MALDI mass spectrometric imaging of biological tissue sections

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Abstract

In biomedical research, the discovery of new biomarkers and new drugs demands analytical techniques with high sensitivity together with increased throughput. The possibility to localize or to follow changes in organisms at the molecular level by imaging component distributions of specific tissues, is of prime importance to unravel biochemical pathways and develop new treatments and drugs. Established molecular imaging techniques such as MRI and PET are already widely used, however their need for molecular probes to report the presence of the analytes of interest precludes the simultaneous exploration of different biomolecules. Matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI MSI) takes full advantage of the high sensitivity of mass spectrometry instrumentation but also of the ability of the latter to simultaneously detect a wide range of compounds, almost regardless from their nature and mass. To perform MALDI MSI, sections of biological tissues are introduced in an MALDI MS instrument, where the UV pulsed laser of the MALDI source is used to raster over a selected area while acquiring mass spectra of the ablated ions at every image point. From this array of spectra, hundreds of analyte-specific images can be generated based on the selected masses. MALDI MSI can be used to track biomarkers such as peptides or proteins but also to map drug/tissue interactions. In this paper, an overview of the possibilities of MSI will be given. As an example, MSI on brain tissue sections for the study of Alzheimer's disease (AD) will be shown. Mapping of amyloid peptides as

a new approach for drug lead optimization will be presented. Target identification thanks to MSI will be introduced and the last part will be dedicated to the molecular scanner approach, which gives access to high-mass range by combining tissue blotting and digestion in a one-step process.

Introduction

The proteomics activities developed in drug discovery are mainly aimed at the search of new diagnostic biomarkers, i.e. proteins and more generally analytes which are correlated to disease states and at the identification of biomolecules involved in different pathways which could be used as drug targets. Since proteomic studies have to cope with very complex mixtures, over an extended mass and concentration range (up to 5 and 9 orders of magnitude, respectively), high-resolution separation techniques combined with highly sensitive detection methods are involved in the discovery phase. Mass spectrometry (MS) stands to be the method of choice for the analysis of proteins, peptides, and metabolites due to its unmatched sensitivity and high specificity. The development of matrix-assisted laser desorption/ionization (MALDI) played an important role in reaching today's high sensitivity. In this approach, the analyte is embedded in an excess of matrix, usually a weak organic acid such as sinapinic acid or 2,5-dihydroxybenzoic acid. For a dissolved analyte, a typical sample preparation involves co-spotting with matrix solution on a metal plate and drying. The sample is then introduced in a mass spectrometer, where short UV laser pulses are fired onto the surface to generate protonated analyte ions. The molecular weight of theses ions are typically determined using a time-of-flight (TOF) mass analyzer. New instrumentation allows to directly fragment molecular ions and to acquire daughter ion spectra. Since the fragmentation follows specific rules, these MS/MS spectra can then be used to identify the analyte. The sensitivity of new instrumentation allows to get mass spectra from proteins and peptides from sample amounts as low as attomols.

Depending on the sample used for a proteomic analysis, the result reflects analyte levels in a body fluid, a specific tissue or a compartment in a tissue. A comprehensive analysis often requires the analysis of a small region in a tissue or a group of cells. With this requirement for specific localization, molecular imaging techniques become increasingly relevant in the drug discovery process. In vivo methods, such as X-ray, magnetic resonance imaging (MRI), positron emission tomography (PET), near-infrared fluorescent (NIRF) or bioluminescence imaging, are based on the tagging of specific biomolecules to give access to their localization in real-time. The use of these probes significantly reduces the number of detectable analytes for one experiment set (in the majority of cases to just one), and requires the development of a tag for each analyte group.

The emerging technology MALDI mass spectrometry imaging (MSI) described in this article offers the flexibility of detection without reporter molecules, as well as sensitivity and resolution needed for biological tissues. This ex vivo assay is based on the concept of applying MALDI TOF MS directly on tissue sections.

Currently developed for many applications (Chaurand et al., 2004a; Schwartz et al., 2003, Stoeckli et al., 1999, Stoeckli et al., 2001, Stoeckli et al., 2002), MALDI MS Imaging is achieved by rastering sequentially the surface of a defined area while acquiring a mass spectrum from every location (see Fig. 1). In a typical procedure for MALDI MSI on tissues, a section is attached on a sample plate, and matrix is deposited, either as a thin layer, or as a spot pattern. The sample is then introduced in the mass spectrometer and from each image position, a mass spectrum is acquired. From a data set, multiple images can be extracted, representing the spatial distribution of the analytes of interest. The same data can be used to compare mass spectra from multiple regions in the same sections.

Reports have shown that mass spectra acquired from biological material contain a large number of signals, which could be correlated to proteins present in the tissues. Examples are profiling of single cells (Li et al., 2000, Kruse et al., 2001), cells selected by laser capture microdissection (Bhattacharya et al., 2003, Xu et al., 2002) and protein profiles obtained from tissues sections (Schwartz et al., 2004). In the drug discovery area, MALDI MSI has also successfully been used for target identification (Pierson et al., 2004) and to unravel proteins or peptides as biomarkers involved in disease states (Chaurand et al., 2004b).

Hence, MALDI MSI permits analysis of a wide mass range of biopolymers, complementary to TOF-SIMS, which is relevant for molecular weights smaller than 1 kDa (McArthur et al., 2004, Nygren et al., 2004). A very important aspect in MALDI MSI on tissue sections is the high number of different analyte classes, which can be simultaneously analyzed, including proteins, lipids, carbohydrates and metal ions. This takes benefit of the high dynamic range of the method but suffers from signal suppression effects. Some analytes are indeed more efficiently ionized during MALDI process, depending hardly on their chemical features but also on their relative amount (Knochenmuss et al., 1996, Wang and Fitzgerald, 2001). Interpretation of complex spectra is not straightforward and filters helping to simplify the information content are hardly desirable. One way to reduce the complexity of the analyzed area is to extract proteins from the tissue section with the help of hydrophobic materials, while preserving their relative location. This principle has been successfully applied by contact blotting fresh cut tissue surfaces on a surface prepared with C18 beads (Caprioli et al., 1997), or on a polymeric conductive membrane (Chaurand et al., 1999).

However, MALDI technique also suffers from mass dependent sensitivity drop-off. Efficient detection and identification of proteins in the high mass range (for MW > 30 kDa) is actually one of the key issues for MALDI MSI to be improved. The molecular scanner approach, which was introduced by Hochstrasser and co-workers, is an interesting way to circumvent this problem, as it combines a blotting step with enzymatic digestion (Bienvenut et al., 1999, Bienvenut et al., 2002, Binz et al., 1999, Binz, 2003). The molecular scanner was initially dedicated to 2D gels, in order to identify separated proteins based on their mass fingerprints, i.e. peptides resulting from tryptic digestion, while keeping their initial relative locations. As schematically presented in Fig. 2, the tissue section is transblotted through a membrane, which contains immobilized trypsin, and the resulting tryptic peptides are captured by a second membrane. This capture membrane is then placed on a sample plate covered with matrix and analyzed by MALDI MSI. Based on the molecular weight of the fragment and the MS/MS spectrum of this fragment, the corresponding protein can be determined.

In this article, we illustrate the various options of applying MS Imaging for drug imaging, biomarker discovery and mapping. Methodologies applied to compound, peptide and protein imaging are here described; their advantages and shortcomings are also discussed.

Section snippets

Instrumentation/software

The acquisition of the mass spectra for peptide and protein imaging has been done in linear mode on a commercial MALDI TOF instrument Voyager sSTR (Applied Biosystems, Framingham, MA) equipped with a Nd:YAG laser (Nanolase, Meylan, France) and using a fast digitizer board (DP211, Acqiris SA, Geneva, Switzerland). With these changes with respect to the initial equipment, the laser can be pulsed up to 300 Hz, which is a 100-fold increase in speed compared to the commercial instrument and which

Brain analysis

To demonstrate the power of MALDI MSI, transverse or coronal mouse brain sections have been mapped after being coated with a matrix solution consisting of 20 mg/ml of SA in 50:50 acetonitrile/0.1% trifluoroacetic acid (v/v). The most intense MS signals have been selected, the distribution of the corresponding analytes being presented in Fig. 3. As illustrated by the different distributions, the scanned area is usually discernible on the displayed distribution (tilted in this case). Besides,

Conclusions

The results presented in this article show that MALDI MSI is a very promising analytical tool to be used in biomedical research. Since it bases on mass spectrometry, the sensitivity and the versatility of this imaging technology are the main advantages with respect to other imaging methods.

From a technical point of view, some improvements are actually under development, e.g. the reduction of the laser spot diameter, which could end up with a higher lateral resolution (Spengler and Hubert, 2002

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