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.     

Imaging mass spectrometry at cellular length scales

Imaging mass spectrometry (IMS) allows the direct investigation of both the identity and the spatial distribution of the molecular content directly in tissue sections, single cells and many other biological surfaces. In this protocol, we present the steps required to retrieve the molecular information from tissue sections using matrix-enhanced (ME) and metal-assisted (MetA) secondary ion mass spectrometry (SIMS) as well as matrix-assisted laser desorption/ionization (MALDI) IMS. These techniques require specific sample preparation steps directed at optimal signal intensity with minimal redistribution or modification of the sample analytes. After careful sample preparation, different IMS methods offer a unique discovery tool in, for example, the investigation of (i) drug transport and uptake, (ii) biological processing steps and (iii) biomarker distributions. To extract the relevant information from the huge datasets produced by IMS, new bioinformatics approaches have been developed. The duration of the protocol is highly dependent on sample size and technique used, but on average takes approximately 5 h.     

Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological tissues at the hundreds to tens of microns scale. When performed under ambient conditions, sample pre-treatment becomes unnecessary, thus simplifying the protocol while maintaining the high quality of information obtained. Desorption electrospray ionization (DESI) is a spray-based ambient MSI technique that allows for the direct sampling of surfaces in the open air, even in vivo. When used with a software-controlled sample stage, the sample is rastered underneath the DESI ionization probe, and through the time domain, m/z information is correlated with the chemical species'' spatial distribution. The fidelity of the DESI-MSI output depends on the source orientation and positioning with respect to the sample surface and mass spectrometer inlet. Herein, we review how to prepare tissue sections for DESI imaging and additional experimental conditions that directly affect image quality. Specifically, we describe the protocol for the imaging of rat brain tissue sections by DESI-MSI.     

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.     

Application of Mass Spectrometry in Proteomics

Mass spectrometry has arguably become the core technology in proteomics. The application of mass spectrometry based techniques for the qualitative and quantitative analysis of global proteome samples derived from complex mixtures has had a big impact in the understanding of cellular function. Here, we give a brief introduction to principles of mass spectrometry and instrumentation currently used in proteomics experiments. In addition, recent developments in the application of mass spectrometry in proteomics are summarised. Strategies allowing high-throughput identification of proteins from highly complex mixtures include accurate mass measurement of peptides derived from total proteome digests and multidimensional peptide separations coupled with mass spectrometry. Mass spectrometric analysis of intact proteins permits the characterisation of protein isoforms. Recent developments in stable isotope labelling techniques and chemical tagging allow the mass spectrometry based differential display and quantitation of proteins, and newly established affinity procedures enable the targeted characterisation of post-translationally modified proteins. Finally, advances in mass spectrometric imaging allow the gathering of specific information on the local molecular composition, relative abundance and spatial distribution of peptides and proteins in thin tissue sections.     

Compositional and molecular species analysis of phospholipids by high performance liquid chromatography coupled with chemical ionization mass spectrometry

High performance liquid chromatography (HPLC) was combined with chemical ionization mass spectrometry (CIMS) by the use of a moving-belt interface. The technique was employed for the analysis of naturally occurring phospholipids. Positive and negative ion mass spectra of various phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and sphingomyelin were obtained in the chemical ionization mode with ammonia or methane as the reagent gas. Specific ions for individual phospholipid "bases" were identified. These ions were used in specific ion monitoring of the phospholipids during HPLC-CIMS. CIMS of each phospholipid also provided extensive information on the molecular species of the individual class of phospholipids. Relative abundance of different molecular species of each phospholipid as determined by CIMS agreed well with the results obtained by gas-liquid chromatography. Rat brain phospholipids were analyzed by HPLC-CIMS in about 15 minutes. Routinely, about 5 micrograms of individual phospholipid was analyzed by HPLC-CIMS, however, with specific ion monitoring the method provides a detection capability at the subnanogram level.     

Proteohistography--direct analysis of tissue with high sensitivity and high spatial resolution using ProteinChip technology.

On the proteomic level, all tissues, tissue constituents, or even single cells are heterogeneous, but the biological relevance of this cannot be adequately investigated with any currently available technique. The analysis of proteins of small tissue areas by any proteomic approach is limited by the number of required cells. Increasing the number of cells only serves to lower the spatial resolution of expressed proteins. To enhance sensitivity and spatial resolution we developed Proteohistography. Laser microdissection was used to mark special areas of interest on tissue sections attached to glass slides. These areas were positioned under microscopic control directly on an affinity chromatographic ProteinChip Array so that cells were lysed and their released proteins bound on a spatially defined point. The ProteinChip System, surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS), allows the laser to be steered to up to 215 distinct positions across the surface of the spot, enabling a high spatial resolution of measured protein profiles for the analyzed tissue area. Protein profiles of the single positions were visually plotted over the used tissue section to visualize distribution proteohistologically. Results show that the spatial distribution of detectable proteins could be used as a Proteohistogram for a given tissue area. Consequently, this procedure can provide additional information to both a matrix-assisted laser desorption/ionization (MALDI)-based approach and immunohistochemistry, as it is more sensitive, highly quantitative, and no specific antibody is needed. Hence, proteomic heterogeneity can be visualized even if proteins are not known or identified.     

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.     

Depicting the Spatial Distribution of Proteins in Human Tumor Tissue Combining SELDI and MALDI Imaging and Immunohistochemistry

Carcinoma tissue consists of not only tumor cells but also fibroblasts, endothelial cells or vascular structures, and inflammatory cells forming the supportive tumor stroma. Therefore, the spatial distribution of proteins that promote growth and proliferation in these complex functional units is of high interest. Matrix-assisted laser desorption/ionization imaging mass spectrometry is a newly developed technique that generates spatially resolved profiles of protein signals directly from thin tissue sections. Surface-enhanced laser desorption/ionization mass spectrometry (MS)combined with tissue microdissection allows analysis of defined parts of the tissue with a higher sensitivity and a broader mass range. Nevertheless, both MS-based techniques have a limited spatial resolution. IHC is a technique that allows a resolution down to the subcellular level. However, the detection and measurement of a specific protein expression level is possible only by semiquantitative methods. Moreover, prior knowledge about the identity of the proteins of interest is necessary. In this study, we combined all three techniques to gain highest spatial resolution, sensitivity, and quantitative information. We used frozen tissue from head and neck tumors and chose two exemplary proteins (HNP1–3 and S100A8) to highlight the advantages and disadvantages of each technique. It could be shown that the combination of these three techniques results in congruent but also synergetic data. (J Histochem Cytochem 58:929–937, 2010)     

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.     

New preparation techniques for molecular and in‐situ analysis of ancient organic micro‐ and nanostructures

Organic microfossils preserved in three dimensions in transparent mineral matrices such as cherts/quartzites, phosphates, or carbonates are best studied in petrographic thin sections. Moreover, microscale mass spectrometry techniques commonly require flat, polished surfaces to minimize analytical bias. However, contamination by epoxy resin in traditional petrographic sections is problematic for the geochemical study of the kerogen in these microfossils and more generally for the in situ analysis of fossil organic matter. Here, we show that epoxy contamination has a molecular signature that is difficult to distinguish from kerogen with time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS). This contamination appears pervasive in organic microstructures embedded in micro‐ to nano‐crystalline carbonate. To solve this problem, a new semi‐thin section preparation protocol without resin medium was developed for micro‐ to nanoscale in situ investigation of insoluble organic matter. We show that these sections are suited for microscopic observation of Proterozoic microfossils in cherts. ToF‐SIMS reveals that these sections are free of pollution after final removal of a <10 nm layer of contamination using low‐dose ion sputtering. ToF‐SIMS maps of fragments from aliphatic and aromatic molecules and organic sulfur are correlated with the spatial distribution of organic microlaminae in a Jurassic stromatolite. Hydrocarbon‐derived ions also appeared correlated with kerogenous microstructures in Archean cherts. These developments in analytical procedures should help future investigations of organic matter and in particular, microfossils, by allowing the spatial correlation of microscopy, spectroscopy, precise isotopic microanalyses, and novel molecular microanalyses such as ToF‐SIMS.     

New developments in profiling and imaging of proteins from tissue sections by MALDI mass spectrometry

Molecular imaging of tissue by MALDI mass spectrometry is a powerful tool for visualizing the spatial distribution of constituent analytes with high molecular specificity. Although the technique is relatively young, it has already contributed to the understanding of many diverse areas of human health. 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. The purpose of this review is to highlight some of the more recent technological advances that have improved the efficiency of imaging mass spectrometry for clinical applications. Advances in the way MALDI mass spectrometry is integrated with histology, improved methods for automation, and better tools for data analysis are outlined in this review. Refined top-down strategies for the identification and validation of candidate biomarkers found in tissue sections are discussed. A clinical example highlighting the application of these methods to a cohort of clinical samples is described.     

Mapping of phospholipids by MALDI imaging (MALDI-MSI): realities and expectations

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has emerged as a novel powerful MS methodology that has the ability to generate both molecular and spatial information within a tissue section. Application of this technology as a new type of biochemical lipid microscopy may lead to new discoveries of the lipid metabolism and biomarkers associated with area-specific alterations or damage under stress/disease conditions such as traumatic brain injury or acute lung injury, among others. However there are limitations in the range of what it can detect as compared with liquid chromatography-MS (LC-MS) of a lipid extract from a tissue section. The goal of the current work was to critically consider remarkable new opportunities along with the limitations and approaches for further improvements of MALDI-MSI. Based on our experimental data and assessments, improvements of the spectral and spatial resolution, sensitivity and specificity towards low abundance species of lipids are proposed. This is followed by a review of the current literature, including methodologies that other laboratories have used to overcome these challenges.     

Layered electrophoretic transfer - A method for pre-analytic processing of histological sections

Current technologies for measuring protein expression across a tissue section are based on MS or in situ detection such as immunohistochemistry. However, due to the inherent molecular complexity of tissue samples and the large dynamic range of protein expression in cells, current approaches are often unable to measure moderate- and low-abundant proteins. In addition, they do not provide information on the physico-chemical properties of the proteins studied. To address these problems, we are developing a new pre-analytic methodology termed layered electrophoretic transfer (LET) that selectively separates and processes proteins from an intact tissue section without compromising important two-dimensional histological information. LET offers two potential advantages over standard techniques: (i) A reduced complexity of the tissue proteome for subsequent analysis; (ii) An opportunity to assess the biochemical status of proteins as they exist in situ. As an initial proof-of-concept, we demonstrate here that the protein content from a mixture of molecular weight standards, human tissue lysates, and tissue sections can be successfully transferred and separated using LET, and further demonstrate that the method can be coupled with immunoblotting or MS for downstream measurements. LET technology represents a new pre-analytic tool for interrogating the proteome in tissue sections while preserving valuable spatial information.     

Assessing protein patterns in disease using imaging mass spectrometry

Direct tissue profiling and imaging mass spectrometry (MS) provides a detailed assessment of the complex protein pattern within a tissue sample. MALDI MS analysis of thin tissue sections results in over of 500 individual protein signals in the mass range of 2 to 70 kDa that directly correlate with protein composition within a specific region of the tissue sample. To date, profiling and imaging MS has been applied to multiple diseased tissues, including human gliomas and nonsmall cell lung cancer. Interrogation of the resulting complex MS data sets has resulted in identification of both disease-state and patient-prognosis specific protein patterns. These results suggest the future usefulness of proteomic information in assessing disease progression, prognosis, and drug efficacy.     

Mass spectrometry image correlation: quantifying colocalization

A typical imaging mass spectrometry data set can contain 100+ images, each describing the distribution of a specific biomolecule. Multivariate and hierarchical clustering techniques have been developed to investigate the correlations within a data set, and have revealed the differential patterns associated with different organs/anatomical features. These methods do not quantify the correlations between the hundreds of molecular distributions produced in an imaging mass spectrometry experiment, and are extremely difficult to apply to multiple tissue section investigations. This latter aspect includes quantifying the correlation between the results of repeat imaging mass spectrometry experiments, a crucial aspect for determining the significance of any measured changes in distribution. To date, the large chemical background and pixel-to-pixel variation in the images has limited the quantification of correlation between imaging mass spectrometry results. Here, we demonstrate how to quantify the correlations between imaging mass spectrometry images, both within a data set and between data sets.     

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.     

Imaging of atomic and molecular species in tissue with Laser-SNMS for pharmaceutical studies

The success of several anti-cancer therapies as well as other therapeutic and diagnostic strategies relies on the ability to selectively deliver compounds to target cells while sparing normal tissue. For many applications, however, current analytical methods lack the sensitivity and selectivity necessary to determine the distribution of pharmaceutical ultra-trace compounds within tissues with sub-cellular resolution. Laser secondary neutral mass spectrometry (Laser-SNMS) and time-of-flight secondary ion mass spectrometry (TOF-SIMS) are capable of detecting atoms and molecules with high sensitivity and a spatial resolution of up to 100 nm. The use of such methods requires special preparation techniques which preserve the morphological and chemical integrity of the living cell. Laser-SNMS was used to verify the effectiveness of the delivery process for various pharmaceutical compounds in animal studies. After injection of the pharmaceuticals, different types of mouse tissue such as brain, kidney and tumors were extracted, then prepared on a special specimen carrier and subsequently plunged with high velocity into LN₂-cooled propane for cryofixation. After trimming, the tissue block was freeze-dried. For postionization of sputtered neutrals, a laser beam with a wavelength of 193 nm was used. Ion-induced electron images showed that the structural and chemical integrity of the cells had been preserved. Cell-specific elemental and molecular signals could be used to identify individual cells and cell nuclei. The obtained data yield information about the distribution of the pharmaceutical products in different kinds of tissue.     

Multiple statistical analysis techniques corroborate intratumor heterogeneity in imaging mass spectrometry datasets of myxofibrosarcoma

MALDI mass spectrometry can generate profiles that contain hundreds of biomolecular ions directly from tissue. Spatially-correlated analysis, MALDI imaging MS, can simultaneously reveal how each of these biomolecular ions varies in clinical tissue samples. The use of statistical data analysis tools to identify regions containing correlated mass spectrometry profiles is referred to as imaging MS-based molecular histology because of its ability to annotate tissues solely on the basis of the imaging MS data. Several reports have indicated that imaging MS-based molecular histology may be able to complement established histological and histochemical techniques by distinguishing between pathologies with overlapping/identical morphologies and revealing biomolecular intratumor heterogeneity. A data analysis pipeline that identifies regions of imaging MS datasets with correlated mass spectrometry profiles could lead to the development of novel methods for improved diagnosis (differentiating subgroups within distinct histological groups) and annotating the spatio-chemical makeup of tumors. Here it is demonstrated that highlighting the regions within imaging MS datasets whose mass spectrometry profiles were found to be correlated by five independent multivariate methods provides a consistently accurate summary of the spatio-chemical heterogeneity. The corroboration provided by using multiple multivariate methods, efficiently applied in an automated routine, provides assurance that the identified regions are indeed characterized by distinct mass spectrometry profiles, a crucial requirement for its development as a complementary histological tool. When simultaneously applied to imaging MS datasets from multiple patient samples of intermediate-grade myxofibrosarcoma, a heterogeneous soft tissue sarcoma, nodules with mass spectrometry profiles found to be distinct by five different multivariate methods were detected within morphologically identical regions of all patient tissue samples. To aid the further development of imaging MS based molecular histology as a complementary histological tool the Matlab code of the agreement analysis, instructions and a reduced dataset are included as supporting information.     

Imaging mass spectrometry in biomarker discovery and validation

Biomarker discovery and validation involves the consideration of many issues and challenges in order to be effectively used for translation from bench to bedside. Imaging mass spectrometry (IMS) is a new technology to assess spatial molecular arrangements in tissue sections, going far beyond microscopy in providing hundreds of different molecular images from a single scan without the need of target-specific reagents. The possibility to correlate distribution maps of multiple analytes with histological and clinical features makes it an ideal tool to discover diagnostic and prognostic markers of diseases. Some recently published studies that show the usefulness and advantages of this technology in the field of cancer research are highlighted.