease research.     

Recent advances in single-cell MALDI mass spectrometry imaging and potential clinical impact

Single-cell analysis is gaining popularity in the field of mass spectrometry as a method for analyzing protein and peptide content in cells. The spatial resolution of MALDI mass spectrometry (MS) imaging is by a large extent limited by the laser focal diameter and the displacement of analytes during matrix deposition. Owing to recent advancements in both laser optics and matrix deposition methods, spatial resolution on the order of a single eukaryotic cell is now achievable by MALDI MS imaging. Provided adequate instrument sensitivity, a lateral resolution of approximately 10 μm is currently attainable with commercial instruments. As a result of these advances, MALDI MS imaging is poised to become a transformative clinical technology. In this article, the crucial steps needed to obtain single-cell resolution are discussed, as well as potential applications to disease research.     

Mass spectrometry imaging with high resolution in mass and space

Mass spectrometry (MS) imaging links molecular information and the spatial distribution of analytes within a sample. In contrast to most histochemical techniques, mass spectrometry imaging can differentiate molecular modifications and does not require labeling of targeted compounds. We have recently introduced the first mass spectrometry imaging method that provides highly specific molecular information (high resolution and accuracy in mass) at cellular dimensions (high resolution in space). This method is based on a matrix-assisted laser desorption/ionization (MALDI) imaging source working at atmospheric pressure which is coupled to an orbital trapping mass spectrometer. Here, we present a number of application examples and demonstrate the benefit of ‘mass spectrometry imaging with high resolution in mass and space.’ Phospholipids, peptides and drug compounds were imaged in a number of tissue samples at a spatial resolution of 5–10 μm. Proteins were analyzed after on-tissue tryptic digestion at 50-μm resolution. Additional applications include the analysis of single cells and of human lung carcinoma tissue as well as the first MALDI imaging measurement of tissue at 3 μm pixel size. MS image analysis for all these experiments showed excellent correlation with histological staining evaluation. The high mass resolution (R = 30,000) and mass accuracy (typically 1 ppm) proved to be essential for specific image generation and reliable identification of analytes in tissue samples. The ability to combine the required high-quality mass analysis with spatial resolution in the range of single cells is a unique feature of our method. With that, it has the potential to supplement classical histochemical protocols and to provide new insights about molecular processes on the cellular level.     

MALDI-based imaging mass spectrometry revealed abnormal distribution of phospholipids in colon cancer liver metastasis

We present the results of matrix-assisted laser desorption/ionization (MALDI) imaging and direct molecular identification using tandem mass spectrometry (MS/MS) in colon cancer liver metastasis. Cancer tissue was removed from a Japanese patient and frozen immediately without any fixations. The sections were sliced to a thickness of 3 microm. The matrix for lipid ionization was 2,6-dihydroxy acetophenone. The matrix solution was applied with an airbrush into a thin uniform matrix layer on the tissue surface. After two-dimensional laser scanning, the images were reconstructed as a function of m/z from a few hundred obtained spectra. In the obtained images, the existence of molecules was represented by a pseudo-color corresponding to the signal intensity. In a feasibility study, we picked up a localized signal, m/z 725 in a cancerous area. The MS/MS result suggested that m/z 725 was sphingomyelin(16:0)+Na. Thus, we successfully show the feasibility of MALDI imaging as a tool for the analysis of pathological specimens.     

Imaging mass spectrometry: a new tool to investigate the spatial organization of peptides and proteins in mammalian tissue sections

MALDI MS imaging mass spectrometry can be used to map the distribution of targeted compounds in tissue sections with a spatial resolution currently of about 50 microm, providing important molecular information in many areas of biological research. After matrix application, a raster of a section by the laser beam yields ions from compounds in a tissue mass-to-charge range from 1000 to over 100000. Two-dimensional intensity maps can then be reconstructed to provide specific molecular images of a tissue.     

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.     

Molecular MALDI imaging: an emerging technology for neuroscience studies

Mass spectrometry (MS) has become an essential tool for the detection, identification, and characterization of the molecular components of biological processes, such as those responsible for the dynamic properties of the nervous system. Generally, the application of these powerful techniques requires the destruction of the specimen under study, but recent technological advances have made it possible to apply the matrix-assisted laser desorption/ionization (MALDI) MS technique directly to tissue sections. The major advantage of direct MALDI analysis is that it enables the acquisition of local molecular expression profiles, while maintaining the topographic integrity of the tissue and avoiding time-consuming extraction, purification, and separation steps, which have the potential for introducing artifacts. With automation and the ability to display complex spectral data using imaging software, it is now possible to create multiple 2D maps of selected biomolecules in register with tissue sections, a method now known as MALDI Imaging, or MSI (for Mass Spectrometry Imaging). This creates, for example, an opportunity to correlate functional states, determined a priori with live recording or imaging, with the corresponding molecular maps obtained at the time the tissue is frozen and analyzed with MSI. We review the increasing application of MALDI Imaging to the analysis of molecular distributions of proteins and peptides in nervous tissues of both vertebrates and invertebrates, focusing in particular on recent studies of neurodegenerative diseases and early efforts to implement assays of neuronal development.     

Mass spectrometry imaging is moving toward drug protein co-localization

Mass spectrometry (MS)-based technology provides label-free localization of molecules in tissue samples. Drugs, proteins, lipids and metabolites can easily be monitored in their environment. Resolution can be achieved down to the cellular level (10-20μm) for conventional matrix-assisted laser desorption/ionization (MALDI) imaging, or even to the subcellular level for more complex technologies such as secondary ionization mass spectrometry (SIMS) imaging. One question remains: are we going to be able to investigate functional relationships between drugs and proteins and compare with localized phenomena? This review describes the various spatial levels of investigation offered by mass spectrometry imaging (MSI), and the advantages and disadvantages compared with other labeling technologies.     

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.     

Simple and robust two-layer matrix/sample preparation method for MALDI MS/MS analysis of peptides

Recent advances in MALDI MS/MS instrumentation allow a high degree of automation in the efficient detection of peptide fragment ions that can be used for protein identification. However, the performance of the technique is dependent on the MALDI sample preparation. We present a simple and robust two-layer sample preparation method tailored for sensitive and reproducible generation of MALDI MS/MS data. This method produces a strong and uniform crystal layer which allows acquisition of high quality MS/MS spectra over the entire sample surface area. Furthermore, due to its crystal strength, the matrix/sample layer can be washed extensively on target, enabling direct analysis of samples containing impurities, such as salts and surfactants. This method is demonstrated to be very useful in routine analysis of in-gel tryptic digests of silver-stained protein gel spots, without the need of desalting steps or hunting for "hot" spots. As an example, seven threonine-phosphorylated proteins involved in signal transduction in response to growth factor stimulation within the lipid raft fractions of the IMR5 neuroblastoma cells have been identified using differential gel display, in-gel digestion and MALDI MS/MS with the new two-layer sample preparation method. Some of these proteins have the functions of maintaining raft structure or cell signaling.     

Exploring proteins in Anopheles gambiae male and female antennae through MALDI mass spectrometry profiling

MALDI profiling and imaging mass spectrometry (IMS) are novel techniques for direct analysis of peptides and small proteins in biological tissues. In this work we applied them to the study of Anopheles gambiae antennae, with the aim of analysing expression of soluble proteins involved in olfaction perireceptor events. MALDI spectra obtained by direct profiling on single antennae and by the analysis of extracts, showed similar profiles, although spectra obtained through profiling had a richer ion population and higher signal to noise ratio. Male and female antennae showed distinct protein profiles. MALDI imaging experiments were also performed and differences were observed in the localization of some proteins. Two proteins were identified through high resolution measurement and top-down MS/MS experiments. A 8 kDa protein only present in the male antennae matched with an unannotated sequence of the An. gambiae genome, while the presence of odorant binding protein 9 (OBP-9) was confirmed through experiments of 2-DE, followed by MS and MS/MS analysis of digested spots. This work shows that MALDI MS profiling is a technique suitable for the analysis of proteins of small and medium MW in insect appendices, and allows obtaining data for several specimens which can be investigated for differences between groups. Proteins of interest can be identified through other complementary MS approaches.     

Matrix‐assisted laser desorption/ionization mass spectrometry imaging: a powerful tool for probing the molecular topology of plant cutin polymer

The cutin polymers of different fruit cuticles (tomato, apple, nectarine) were examined using matrix‐assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) after in situ release of the lipid monomers by alkaline hydrolysis. The mass spectra were acquired from each coordinate with a lateral spatial resolution of approximately 100 μm. Specific monomers were released at their original location in the tissue, suggesting that post‐hydrolysis diffusion can be neglected. Relative quantification of the species was achieved by introducing an internal standard, and the collection of data was subjected to non‐supervised and supervised statistical treatments. The molecular images obtained showed a specific distribution of ions that could unambiguously be ascribed to cutinized and suberized regions observed at the surface of fruit cuticles, thus demonstrating that the method is able to probe some structural changes that affect hydrophobic cuticle polymers. Subsequent chemical assignment of the differentiating ions was performed, and all of these ions could be matched to cutin and suberin molecular markers. Therefore, this MALDI‐MSI procedure provides a powerful tool for probing the surface heterogeneity of plant lipid polymers. This method should facilitate rapid investigation of the relationships between cuticle phenotypes and the structure of cutin within a large population of mutants.     

Peptide and protein imaging mass spectrometry in cancer research

MALDI mass spectrometry is able to acquire protein profiles directly from tissue that can describe the levels of hundreds of distinct proteins. MALDI imaging MS can simultaneously reveal how each of these proteins varies in heterogeneous tissues. Numerous studies have now demonstrated how MALDI imaging MS can generate different protein profiles from the different cell types in a tumor, which can act as biomarker profiles or enable specific candidate protein biomarkers to be identified.     

Visualizing spatial lipid distribution in porcine lens by MALDI imaging high-resolution mass spectrometry

The intraocular lens contains high levels of both cholesterol and sphingolipids, which are believed to be functionally important for normal lens physiology. The aim of this study was to explore the spatial distribution of sphingolipids in the ocular lens using mass spectrometry imaging (MSI). Matrix-assisted laser desorption/ionization (MALDI) imaging with ultra high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) was used to visualize the lipid spatial distribution. Equatorially-cryosectioned, 12 μm thick slices of tissue were thaw-mounted to an indium-tin oxide (ITO) glass slide by soft-landing to an ethanol layer. This procedure maintained the tissue integrity. After the automated MALDI matrix deposition, the entire lens section was examined by MALDI MSI in a 150 μm raster. We obtained spatial- and concentration-dependent distributions of seven lens sphingomyelins (SM) and two ceramide-1-phosphates (CerP), which are important lipid second messengers. Glycosylated sphingolipids or sphingolipid breakdown products were not observed. Owing to ultra high resolution MS, all lipids were identified with high confidence, and distinct distribution patterns for each of them are presented. The distribution patterns of SMs provide an understanding of the physiological functioning of these lipids in clear lenses and offer a novel pathophysiological means for understanding diseases of the lens.     

Natural products in Glycyrrhiza glabra (licorice) rhizome imaged at the cellular level by atmospheric pressure matrix‐assisted laser desorption/ionization tandem mass spectrometry imaging

The rhizome of Glycyrrhiza glabra (licorice) was analyzed by high‐resolution mass spectrometry imaging and tandem mass spectrometry imaging. An atmospheric pressure matrix‐assisted laser desorption/ionization imaging ion source was combined with an orbital trapping mass spectrometer in order to obtain high‐resolution imaging in mass and space. Sections of the rhizome were imaged with a spatial resolution of 10 μm in the positive ion mode, and a large number of secondary metabolites were localized and identified based on their accurate mass and MS/MS fragmentation patterns. Major tissue‐specific metabolites, including free flavonoids, flavonoid glycosides and saponins, were successfully detected and visualized in images, showing their distributions at the cellular level. The analytical power of the technique was tested in the imaging of two isobaric licorice saponins with a mass difference of only 0.02 Da. With a mass resolving power of 140 000 and a bin width of 5 ppm in the image processing, the two compounds were well resolved in full‐scan mode, and appeared with different distributions in the tissue sections. The identities of the compounds and their distributions were validated in a subsequent MS/MS imaging experiment, thereby confirming their identities and excluding possible analyte interference. The use of high spatial resolution, high mass resolution and tandem mass spectrometry in imaging experiments provides significant information about the biosynthetic pathway of flavonoids and saponins in legume species, combing the spatially resolved chemical information with morphological details at the microscopic level. Furthermore, the technique offers a scheme capable of high‐throughput profiling of metabolites in plant tissues.     

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.     

Matrix‐free UV‐laser desorption/ionization (LDI) mass spectrometric imaging at the single‐cell level: distribution of secondary metabolites of Arabidopsis thaliana and Hypericum species

The present paper describes matrix‐free laser desorption/ionisation mass spectrometric imaging (LDI‐MSI) of highly localized UV‐absorbing secondary metabolites in plant tissues at single‐cell resolution. The scope and limitations of the method are discussed with regard to plants of the genus Hypericum. Naphthodianthrones such as hypericin and pseudohypericin are traceable in dark glands on Hypericum leaves, placenta, stamens and styli; biflavonoids are also traceable in the pollen of this important phytomedical plant. The highest spatial resolution achieved, 10 μm, was much higher than that achieved by commonly used matrix‐assisted laser desorption/ionization (MALDI) imaging protocols. The data from imaging experiments were supported by independent LDI‐TOF/MS analysis of cryo‐sectioned, laser‐microdissected and freshly cut plant material. The results confirmed the suitability of combining laser microdissection (LMD) and LDI‐TOF/MS or LDI‐MSI to analyse localized plant secondary metabolites. Furthermore, Arabidopsis thaliana was analysed to demonstrate the feasibility of LDI‐MSI for other commonly occurring compounds such as flavonoids. The organ‐specific distribution of kaempferol, quercetin and isorhamnetin, and their glycosides, was imaged at the cellular level.     

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.     

Specific MALDI imaging and profiling for biomarker hunting and validation: fragment of the 11S proteasome activator complex, Reg alpha fragment, is a new potential ovary cancer biomarker

MALDI imaging mass spectrometry represents a new analytical tool to directly provide the spatial distribution and relative abundance of proteins in tissue. Twenty-five ovary carcinomas (stages III and IV) and 23 benign ovaries were directly analyzed using MALDI-TOF MS. The biomarker with the major prevalence (80%) has been fully identified using MALDI MS and nanoESI MS and MS/MS after separation by RP-HPLC and trypsin enzymatic digestion. This marker with an m/z of 9744 corresponds to 84 amino acid residues from the 11S proteasome activator complex, named PA28 or Reg-alpha. Validation of this marker has been performed using MALDI imaging, classical immunocytochemistry with an antibody raised against the C-terminal part of the protein, specific MALDI imaging, and Western blot analysis. The validation, using immunocytochemistry, confirmed the epithelial localization of this fragment with nucleus localization in benign epithelial cells and a cytoplasmic localization in carcinoma cells. This indicates that this antibody could be used to discriminate the borderline tumor cases. At this point, a multicentric study needs to be conducted in order to clearly establish the potential of this biomarker. Taken together these studies reflect that direct tissue analysis and specific MALDI imaging strategies facilitate biomarker hunting and validation which can be named pathological proteomics.