Profiling and Imaging Proteins
in Tissue Sections by MS MALDI imaging MS allows simultaneous mapping of hundreds of peptides and proteins in thin tissue sections with a lateral resolution of ~30–50 µm.
PIERRE CHAURAND SARAH A. SCHWARTZ RICHARD M. CAPRIOLI
D
Vanderbilt University
esorption and ionization techniques such as MALDI MS and significant improvements in TOF mass spectrometers have literally revolutionized our ability to analyze proteins. They offer levels of sensitivity and mass accuracy never before achieved for detecting, identifying, and characterizing proteins (1–4). Molecular weights >200 kDa and mass measurements accurate to low parts per million can now be routinely determined. The rapid expansion of protein and gene databases has greatly facilitated protein identification. Modern mass spectrometers can rapidly map and fragment peptides that result from protease digestion and obtain sequence information to identify proteins. MALDI MS utilizes a matrix that is generally a small acidic aromatic molecule that absorbs energy at the wavelength of the irradiating laser. The analyte molecule is mixed with the matrix in a ratio of typically 1/5000, deposited on a target plate, and allowed to dry. During drying, matrix–analyte co-crystals form. These crystals are subjected to very short laser pulses (usually UV laser light), which results in desorption and ionization of the analyte. Mostly intact protonated molecular ions ([M+H]+, in which M is the molecular weight of the analyte) are formed; the ions’ m/z are measured in a TOF mass analyzer. A recent application of MALDI MS is profiling and imaging proteins directly from thin tissue sections (5, 6; Figure 1). Tissue sections ~10–20-µm thick are cut on a cryostat from frozen block tissue and transferred to target plates. Matrix is applied, either as discrete droplets (spots) or as an even coating, and the sample is then placed into the © 2004 AMERICAN CHEMICAL SOCIETY
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Slice frozen tissue on cryostat (~12 mm thick)
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FIGURE 1. Steps involved in profiling and imaging MS of mammalian tissue samples.
MS source. A laser beam irradiates the sample at discrete spots with 30–200 shots/spot. An array of spots, or pixels, covers the surface at intervals that define the resolution of the image that will be generated. In the profiling experiment, the focus is comparing protein from a discrete number of matrix spots or areas. Although no precise definition exists, typically up to 10 spots on a given tissue are compared. In the imaging experiment, the goal is to get full coverage and display a molecular image of the proteins within an entire sample or a defined area. The resolution of the image depends primarily on the size and spacing of the pixels. In a high-resolution image, many thousands of pixels can define the array, and each one is associated with a full mass spectrum, for example, m/z 1000–100,000. Typically, each pixel is ~30–50 µm in diameter and pixels are 50–150 µm from center to center, depending on the imaging resolution required. The MS data are then acquired by using a predetermined number of laser shots per grid coordinate. The signal intensity for each m/z value at every acquisition coordinate is integrated, and a 2-D ion density map, or image, is reconstructed. An image can be generated for each mass signal detected throughout the section.
From a single acquisition, several hundred images, each at a specific molecular weight, can be reconstructed. Recently, several methodologies have been developed to investigate cells, small tissue fragments, and microorganisms (7– 16). However, in most cases, only peptides and low-molecularweight proteins were observed. In early experiments, Caprioli and collaborators used a novel approach to transfer proteins by blotting fresh tissues onto an active C18-coated surface (17 ). Matrix was then applied by electrospray over the blotted area prior to analysis. Blotting establishes physical contact between the tissue and the C18 surface that maintains tissue orientation and geometry, allowing an MS “image” of the imprint to be obtained. Regions of protein localization across the sample were observed by monitoring the detected m/z signals. In this experiment, numerous images of neuropeptides and proteins from the rat pituitary gland were obtained with a spatial resolution of ~25 µm. Other organic surfaces have been successfully tested for protein transfer from fresh tissue sections. Chaurand et al. used polyethylene as a transfer medium to profile proteins in healthy (18) and cancerous (19) colon tissue. This approach was also used to profile proteins in healthy and cancerous mouse prostates, an experiment in which a number of protein cancer markers were identified (20). From these profiles, typically >300 distinct mass signals were observed in an m/z range of 2000– 100,000 (6, 21). A major advance in MALDI MS imaging was the direct analysis of thin tissue sections (22). For high-resolution ion images, the matrix must be evenly deposited over the entire surface of the section. Specialized instrument-control software programs are used to set a data acquisition grid that defines a discrete Cartesian pattern across the sample surface (23 –26); these same software programs are also used for data acquisition and image reconstruction. Stoeckli et al. have mapped and identified several proteins found to have sharp localization patterns in a human glioma (22). Amyloid peptides have been mapped in mouse brains that show features similar to those present in Alzheimer’s disease (26). The A-NTP neuropeptide has been mapped in the atrial gland of Aplysia california (27). Yanagisawa et al. have identified protein patterns in tumor subsets of non-small-cell lung cancer that accurately classify and predict histological groups and survival (28). Chaurand and colleagues profiled and mapped proteins along the mouse epididymis (29).
Sample preparation After dissection, the tissue sample should be loosely wrapped in aluminum foil, immediately snap-frozen in liquid nitrogen, and stored at –80 °C until needed. Thin frozen sections are typically
Imaging MS is an extraordinary discovery tool because it eliminates the need to know in advance the specific proteins that may have changed in a comparative study.
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cut at –15 °C by using a cryostat (optimum cutting temperature is tissue-dependent). The tissue sample is maintained with the desired orientation on the cutting block by using a medium such as optimum cutting temperature (OCT) polymer. However, it is important to note that OCT is only used to mount the tissue on the cryostat block, leaving the surface of tissue free of polymers and available for sectioning. We have found that if the tissue is embedded in or comes in contact with OCT during sectioning, a film of the polymer is deposited on the surface of the sections, which degrades the MALDI MS signal quality (30). Tissue sections are typically cut at a thickness of 10–20 µm because they can usually be manipulated with minimal tearing. Thinner sections are difficult to handle, and thicker sections can warp and crack as they dry. Sections are thawmounted on flat MALDI target plates (Figure 2a) and allowed to dehydrate for 1–2 h in a vacuum desiccator prior to matrix deposition and analysis. Tissue sections may also be kept sealed under nitrogen and stored at –80 °C for many months with minimal signal loss (30). Depending on the analytical task at hand, the MALDI matrix can either be deposited as individual droplets (spotted) or as a homogeneous layer (coated) onto the tissue section (Figure 1). We have found sinapinic acid (saturated in 50/ 50/0.1 by volume acetonitrile/ H2O/trifluoroacetic acid) to be the matrix of choice for the analysis of proteins (30), although -cyano-4-hydroxycinnamic acid may also be used for peptides and lower-molecularweight proteins (27, 31). With this solvent system, essentially
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FIGURE 2. MALDI MS profiling of a healthy mouse brain section. (a) Photomicrograph of a 12-µm coronal mouse brain section (bregma +0.70 mm) mounted on a MALDI target plate and spotted with sinapinic acid matrix; 1, cerebral cortex; 2, corpus callosum; 3, striatum. (Bregma is the point in the skull where the frontal bone meets the parietal bones; +0.70 mm designates the location of the section.) (b) Close-up of a MALDI droplet. Spectra of protein profiles from (c) cerebral cortex, (d) striatum, and (e) corpus callosum. Only the most intense signals are labeled. Colored inset details the protein profiles in the m/z range 7200–8500 for (top to bottom) cerebral cortex, striatum, and corpus callosum.
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Small volumes of matrix (200–500 nL) can be directly deposited onto a tissue section by using an automatic pipette. Smaller droplet sizes (5–50 nL) can be applied with a fine capillary attached to a Hamiltontype syringe that contains the 100% 0 matrix; deposition is performed (e) (f) (g) under low to medium magnification to deposit the matrix droplets precisely at the desired tissue coordinates. The resulting matrix spots have a crystal density that covers ~50% of the area with individual crystals ~20–200 µm in length (Figm/z 11,308 m/z 18,412 m/z 9978 ures 2a and b). Upon analysis, the mass spectra typically (b) yield 300–1000 or more sig100 nals of various intensities over 3–4 orders of magnitude in a m/z range that starts at 2000 80 and may, in some cases, exceed 200,000 (6). However, because of the difficulty that MALDI 60 TOF mass spectrometers have in resolving and detecting X30 higher-molecular-weight com40 pounds (32–34), most of the signals detected are below m/z 30,000. It is also presumed 20 that the most intense signals come from the most abundant protein species. A key goal of sample prepa0 ration for high-resolution im2000 8600 15,200 21,800 28,400 35,000 aging is to deposit the MALDI Mass (m/z) matrix on the sample in an even coating without causing major lateral migration of the proFIGURE 3. Imaging MS of a healthy mouse brain section. teins on the surface of the sec(a) Photomicrograph of a 12-µm hematoxylin- and eosin-stained section (bregma +0.74 mm) that shows diftion. There are many methods ferent anatomic brain substructures; 1, cerebral cortex; 2, corpus callosum; 3, striatum. (b) Survey protein profile obtained after homogeneous matrix deposition and image acquisition from a subsequent serial secof coating the tissue, including tion. Colored inset displays the profile between m/z 8000 and 10,500. (c–g) Ion density maps obtained at dried droplet addition, nebudifferent m/z values with an imaging resolution of 50 µm. The ion density maps are depicted as pseudolization, and acoustic and eleccolor images with white representing the highest protein concentration and black the lowest. trospray deposition. No method is best for all cases, but what follows is generally suitable for soluble (hydrophilic) proteins are rendered accessible for MALDI many tissues. When droplets of matrix solution are placed on the MS. The data presented in this article were acquired on an tissue, solubilized proteins from the section may migrate within Applied Biosystems DE-STR MALDI TOF mass spectrometer. the volume of the droplet and precise localization of these comAcquisition was performed in the linear mode under 25 kV of pounds within the covered area is not possible. In an attempt to accelerating voltage and optimized delayed extraction conditions, “fix” the proteins during the coating process, the sections mountwhich allowed for optimum sensitivity and resolution across the ed on the target plate are soaked in an ethanol solution that contains matrix (sinapinic acid, 20 mg/mL in 90/10/0.1 ethanol/ entire observed mass range. A N A LY T I C A L C H E M I S T R Y / M A R C H 1 , 2 0 0 4
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corpus callosum differs substantially from the other two. Signals such as m/z 6488, 12,168, and 21,888 were more abundant or unique to the corpus callosum. Figure 3a is a photomicrograph of another 12-µm coronal mouse brain section mounted on a glass slide and stained with hematoxylin and eosin. This stained section is shown to high-
H2O/trifluoroacetic acid) for 10 min and allowed to dry at room temperature. Researchers have analyzed by MALDI MS juxtaposed cells isolated by laser-captured microdissection (35–37) from ethanol-dehydrated thin tissue sections and determined that protein migration is minimal (29, 38). Fixing with an ethanol/matrix solution benefits coating by seeding small matrix crystals on the sections, which are then coated with matrix solution (sinapinic acid, 20 mg/mL in 50/50/0.1 acetonitrile/H2O/trifluoroacetic acid) by using a pneumatic handheld Venturi glass sprayer. At this stage, special care must be taken not to “overwet” the section. On average, 10 spray cycles are necessary to achieve a thin, relatively homogeneous matrix coating that covers the entire tissue section. The coating procedure may be monitored under a microscope to assess crystal size and density (30).
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We have imaged the mouse brain, not only because of its physiological importance in health and disease, but also because it is an excellent model with wellmapped anatomy and bilateral symmetry (5, 21, 22). On coronal brain sections, symmetric features from the left and right hemispheres should display very similar protein profiles. Furthermore, protein migration caused by matrix deposition can be assessed when the substructure-specific protein signals are monitored. Figure 2a is a photomicrograph of a 12-µm coronal brain section from a healthy adult mouse, thaw-mounted on a gold-coated metallic target plate and spotted with sinapinic acid matrix in three different anatomical regions. Spot 1 was deposited on the cerebral cortex, spot 2 on the corpus callosum, and spot 3 on the striatum. Figures 2c–2e present the corresponding protein profiles in the m/z range 2000–25,000. Each profile is the result of the average of 1000 laser shots randomly acquired over the surface within a given matrix spot. The inset details the different protein profiles in the m/z range 7200–8500. Significant differences in protein expression or relative abundances as a function of location were observed. Some signals observed in the striatum, for example, m/z 7339 and 7414, were not found at similar levels in the cerebral cortex, although the spectra are similar. The profile obtained from the
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FIGURE 4. MALDI MS profiling of a mouse brain with a tumor. Photomicrographs of serial 12-µm coronal mouse brain sections (a) mounted on a glass slide stained with hematoxylin and eosin (bregma +0.92 mm) and (b) mounted on a MALDI target plate and spotted with matrix. (c) Spectra are the protein profiles from the tumor (T) and nontumor (N) areas, respectively. Only the most intense signals have been labeled.
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FIGURE 5. Imaging MS of a mouse brain with a tumor. (a) Photomicrograph of the section (12-µm thick) before matrix application. The area that contains the tumor is outlined in red. (b–l) Ion density maps obtained at different m/z values with an imaging resolution of 110 µm. The ion density maps are depicted as pseudocolor images with white representing the highest protein concentration and black the lowest.
light substructures such as the cerebral cortex, the corpus callosum, and the striatum. The next serial section was imaged by MS, analyzing a 142 199 data point grid (representing 28,258 mass spectra) with a resolution of 50 µm. Each spectrum is the result of the average of 40 laser shots. Spot-to-spot repositioning and data transfer time takes ~0.5 s, and the total imaging time was 19.6 h. A signal threshold was applied to remove background noise from each spectrum. Figure 3b is a survey protein profile that was obtained by averaging all of the mass spectra acquired from the area of the section. From this profile, hundreds of distinct mass signals, some with very low signal intensities, were observed in the m/z range 2000–35,000. The inset in Figure 3b illustrates the complexity of the data by displaying the profile in the m/z range 8000–10,500, where >50 distinct mass signals were detected. Figures 3c–3g are five ion density maps for different protein signals detected in the survey scan (signals labeled c–g in Figure 3b). As expected, certain signals were expressed in very specific regions of the section. For example, the signal at m/z 18,412 is almost exclusively found in the corpus callosum, whereas the signal at m/z 6720 is most abundant in the striatum. In another set of experiments, GL261 brain cancer cells were injected in vivo into the left hemisphere of a mouse’s brain (39, 40). Two weeks later, the brain was sectioned and analyzed. A 92 A
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tumor of several millimeters developed in the brain’s left lateral ventricle (Figure 4a). Evidence of metastatic migration is seen in the right lateral ventricle (Figure 4a). MS profiles obtained from a serial coronal section (Figure 4b) are shown in Figure 4c (tumor and nontumor tissue areas are labeled T and N, respectively). Each profile is the result of the average of 500 laser shots randomly acquired over the surface of the corresponding matrix spot. Significant differences in the profiles can be observed. For example, the signals at m/z 4965, 6719, 8565, and 12,134 were consistently expressed at relatively higher levels in normal tissue, whereas at m/z 9737, 9910, 11,641, and 12,372 they were consistently expressed at relatively higher levels in the tumor. A high-resolution image analysis of a serial section from the same brain was also performed. Figure 5a is a photomicrograph of a 12-µm section mounted on a target plate prior to matrix application, with the perimeter of the tumor outlined. Imaging was performed with a resolution of 110 µm, averaging 20 laser shots/spectrum, and the only data processing was the removal of background noise. Figures 5b–5l are different protein expression maps across the section. For example, those that show the histones are consistent with the presence of fast-developing tumor cells in a given area (20). Identification of the molecular weight markers of interest is performed by wellestablished methods that extract the proteins from the tissue and separate them by HPLC. After screening by MALDI MS, the HPLC fractions found to contain the targeted molecular weight markers are digested by trypsin; the resulting peptides are sequenced by tandem MS. The proteins are identified by interrogating gene or protein databases with the experimentally recovered sequences (19, 22, 29).
Perspectives Imaging MS needs further development to make it routinely accessible to users. Imaging time depends on several instrumental parameters, such as the laser repetition rate, spot-to-spot sample repositioning, and data processing. Lasers with repetition rates ≥1 kHz and improved electronics will reduce acquisition times from hours to minutes. Data mining tools as well as acquisition algorithms capable of recording high-throughput data are also being developed. Imaging resolution, currently in the 25–50-µm range for tissues, may be increased to 1–5 µm for applications that require subcellular analyses (25). Sample preparation and matrix coating procedures are being developed to achieve homogeneous fields of small crystals (
