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.     

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.     

High resolution spatial distribution of neuropeptides by MALDI imaging mass spectrometry in the terrestrial snail, Helix pomatia

Imaging mass spectrometry (IMS) is a powerful technique that combines the chemical and spatial analysis of surface materials. It allows spatial localization of peptides, proteins or lipids that are recorded in parallel without the need of a label. It is currently one of the most rapidly developing techniques in the proteomics toolbox. In the present study, accurate mass matrix-assisted laser desorption/ionization imaging mass spectrometry (MALD IMS) was used for direct molecular mapping of nervous tissue at micrometer spatial resolution. Cryosections of the whole brain of the terrestrial snail, Helix pomatia, were placed on indium-tin-oxide (ITO)-coated conductive glass slides and covered with a thin layer of α-cyano-4-hydroxycinnamic acid (CHCA) matrix by electro spray deposition. High-resolution molecular ion maps of well-known neuropeptides, such as FMRFamide were constructed. FMRFamide is known to exert powerful modulatory effect on synaptic transmission in molluscs. FMRFamide was predominantly localized in the cluster of neurons in the pro-, meso- and postcerebral regions of cerebral ganglia, pedal ganglia and right parietal ganglia of the central nervous system. Our present study, using MALDI IMS confirmed the distribution of FMRFamide containing cells in the Helix central nervous system previously detected by antibody dependent immunohistochemistry.     

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.     

Comparative proteomics using 2-D gel electrophoresis and mass spectrometry as tools to dissect stimulons and regulons in bacteria with sequenced or partially sequenced genomes

We propose two-dimensional gel electrophoresis (2-DE) and mass spectrometry to define the protein components of regulons and stimulons in bacteria, including those organisms where genome sequencing is still in progress. The basic 2-DE protocol allows high resolution and reproducibility and enables the direct comparison of hundreds or even thousands of proteins simultaneously. To identify proteins that comprise stimulons and regulons, peptide mass fingerprint (PMF) with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF-MS) analysis is the first option and, if results from this tool are insufficient, complementary data obtained with electrospray ionization tandem-MS (ESI-MS/MS) may permit successful protein identification. ESI-MS/MS and MALDI-TOF-MS provide complementary data sets, and so a more comprehensive coverage of a proteome can be obtained using both techniques with the same sample, especially when few sequenced proteins of a particular organism exist or genome sequencing is still in progress.     

Imaging techniques for elements and element species in plant science

Revealing the uptake, transport, localization and speciation of both essential and toxic elements in plants is important for understanding plant homeostasis and metabolism, subsequently, providing information for food and nutrient studies, agriculture activities, as well as environmental research. In the last decade, emerging techniques for elemental imaging and speciation analysis allowed us to obtain increasing knowledge of elemental distribution and availabilities in plants. Chemical imaging techniques include mass spectrometric methods such as secondary ionization mass spectrometry (SIMS), laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and synchrotron-based techniques such as X-ray fluorescence spectroscopy (SRXRF), and so forth. On the other hand, X-ray absorption spectroscopy (XAS) based on synchrotron radiation is capable of in situ investigation of local atomic structure around the central element of interest. This technique can also be operated in tandem with SRXRF to image each element species of interest within plant tissue. In this review, the principles and state-of-the-art of these techniques regarding sample preparation, advantages and limitations, and improvement of sensitivity and spatial resolution are discussed. New results with respect to elemental distribution and speciation in plants revealed by these techniques are presented.     

Mass spectrometry imaging: Towards a lipid microscope?

Biological imaging techniques are the most efficient way to locally measure the variation of different parameters on tissue sections. These analyses are gaining increasing interest since 20 years and allow observing extremely complex biological phenomena at lower and lower time and resolution scale. Nevertheless, most of them only target very few compounds of interest, which are chosen a priori, due to their low resolution power and sensitivity. New chemical imaging technique has to be introduced in order to overcome these limitations, leading to more informative and sensitive analyses for biologists and physicians.Two major mass spectrometry methods can be efficiently used to generate the distribution of biological compounds over a tissue section. Matrix-Assisted Laser Desorption/Ionisation-Mass Spectrometry (MALDI-MS) needs the co-crystallization of the sample with a matrix before to be irradiated by a laser, whereas the analyte is directly desorbed by a primary ion bombardment for Secondary Ion Mass Spectrometry (SIMS) experiments. In both cases, energy used for desorption/ionization is locally deposited -some tens of microns for the laser and some hundreds of nanometers for the ion beam- meaning that small areas over the surface sample can be separately analyzed. Step by step analysis allows spectrum acquisitions over the tissue sections and the data are treated by modern informatics software in order to create ion density maps, i.e., the intensity plot of one specific ion versus the (x,y) position.Main advantages of SIMS and MALDI compared to other chemical imaging techniques lie in the simultaneous acquisition of a large number of biological compounds in mixture with an excellent sensitivity obtained by Time-of-Flight (ToF) mass analyzer. Moreover, data treatment is done a posteriori, due to the fact that no compound is selectively marked, and let us access to the localization of different lipid classes in only one complete acquisition.     

Identification of enzymes and activity from two-dimensional gel electrophoresis

Identification of proteins with enzymatic activity by mass spectrometry (MS) and concomitant determination of function by screening enzyme activity from two-dimensional gel electrophoresis (2DE) is one of the challenges of gel-based proteomics. In this protocol, proteins are extracted from spinal cord tissue followed by 2DE with in-gel digestion and identification by matrix-assisted laser desorption/ionization. Protein spots identified as possible enzyme of interest are punched, eluted by SDS-containing Tris buffer and renatured by buffers under reductive conditions. Enzyme activity is determined using micromethods. Within about 4 weeks, a structural and functional map can be generated and MS identification can be validated, complementing immunochemical methods. 2DE separation can be seen as a prepurification step and therefore allows activity assays from minute amounts of protein as provided in a 2DE gel spot; the method may be an alternative to the time-consuming use of recombinant enzyme techniques.     

Tissue-specific microdissection coupled with ProteinChip array technologies: applications in cancer research

Analysis of whole genomes to monitor specific changes in gene activation or changes in gene copy number due to perturbation has recently become possible using DNA chip technologies. It is now becoming apparent, however, that knowing the genetic sequence encoding a protein is not sufficient to predict the size or biological nature of a protein. This can be particularly important in cancer research where posttranslational modifications of a protein can specifically lead to the disease. To address this area, several proteomic tools have been developed. Currently the most widely used proteomics tool is two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), which can display protein expression patterns to a high degree of resolution. However, 2D-PAGE can be time consuming; the analysis is complicated and, compared with DNA techniques, is not very sensitive. Although some of these problems can be alleviated by using high-quality homogeneous samples, such as those generated using microdissection techniques, the quantity of sample is often limited and may take several days to generate sufficient material for a single 2D-PAGE analysis. As an alternative to 2D-PAGE, a preliminary study using a new technique was used to generate protein expression patterns from either whole tissue extracts or microdissected material. Surface-enhanced laser desorption and ionization allows the retention of proteins on a solid-phase chromatographic surface or ProteinChip Array with direct detection of retained proteins by time-of-flight mass spectrometry. Using this system, we analyzed tumor and normal tissue from head and neck cancer and microdissected melanoma to determine differentially expressed proteins. In particular, comparisons of the protein expression patterns from microdissected normal and tumor tissues indicated several differences, highlighting the importance of extremely defined tissue lysates for protein profiling.     

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.     

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.     

Mass spectrometry: m/z 1983-2008

While definitely not a new technology, mass spectrometry (MS) has seen incredible growth over the past 25 years. Mass spectrometry has rapidly evolved to the forefront of analytical techniques; its ability to analyze proteins is the major driving force in the field of proteomics. MS instrumentation has increased approximately 5-fold in sensitivity every three years. The level of performance that is achievable with MS today allows scientists to study proteins in ways that were inconceivable a quarter century ago. This review of the history of MS over the past 25 years is timely in that it encompasses two of the biggest developments, electrospray and matrix-assisted laser desorption/ionization (MALDI), which have enabled many of the uses of this technology today.     

Direct protein detection from biological media through electrospray-assisted laser desorption ionization/mass spectrometry

We report here using a novel technology-electrospray-assisted laser desorption ionization (ELDI)/mass spectrometry-for the rapid and sensitive detection of the major proteins that exist in dried biological fluids (e.g., blood, tears, saliva, serum), bacterial cultures, and tissues (e.g., porcine liver and heart) under ambient conditions. This technique required essentially no sample pretreatment. The proteins in the samples were desorbed using a pulsed nitrogen laser without the assistance of an organic matrix. The desorbed protein molecules were then post-ionized through their fusion into the charged solvent droplets produced from the electrospray of an acidic methanol solution; electrospray ionization (ESI) proceeded from the newly formed droplets to generate the ESI-like protein ions. This new ionization approach combines some of the features of electrospray ionization with those of matrix-assisted laser desorption ionization (MALDI), that is, sampling of a solid surface with spatial resolution, generating ESI-like mass spectra of the desorbed proteins, and operating under ambient conditions.     

Surface-assisted laser desorption/ionization mass spectrometry

Laser desorption/ionization mass spectrometry (MS) is rapidly growing in popularity as an analytical characterization method in several fields. The technique shot to prominence using matrix-assisted desorption/ionization for large biomolecules (>700 Da), such as proteins, peptides and nucleic acids. However, because the matrix, which consists of small organic molecules, is also ionized, the technique is of limited use in the low-molecular-mass range (<700 Da). Recent advances in surface science have facilitated the development of matrix-free laser desorption/ionization MS approaches, which are referred to here as surface-assisted laser desorption/ionization (SALDI) MS. In contrast to traditional matrix-assisted techniques, the materials used for SALDI-MS are not ionized, which expands the usefulness of this technique to small-molecule analyses. This review discusses the current status of SALDI-MS as a standard analytical technique, with an emphasis on potential applications in proteomics.     

Proteomic analysis of glycosylation: structural determination of N- and O-linked glycans by mass spectrometry

This review summarizes the methods, mainly based on mass spectrometry, for the structural determination of N- and O-linked carbohydrates that are post-translationally attached to a large number of proteins and which play a key role in determining the function and biophysical properties of these compounds. Analysis of these carbohydrates has proved difficult in the past due to their structural complexity. However, modern analytical methods such as mass spectrometry have the ability to elucidate most structural details at the concentration levels required for proteomics. This review describes methods for direct examination of glycoproteins by mass spectrometry, the release of N- and O-linked glycans from glycoproteins separated in sodium dodecyl sulfate polyacrylamide electrophoresis gels, and the analysis of these compounds by techniques such as matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry. Matrix-assisted laser desorption/ionization mass spectrometry provides the most rapid method for comparing glycan profiles and is probably most appropriate for clinical studies. One of the most promising techniques for determining the structures of N-glycans in proteomic studies is negative ion fragmentation of electrosprayed ions. This technique combines high throughput with ease of structural interpretation and provides structural details that are difficult to obtain by classical methods.     

Surface mass spectrometry — application to biosensor characterization

In order to allow a detailed control and specific tailoring of interfaces a surface analytical tool is required that localizes, identifies, and quantifies the molecular structures of interest. Surface mass spectrometry techniques (secondary ion mass spectrometry (SIMS) and laser postionization of sputtered neutrals (SNMS) as well as matrix assisted laser desorption (MALDI)) are such analytical tools.

SIMS is the method of choice for a detailed characterization of the uppermost monolayer. With time-of-flight analysers (TOF-SIMS) elements as well as molecules up to 15,000 u can be identified with a sensitivity down to the ppb and fmol range, respectively. Larger molecules can be investigated by their characteristic fragments in so called fingerprint spectra. By rastering the ion beam across the surface ion images with a lateral resolution of <100 nm (elements) and about 1 μm (molecules) can be obtained. The additional use of lasers for the postionization of sputtered neutrals (laser-SNMS) allows the quantification of the results.

By means of MALDI molecules up to several 100,000 u can be desorbed without fragmentation. In particular, proteins, saccharides and polymer molecules can uniquely be analyzed by this technique.     

Surface-assisted laser desorption/ionization mass spectrometry

Laser desorption/ionization mass spectrometry (MS) is rapidly growing in popularity as an analytical characterization method in several fields. The technique shot to prominence using matrix-assisted desorption/ionization for large biomolecules (>700 Da), such as proteins, peptides and nucleic acids. However, because the matrix, which consists of small organic molecules, is also ionized, the technique is of limited use in the low-molecular-mass range (<700 Da). Recent advances in surface science have facilitated the development of matrix-free laser desorption/ionization MS approaches, which are referred to here as surface-assisted laser desorption/ionization (SALDI) MS. In contrast to traditional matrix-assisted techniques, the materials used for SALDI-MS are not ionized, which expands the usefulness of this technique to small-molecule analyses. This review discusses the current status of SALDI-MS as a standard analytical technique, with an emphasis on potential applications in proteomics.     

Alternative profiling platform based on MELDI and its applicability in clinical proteomics

The presence of numerous proteomics data and their results in literature reveal the importance and influence of proteins and peptides on human cell cycle. For instance, the proteomic profiling of biological samples, such as serum, plasma or cells, and their organelles, carried out by surface-enhanced laser desorption/ionization mass spectrometry, has led to the discovery of numerous key proteins involved in many biological disease processes. However, questions still remain regarding the reproducibility, bioinformatic artifacts and cross-validations of such experimental set-ups. The authors have developed a material-based approach, termed material-enhanced laser desorption/ionization mass spectrometry (MELDI-MS), to facilitate and improve the robustness of large-scale proteomic experiments. MELDI-MS includes a fully automated protein-profiling platform, from sample preparation and analysis to data processing involving state-of-the-art methods, which can be further improved. Multiplexed protein pattern analysis, based on material morphology, physical characteristics and chemical functionalities provides a multitude of protein patterns and allows prostate cancer samples to be distinguished from non-prostate cancer samples. Furthermore, MELDI-MS enables not only the analysis of protein signatures, but also the identification of potential discriminating peaks via capillary liquid chromatography mass spectrometry. The optimized MELDI approach offers a complete proteomics platform with improved sensitivity, selectivity and short sample preparation times.     

Alternative profiling platform based on MELDI and its applicability in clinical proteomics

The presence of numerous proteomics data and their results in literature reveal the importance and influence of proteins and peptides on human cell cycle. For instance, the proteomic profiling of biological samples, such as serum, plasma or cells, and their organelles, carried out by surface-enhanced laser desorption/ionization mass spectrometry, has led to the discovery of numerous key proteins involved in many biological disease processes. However, questions still remain regarding the reproducibility, bioinformatic artifacts and cross-validations of such experimental set-ups. The authors have developed a material-based approach, termed material-enhanced laser desorption/ionization mass spectrometry (MELDI-MS), to facilitate and improve the robustness of large-scale proteomic experiments. MELDI-MS includes a fully automated protein-profiling platform, from sample preparation and analysis to data processing involving state-of-the-art methods, which can be further improved. Multiplexed protein pattern analysis, based on material morphology, physical characteristics and chemical functionalities provides a multitude of protein patterns and allows prostate cancer samples to be distinguished from non-prostate cancer samples. Furthermore, MELDI-MS enables not only the analysis of protein signatures, but also the identification of potential discriminating peaks via capillary liquid chromatography mass spectrometry. The optimized MELDI approach offers a complete proteomics platform with improved sensitivity, selectivity and short sample preparation times.     

Molecular mass spectrometry imaging in biomedical and life science research

This review describes the current state of mass spectrometry imaging (MSI) in life sciences. A brief overview of mass spectrometry principles is presented followed by a thorough introduction to the MSI workflows, principles and areas of application. Three major desorption-ionization techniques used in MSI, namely, secondary ion mass spectrometry (SIMS), matrix-assisted laser desorption ionization (MALDI), and desorption electrospray ionization (DESI) are described, and biomedical and life science imaging applications of each ionization technique are reviewed. A separate section is devoted to data handling and current challenges and future perspectives are briefly discussed at the end.