- Email: [email protected]
Molecular imaging of thin mammalian tissue sections by mass spectrometry
Molecular imaging of thin mammalian tissue sections by mass spectrometry Pierre Chaurand, D Shannon Cornett and Richard M Caprioli Imaging of tissue sections by mass spectrometry provides a detailed molecular picture containing information on both the abundance and distribution of many constituent compounds. Mass spectra are acquired directly from fresh frozen tissue sections using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS); sample preparation and data collection mode determine the spatial resolution or surface area of the section represented in each mass spectrum. Statistical analyses of the individual ion signatures yield biomarkers whose abundances correlate to cell development processes, tumorigenesis and/or drug treatment. In an alternate mode, the generation of intensity maps for individual ions provides a visual representation of the distribution of each species throughout the section at spatial resolutions as small as 50 mm. The availability of this molecular information is likely to be of great value to clinicians and should lead to improved therapeutic efficacy in the future. Addresses Mass Spectrometry Research Center and Department of Biochemistry, Vanderbilt University, Nashville TN, USA Corresponding author: Caprioli, Richard M ([email protected])
Current Opinion in Biotechnology 2006, 17:431–436 This review comes from a themed issue on Protein technologies Edited by Deb K Chatterjee and Joshua LaBaer Available online 16th June 2006 0958-1669/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2006.06.002
Introduction In the past decade, mass spectrometry (MS) has become an indispensable tool for proteomic studies, that is, the detection, identification and characterization of the protein content of cells, tissues and organs at any time point in both health and disease. For protein analysis, several different types of instruments and protocols allow for the determination of molecular weight, primary and higher order structure, post-translational modifications, quantitation, and localization. Desorption and ionization techniques such as matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) [1] have literally revolutionized our ability to analyze proteins. Improvements in MS instrumentation now offer levels of sensitivity and mass accuracy never before achieved for the detection, identification and structural characterization of proteins. www.sciencedirect.com
It is now possible to routinely measure molecular weights above 200 kDa, as well as to obtain low parts per million mass measurement accuracies for the detection of low femtomolar amounts of peptides. Modern MALDI mass spectrometers can now rapidly map and fragment peptides that result from protease digestion in order to identify and further characterize proteins. Protein identification has also been greatly facilitated owing to the rapid expansion of protein and gene databases and associated search tools, some of which are readily available over the internet. MALDI-MS is particularly useful because of its potential for high-throughput data acquisition together with its ability to provide information on the localization of molecules in a sample [2,3]. Imaging mass spectrometry (IMS) is a MALDI-MS based technology developed in our laboratory for the direct analysis of peptides and proteins from thin tissue sections [4,5]. Sections investigated by IMS clearly show retention of spatial and anatomic relationships of the MS signals, permitting the complex interaction between cells and their environment to be studied at the molecular level [4,6]. From the systematic analysis of a tissue section, protein-specific maps directly correlated with tissue architecture or morphology can be simultaneously obtained from hundreds of different protein species [4,6]. Similar approaches for mapping other types of biocompounds, such as cholesterol and phospholipids, have also been reported [7]. The potential for this type of analysis is particularly exciting for the study of diseases such as cancer [6,8], Duchenne myopathy [9], muscle dystrophy [10], Alzheimer’s [11] and Parkinson’s [12,13]. Although immunohistochemical techniques are routinely used to target known individual proteins, IMS offers the potential for the simultaneous analysis of many molecular species present in a single tumor regardless of the availability of specific antibodies. In addition, IMS is also capable of imaging the distribution of administered pharmaceutical agents [14–17] and resulting metabolites [18] within tissue sections, opening new possibilities for the measurement of concomitant protein changes in specific tissues after systemic drug administration [18]. In this review, we present the basic methodologies for processing tissue specimens, and describe the acquisition and subsequent processing required to visualize IMS measurements.
Methodologies In IMS, protein profiles and images are obtained directly from thin tissue sections cut from fresh frozen tissue specimens [19]. Tissue biopsies or other relevant tissue Current Opinion in Biotechnology 2006, 17:431–436
432 Protein technologies
samples are frozen in liquid nitrogen immediately after sampling to minimize protein degradation through enzymatic proteolysis and taking special care to preserve the sample’s morphology. Typically, for most applications, 10–12 mm thick sections are cut using a cryostat and thawmounted on a flat electrically conductive sample plate. The sections can then be dried in a dessicator for several minutes prior to MALDI matrix deposition. After mounting the sections on the target plates, rinsing procedures have been established to increase the MS data quality. A typical rinsing procedure consists of immersing the sections for 30 s in 70% ethanol followed by a second rinse for 30 s in a mixture of 90% ethanol, 9% acetic acid and 1% deionized water. For most applications, we have found that sinapinic acid (25 mg/ml in 50/50/0.1 acetonitrile/H2O/trifluoroacetic acid) is an excellent matrix for the analysis of proteins [19]. Other matrices have also been successfully used for the analysis of peptides [20,21,22] and lipids [7,23]. Small volumes of matrix (typically 200–500 nL) can be directly spotted onto a tissue section using an automatic pipette. Even smaller volumes (5–50 nL) can be applied with a finely pulled capillary loaded with matrix. In this case, matrix deposition must be performed under low to medium magnification to precisely deposit the matrix droplets at the desired tissue coordinates. Upon analysis, the resulting mass spectra typically yield from 300 to as many as 1000 protein signals within an intensity range of one to three orders of magnitude and in a mass-to-charge (m/z) range from m/z 1000 up to, and in some cases over, m/z 200 000 [24]; however, a large majority of these signals are observed in the m/z range below 30 000. It is believed that the most intense signals come from the most abundant protein species. The exact sensitivity of the technology is hard to estimate, however, because exact quantities of proteins within a specific tissue are generally not well known. The profiles recovered have been found to be highly specific for a given tissue or cell type (Figure 1) and, when analyzing serial sections, are highly reproducible [25]. High reproducibility has also been observed when analyzing sections of the same tissue sampled from different animals or individuals. One of the latest tissue analysis protocols developed in our laboratory utilizes conductive optically transparent glass slides as target plates to accommodate for MALDI-MS friendly tissue staining protocols [25]. This makes possible the microscopic evaluation of a tissue section followed by the molecular imaging of the same section by MS. If desired, these sections can then be stained after matrix removal to exactly correlate the observed protein signals with the local cellular composition observed under the spot [26]. On the basis of protein profiles recovered from control and cancerous human biopsies, specific patterns of protein expression have found to correlate well with histology-based diagnosis and prognosis [27,28,29]. Current Opinion in Biotechnology 2006, 17:431–436
An imaging experiment becomes necessary when highresolution protein distribution information within a tissue section is of importance. This is particularly true when analyzing tumor biopsies, where the precise architectural arrangement of the various cell types present in the section and the differentially expressed molecular weight signals could lead to a better understanding of molecular interactions between a tumor and its surrounding tissue. Highresolution images are also desirable when there is a defined substructure present, such as in brain coronal sections [4,6] or kidney (Figures 1 and 2) [8]. In the imaging experiment, a critical aspect of sample preparation is the homogeneous deposition of the MALDI matrix in such a way as to avoid significant lateral migration of proteins on the surface of the section. Several methods allow for the homogeneous deposition of matrix on tissue sections (i.e. the addition of large individual drops [4,11], spraying by nebulization [19,24,30], electrospray deposition [20], and automated microdispensing of the matrix [31]). The automated microdispensing technique (developed in our laboratory in collaboration with Labcyte Inc. Sunnyvale, CA) utilizes acoustic waves to vertically eject 120 pL nanodroplets of matrix from a reservoir on to the tissue section (facing the reservoir). Some 50 to 70 droplets are typically collected per tissue coordinate to form a matrix spot with a diameter of 200 mm. Cartesian arrays of droplets are deposited to fully cover the section to be analyzed (Figure 1). In this case, if protein delocalization does occur it is limited to the tissue area covered by a droplet. Other spotters are also available, in particular the CHIP chemical spotter from Shimadzu Biotech (Columbia, MD), which is currently being tested in our laboratory for matrix array deposition. For IMS, specialized instrument control software is used to set a data acquisition grid consisting of a discrete Cartesian pattern over the area to be imaged and to control the x/y movement of the sample stage [32]. This pattern has a fixed center-to-center distance between spots, typically 50–300 mm depending on the dimensions of the section and the imaging resolution required. MALDI-MS imaging at higher resolution in the micron range is technically feasible and has been described [33]. The mass spectrometric data is then acquired utilizing this grid pattern with a predetermined number of laser shots per grid coordinate. In the case of microspotted tissue sections, the grid pattern is aligned with the droplet array. Data processing (image reconstruction) is also performed using specialized software by integrating signal intensities at desired m/z values across the dataset (Figure 2) [11]. The time required for data acquisition depends on the area of the tissue to be analyzed, the resolution (number of pixels) required, and the overall acquisition speed of the instrument used. Although the local protein composition can affect the ionization and therefore detection of a monitored protein species through ion suppression effects, to date, good agreements www.sciencedirect.com
Imaging thin tissue sections by MS Chaurand, Cornett and Caprioli 433
Figure 1
Analysis by MALDI imaging MS of a 12 mm thick sagittal section from a rat kidney. (a) Photomicrograph of an H&E (hematoxylin and eosin) stained serial kidney section. The capsule, cortex, medulla and calyx tissue layers are visible and outlined on the image. (b) The upper part of the section (boxed area) was coated with sinapinic acid using an automated matrix spotter at a pitch of 300 mm (see text for details). (c) Unique protein profiles were obtained upon analysis of the different kidney tissues. The color-coded circles indicate the areas from which the spectra were acquired.
have been observed between images obtained by IMS and other protein imaging techniques such as immunohistochemistry [8,34]. With modern MALDI time-offlight mass spectrometers equipped with solid-state UV lasers that operate at repetition rates of several hundreds of Hertz, a 1 cm2 tissue biopsy can be imaged at a resolution of 250 mm in a matter of hours. Other instrument platforms have been described for imaging peptides and proteins from tissue sections. Of noticeable interest is the use of a position-sensitive mass spectrometer [35]. In this case, after homogeneous matrix spray deposition, ion images are acquired in the so-called microscope mode where the requirement for a highly focused ionization beam is removed. The defocused laser beam irradiates a large sample surface of the section and images are reconstructed based on the point of impact of ions with the same m/z on the detector. The broad concepts used for profiling and imaging proteins directly from tissue sections using MALDI-MS have www.sciencedirect.com
been extended to the examination of low molecular weight compounds, particularly pharmaceuticals [14,15,17]. Section handling and preparation techniques are nearly identical to those used for protein imaging. Some specific concerns, however, are unique to the analysis of low molecular weight compounds by MALDI-MS, namely significant matrix-related spectral interferences. Imaging of drugs in tissues is accomplished in an analogous fashion to imaging of proteins, but to increase the specificity and sensitivity, collision-activated dissociation (CAD) is employed [15]. For lower molecular weight compounds, direct imaging of the molecular ion is unreliable, because numerous endogenous compounds as well as potential matrix cluster ions can be observed at the same nominal molecular weight. The general strategy involves selection of the intact drug parent ion, fragmentation of this ion by CAD, and the detection of drugspecific signature m/z fragment ions. In parallel to drug localization, variations of the proteome induced by the drug can be investigated by IMS from a serial section as a Current Opinion in Biotechnology 2006, 17:431–436
434 Protein technologies
Figure 2
Ion density maps constructed after MALDI imaging MS of a 12 mm thick sagittal section from a rat kidney. Twelve different protein signals that are labeled in Figure 1c are depicted as gray-scale images with white representing the highest relative intensity for each m/z value.
function of dosage or time to determine their efficacy [18]. This strategy could be extremely useful to assess positive (or negative) reactions to drug therapy at very early time points.
Conclusions and future perspectives In the coming years, advances in molecular biology and molecular technologies are likely to have a profound impact on diagnostic pathology [8,36]. Molecular information derived from genomics, proteomics and metabolomics will be of crucial importance for the understanding of normal development and pathogenesis at the molecular level, therefore improving therapeutic efficacy and enhancing the quality of information available to clinicians. IMS offers an entirely new and highly precise means of analyzing disease tissue at the molecular level. In a broader context, with continued advances in instrumentation, tissue and data processing, it should be possible to acquire the proteomic profile of a tumor within the time frame currently utilized for interoperative section examination. Such measurements will generate important information that is complementary to traditional methods of histopathologic analysis [8,37]. It is believed that these new approaches will markedly impact three major areas: tissue-based diagnosis, prognosis determination, and prediction of response to specific modes of therapy [8,36]. In addition, the identification of molecular Current Opinion in Biotechnology 2006, 17:431–436
signatures that are precursors or early markers of disease should provide reliable methods of early detection for a broad spectrum of disease processes. The sum of molecular information obtained from a biopsy is likely to significantly impact the course of therapy for a particular tumor and would be available before the patient leaves the operating room.
Acknowledgements The authors would like to thank Serrine Lau and Barbara Leinweb (University of Arizona) for supplying the mouse kidney. The authors also acknowledge support from the NIH (GM 58008-08), and NCI (CA 86243-03 and CA 116123-01).
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
Hillenkamp F, Karas M, Beavis RC, Chait BT: Matrix-assisted laser desorption ionization mass-spectrometry of biopolymers. Anal Chem 1991, 63:1193A-1202A.
2.
Gusev AI, Vasseur OJ, Proctor A, Sharkey AG, Hercules MH: Imaging of thin-layer chromatograms using matrix-assisted laser desorption/ionization mass spectrometry. Anal Chem 1995, 67:4565-4570.
3.
Caprioli RM, Farmer TB, Gile J: Molecular imaging of biological samples: localization of peptides and proteins using MALDITOF MS. Anal Chem 1997, 69:4751-4760. www.sciencedirect.com
Imaging thin tissue sections by MS Chaurand, Cornett and Caprioli 435
4.
5.
Stoeckli M, Chaurand P, Hallahan DE, Caprioli RM: Imaging mass spectrometry: a new technology for the analysis of protein expression in mammalian tissues. Nat Med 2001, 7:493-496. Todd PJ, Schhaaff TG, Chaurand P, Caprioli RM: Organic ion imaging of biological tissue with SIMS and MALDI. J Mass Spectrom 2001, 36:355-369.
6.
Chaurand P, Schwartz SA, Caprioli RM: Profiling and imaging proteins in tissue sections by mass spectrometry. Anal Chem 2004, 76:86A-93A. Review article summarizing tissue section imaging by MALDI-MS. 7.
Jackson SN, Wang HYJ, Woods AS: Direct profiling of lipid distribution in brain tissue using MALDI-TOF MS. Anal Chem 2005, 77:4523-4527.
8.
Chaurand P, Sanders ME, Jensen RA, Caprioli RM: Proteomics in diagnostic pathology: profiling and imaging proteins directly in tissue sections. Am J Pathol 2004, 165:1057-1068. Review article summarizing the clinical potential of protein profiles and images. 9.
Brunelle A, Touboul D, Piednoel H, Voisin V, De La Porte S, Tallarek E, Hagenhoff B, Halgand F, Laprevote O: Direct mapping on muscle tissues of duchenne myopathy biomarkers using both MALDI and cluster-SIMS imaging mass spectrometry. Mol Biol Cell 2004, 15:103A-104A.
10. Touboul D, Piednoel H, Voisin V, De La Porte S, Brunelle A, Halgand F, Laprevote O: Changes in phospholipid composition within the dystrophic muscle by matrix-assisted laser desorption/ionization mass spectrometry and mass spectrometry imaging. Eur J Mass Spectrom 2004, 10:657-664. 11. Stoeckli M, Staab D, Staufenbiel M, Wiederhold K-H, Signor L: Molecular imaging of amyloid beta peptides in mouse brain sections using mass spectrometry. Anal Biochem 2002, 311:33-39. 12. Pierson J, Norris JL, Aerni HR, Svenningsson P, Caprioli RM, Andren PE: Molecular profiling of experimental Parkinson’s disease: direct analysis of peptides and proteins on brain tissue sections by MALDI mass spectrometry. J Proteome Res 2004, 3:289-295. 13. Pierson J, Svenningsson P, Caprioli RM, Andren PE: Increased levels of ubiquitin in the 6-OHDA-lesioned striatum of rats. J Proteome Res 2005, 4:223-226. 14. Troendle FJ, Reddick CD, Yost RA: Detection of pharmaceutical compounds in tissue by matrix-assisted laser desorption/ ionization and laser desorption/chemical ionization tandem mass spectrometry with a quadrupole ion trap. J Am Soc Mass Spectrom 1999, 10:1315-1321. 15. Reyzer ML, Hsieh Y, Ng K, Korfmacher WA, Caprioli RM: Direct analysis of drug candidates in tissue by matrix-assisted laser desorption/ionization mass spectrometry. J Mass Spectrom 2003, 38:1081-1092. 16. Rohner TC, Staab D, Stoeckli M: MALDI mass spectrometric imaging of biological tissue sections. Mech Ageing Dev 2005, 126:177-185. 17. Wang HYJ, Jackson SN, McEuen J, Woods AS: Localization and analyses of small drug molecules in rat brain tissue sections. Anal Chem 2005, 77:6682-6686. 18. Reyzer ML, Caldwell RL, Dugger TC, Forbes JT, Ritter CA, Guix M, Arteaga CL, Caprioli RM: Early changes in protein expression detected by mass spectrometry predict tumor response to molecular therapeutics. Cancer Res 2004, 64:9093-9100. Key reference demonstrating the correlation between the presence of a drug in tumor tissue and its influence on the proteome monitored by imaging mass spectrometry. 19. Schwartz SA, Reyzer ML, Caprioli RM: Direct tissue analysis using matrix-assisted laser desorption/ionization mass spectrometry: practical aspects of sample preparation. J Mass Spectrom 2003, 38:699-708. www.sciencedirect.com
Methodology article detailing the steps necessary for the successful analysis of tissue specimens by MALDI-MS. 20. Kruse R, Sweedler JV: Spatial profiling invertebrate ganglia using MALDI MS. J Am Soc Mass Spectrom 2003, 14:752-759. 21. Fournier I, Day R, Salzet M: Direct analysis of neuropeptides by in situ MALDI-TOF mass spectrometry in the rat brain. Neuroendocrinol Lett 2003, 24:9-14. 22. Lemaire R, Tabet JC, Ducoroy P, Hendra JB, Salzet M, Fournier I: Solid ionic matrixes for direct tissue analysis and MALDI imaging. Anal Chem 2006, 78:809-819. First use of ionic matrices for profiling and imaging peptides and proteins from tissue sections by MALDI-MS. 23. Rujoi M, Estrada R, Yappert MC: In situ MALDI-TOF MS regional analysis of neutral phospholipids in lens tissue. Anal Chem 2004, 76:1657-1663. 24. Chaurand P, Caprioli RM: Direct profiling and imaging of peptides and proteins from mammalian cells and tissue sections by mass spectrometry. Electrophoresis 2002, 23:3125-3135. 25. Chaurand P, Schwartz SA, Billheimer D, Xu BJ, Crecelius A, Caprioli RM: Integrating histology and imaging mass spectrometry. Anal Chem 2004, 76:1145-1155. First use of MALDI-compatible tissue staining procedures and conductive glass slides as section support for profiling and imaging mass spectrometry. 26. Crecelius AC, Cornett DS, Caprioli RM, Williams B, Dawant BM, Bodenheimer B: Three-dimensional visualization of protein expression in mouse brain structures using imaging mass spectrometry. J Am Soc Mass Spectrom 2005, 16:1093-1099. 27. Yanagisawa K, Shyr Y, Xu BJ, Massion PP, Larsen PH, White BC, Roberts JR, Edgerton M, Gonzalez A, Nadaf S et al.: Proteomic patterns of tumour subsets in non-small-cell lung cancer. Lancet 2003, 362:433-439. First demonstration that protein profiles generated by MALDI-MS from lung cancer and control tissue sections can be correlated with patient diagnosis and outcome. 28. Schwartz SA, Weil RJ, Johnson MD, Toms SA, Caprioli RM: Protein profiling in brain tumors using mass spectrometry: feasibility of a new technique for the analysis of protein expression. Clin Cancer Res 2004, 10:981-987. 29. Schwartz SA, Weil RJ, Thompson RC, Shyr Y, Moore JH, Toms SA, Johnson MD, Caprioli RM: Proteomic-based prognosis of brain tumor patients using direct-tissue matrix-assisted laser desorption ionization mass spectrometry. Cancer Res 2005, 65:7674-7681. Demonstration that protein profiles generated by MALDI-MS from brain gliomas and control tissue sections can be correlated with patient diagnosis and outcome. 30. Chaurand P, Schwartz SA, Caprioli RM: Imaging mass spectrometry: a new tool to investigate the spatial organization of peptides and proteins in mammalian tissue sections. Curr Opin Chem Biol 2002, 6:676-681. 31. Aerni HR, Cornett DS, Caprioli RM: Automated acoustic matrix deposition for MALDI sample preparation. Anal Chem 2006, 78:827-834. Key reference showing that arrays of small matrix droplets can be automatically printed on tissue sections for MALDI imaging mass spectrometry. 32. Stoeckli M, Farmer TB, Caprioli RM: Automated mass spectrometry imaging with a matrix-assisted laser desorption ionization time-of-flight instrument. J Am Soc Mass Spectrom 1999, 10:67-71. 33. Spengler B, Hubert M: Scanning microprobe matrix-assisted laser desorption ionization (SMALDI) mass spectrometry: instrumentation for sub-micrometer resolved LDI and MALDI surface analysis. J Am Soc Mass Spectrom 2002, 13:735-748. 34. Chaurand P, Fouchecourt S, DaGue BB, Xu BJ, Reyzer ML, Orgebin-Crist MC, Caprioli RM: Profiling and imaging proteins in Current Opinion in Biotechnology 2006, 17:431–436
436 Protein technologies
the mouse epididymis by imaging mass spectrometry. Proteomics 2003, 3:2221-2239. 35. Luxembourg SL, Mize TH, McDonnell LA, Heeren RMA: High-spatial resolution mass spectrometric imaging of peptide and protein distributions on a surface. Anal Chem 2004, 76:5339-5344. Interesting instrumentation and methodology for imaging by MALDI-MS using a position-sensitive mass spectrometer.
Current Opinion in Biotechnology 2006, 17:431–436
36. Chaurand P, Schwartz SA, Caprioli RM: Assessing protein patterns in disease using imaging mass spectrometry. J Proteome Res 2004, 3:245-252. 37. Caprioli RM: Deciphering protein molecular signatures in cancer tissues to aid in diagnosis, prognosis, and therapy. Cancer Res 2005, 65:10642-10645. Perspective on the future role of profiling and imaging mass spectrometry in the clinical setting.
www.sciencedirect.com
Current Opinion in Biotechnology 2006, 17:431–436 This review comes from a themed issue on Protein technologies Edited by Deb K Chatterjee and Joshua LaBaer Available online 16th June 2006 0958-1669/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2006.06.002
Introduction In the past decade, mass spectrometry (MS) has become an indispensable tool for proteomic studies, that is, the detection, identification and characterization of the protein content of cells, tissues and organs at any time point in both health and disease. For protein analysis, several different types of instruments and protocols allow for the determination of molecular weight, primary and higher order structure, post-translational modifications, quantitation, and localization. Desorption and ionization techniques such as matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) [1] have literally revolutionized our ability to analyze proteins. Improvements in MS instrumentation now offer levels of sensitivity and mass accuracy never before achieved for the detection, identification and structural characterization of proteins. www.sciencedirect.com
It is now possible to routinely measure molecular weights above 200 kDa, as well as to obtain low parts per million mass measurement accuracies for the detection of low femtomolar amounts of peptides. Modern MALDI mass spectrometers can now rapidly map and fragment peptides that result from protease digestion in order to identify and further characterize proteins. Protein identification has also been greatly facilitated owing to the rapid expansion of protein and gene databases and associated search tools, some of which are readily available over the internet. MALDI-MS is particularly useful because of its potential for high-throughput data acquisition together with its ability to provide information on the localization of molecules in a sample [2,3]. Imaging mass spectrometry (IMS) is a MALDI-MS based technology developed in our laboratory for the direct analysis of peptides and proteins from thin tissue sections [4,5]. Sections investigated by IMS clearly show retention of spatial and anatomic relationships of the MS signals, permitting the complex interaction between cells and their environment to be studied at the molecular level [4,6]. From the systematic analysis of a tissue section, protein-specific maps directly correlated with tissue architecture or morphology can be simultaneously obtained from hundreds of different protein species [4,6]. Similar approaches for mapping other types of biocompounds, such as cholesterol and phospholipids, have also been reported [7]. The potential for this type of analysis is particularly exciting for the study of diseases such as cancer [6,8], Duchenne myopathy [9], muscle dystrophy [10], Alzheimer’s [11] and Parkinson’s [12,13]. Although immunohistochemical techniques are routinely used to target known individual proteins, IMS offers the potential for the simultaneous analysis of many molecular species present in a single tumor regardless of the availability of specific antibodies. In addition, IMS is also capable of imaging the distribution of administered pharmaceutical agents [14–17] and resulting metabolites [18] within tissue sections, opening new possibilities for the measurement of concomitant protein changes in specific tissues after systemic drug administration [18]. In this review, we present the basic methodologies for processing tissue specimens, and describe the acquisition and subsequent processing required to visualize IMS measurements.
Methodologies In IMS, protein profiles and images are obtained directly from thin tissue sections cut from fresh frozen tissue specimens [19]. Tissue biopsies or other relevant tissue Current Opinion in Biotechnology 2006, 17:431–436
432 Protein technologies
samples are frozen in liquid nitrogen immediately after sampling to minimize protein degradation through enzymatic proteolysis and taking special care to preserve the sample’s morphology. Typically, for most applications, 10–12 mm thick sections are cut using a cryostat and thawmounted on a flat electrically conductive sample plate. The sections can then be dried in a dessicator for several minutes prior to MALDI matrix deposition. After mounting the sections on the target plates, rinsing procedures have been established to increase the MS data quality. A typical rinsing procedure consists of immersing the sections for 30 s in 70% ethanol followed by a second rinse for 30 s in a mixture of 90% ethanol, 9% acetic acid and 1% deionized water. For most applications, we have found that sinapinic acid (25 mg/ml in 50/50/0.1 acetonitrile/H2O/trifluoroacetic acid) is an excellent matrix for the analysis of proteins [19]. Other matrices have also been successfully used for the analysis of peptides [20,21,22] and lipids [7,23]. Small volumes of matrix (typically 200–500 nL) can be directly spotted onto a tissue section using an automatic pipette. Even smaller volumes (5–50 nL) can be applied with a finely pulled capillary loaded with matrix. In this case, matrix deposition must be performed under low to medium magnification to precisely deposit the matrix droplets at the desired tissue coordinates. Upon analysis, the resulting mass spectra typically yield from 300 to as many as 1000 protein signals within an intensity range of one to three orders of magnitude and in a mass-to-charge (m/z) range from m/z 1000 up to, and in some cases over, m/z 200 000 [24]; however, a large majority of these signals are observed in the m/z range below 30 000. It is believed that the most intense signals come from the most abundant protein species. The exact sensitivity of the technology is hard to estimate, however, because exact quantities of proteins within a specific tissue are generally not well known. The profiles recovered have been found to be highly specific for a given tissue or cell type (Figure 1) and, when analyzing serial sections, are highly reproducible [25]. High reproducibility has also been observed when analyzing sections of the same tissue sampled from different animals or individuals. One of the latest tissue analysis protocols developed in our laboratory utilizes conductive optically transparent glass slides as target plates to accommodate for MALDI-MS friendly tissue staining protocols [25]. This makes possible the microscopic evaluation of a tissue section followed by the molecular imaging of the same section by MS. If desired, these sections can then be stained after matrix removal to exactly correlate the observed protein signals with the local cellular composition observed under the spot [26]. On the basis of protein profiles recovered from control and cancerous human biopsies, specific patterns of protein expression have found to correlate well with histology-based diagnosis and prognosis [27,28,29]. Current Opinion in Biotechnology 2006, 17:431–436
An imaging experiment becomes necessary when highresolution protein distribution information within a tissue section is of importance. This is particularly true when analyzing tumor biopsies, where the precise architectural arrangement of the various cell types present in the section and the differentially expressed molecular weight signals could lead to a better understanding of molecular interactions between a tumor and its surrounding tissue. Highresolution images are also desirable when there is a defined substructure present, such as in brain coronal sections [4,6] or kidney (Figures 1 and 2) [8]. In the imaging experiment, a critical aspect of sample preparation is the homogeneous deposition of the MALDI matrix in such a way as to avoid significant lateral migration of proteins on the surface of the section. Several methods allow for the homogeneous deposition of matrix on tissue sections (i.e. the addition of large individual drops [4,11], spraying by nebulization [19,24,30], electrospray deposition [20], and automated microdispensing of the matrix [31]). The automated microdispensing technique (developed in our laboratory in collaboration with Labcyte Inc. Sunnyvale, CA) utilizes acoustic waves to vertically eject 120 pL nanodroplets of matrix from a reservoir on to the tissue section (facing the reservoir). Some 50 to 70 droplets are typically collected per tissue coordinate to form a matrix spot with a diameter of 200 mm. Cartesian arrays of droplets are deposited to fully cover the section to be analyzed (Figure 1). In this case, if protein delocalization does occur it is limited to the tissue area covered by a droplet. Other spotters are also available, in particular the CHIP chemical spotter from Shimadzu Biotech (Columbia, MD), which is currently being tested in our laboratory for matrix array deposition. For IMS, specialized instrument control software is used to set a data acquisition grid consisting of a discrete Cartesian pattern over the area to be imaged and to control the x/y movement of the sample stage [32]. This pattern has a fixed center-to-center distance between spots, typically 50–300 mm depending on the dimensions of the section and the imaging resolution required. MALDI-MS imaging at higher resolution in the micron range is technically feasible and has been described [33]. The mass spectrometric data is then acquired utilizing this grid pattern with a predetermined number of laser shots per grid coordinate. In the case of microspotted tissue sections, the grid pattern is aligned with the droplet array. Data processing (image reconstruction) is also performed using specialized software by integrating signal intensities at desired m/z values across the dataset (Figure 2) [11]. The time required for data acquisition depends on the area of the tissue to be analyzed, the resolution (number of pixels) required, and the overall acquisition speed of the instrument used. Although the local protein composition can affect the ionization and therefore detection of a monitored protein species through ion suppression effects, to date, good agreements www.sciencedirect.com
Imaging thin tissue sections by MS Chaurand, Cornett and Caprioli 433
Figure 1
Analysis by MALDI imaging MS of a 12 mm thick sagittal section from a rat kidney. (a) Photomicrograph of an H&E (hematoxylin and eosin) stained serial kidney section. The capsule, cortex, medulla and calyx tissue layers are visible and outlined on the image. (b) The upper part of the section (boxed area) was coated with sinapinic acid using an automated matrix spotter at a pitch of 300 mm (see text for details). (c) Unique protein profiles were obtained upon analysis of the different kidney tissues. The color-coded circles indicate the areas from which the spectra were acquired.
have been observed between images obtained by IMS and other protein imaging techniques such as immunohistochemistry [8,34]. With modern MALDI time-offlight mass spectrometers equipped with solid-state UV lasers that operate at repetition rates of several hundreds of Hertz, a 1 cm2 tissue biopsy can be imaged at a resolution of 250 mm in a matter of hours. Other instrument platforms have been described for imaging peptides and proteins from tissue sections. Of noticeable interest is the use of a position-sensitive mass spectrometer [35]. In this case, after homogeneous matrix spray deposition, ion images are acquired in the so-called microscope mode where the requirement for a highly focused ionization beam is removed. The defocused laser beam irradiates a large sample surface of the section and images are reconstructed based on the point of impact of ions with the same m/z on the detector. The broad concepts used for profiling and imaging proteins directly from tissue sections using MALDI-MS have www.sciencedirect.com
been extended to the examination of low molecular weight compounds, particularly pharmaceuticals [14,15,17]. Section handling and preparation techniques are nearly identical to those used for protein imaging. Some specific concerns, however, are unique to the analysis of low molecular weight compounds by MALDI-MS, namely significant matrix-related spectral interferences. Imaging of drugs in tissues is accomplished in an analogous fashion to imaging of proteins, but to increase the specificity and sensitivity, collision-activated dissociation (CAD) is employed [15]. For lower molecular weight compounds, direct imaging of the molecular ion is unreliable, because numerous endogenous compounds as well as potential matrix cluster ions can be observed at the same nominal molecular weight. The general strategy involves selection of the intact drug parent ion, fragmentation of this ion by CAD, and the detection of drugspecific signature m/z fragment ions. In parallel to drug localization, variations of the proteome induced by the drug can be investigated by IMS from a serial section as a Current Opinion in Biotechnology 2006, 17:431–436
434 Protein technologies
Figure 2
Ion density maps constructed after MALDI imaging MS of a 12 mm thick sagittal section from a rat kidney. Twelve different protein signals that are labeled in Figure 1c are depicted as gray-scale images with white representing the highest relative intensity for each m/z value.
function of dosage or time to determine their efficacy [18]. This strategy could be extremely useful to assess positive (or negative) reactions to drug therapy at very early time points.
Conclusions and future perspectives In the coming years, advances in molecular biology and molecular technologies are likely to have a profound impact on diagnostic pathology [8,36]. Molecular information derived from genomics, proteomics and metabolomics will be of crucial importance for the understanding of normal development and pathogenesis at the molecular level, therefore improving therapeutic efficacy and enhancing the quality of information available to clinicians. IMS offers an entirely new and highly precise means of analyzing disease tissue at the molecular level. In a broader context, with continued advances in instrumentation, tissue and data processing, it should be possible to acquire the proteomic profile of a tumor within the time frame currently utilized for interoperative section examination. Such measurements will generate important information that is complementary to traditional methods of histopathologic analysis [8,37]. It is believed that these new approaches will markedly impact three major areas: tissue-based diagnosis, prognosis determination, and prediction of response to specific modes of therapy [8,36]. In addition, the identification of molecular Current Opinion in Biotechnology 2006, 17:431–436
signatures that are precursors or early markers of disease should provide reliable methods of early detection for a broad spectrum of disease processes. The sum of molecular information obtained from a biopsy is likely to significantly impact the course of therapy for a particular tumor and would be available before the patient leaves the operating room.
Acknowledgements The authors would like to thank Serrine Lau and Barbara Leinweb (University of Arizona) for supplying the mouse kidney. The authors also acknowledge support from the NIH (GM 58008-08), and NCI (CA 86243-03 and CA 116123-01).
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
Hillenkamp F, Karas M, Beavis RC, Chait BT: Matrix-assisted laser desorption ionization mass-spectrometry of biopolymers. Anal Chem 1991, 63:1193A-1202A.
2.
Gusev AI, Vasseur OJ, Proctor A, Sharkey AG, Hercules MH: Imaging of thin-layer chromatograms using matrix-assisted laser desorption/ionization mass spectrometry. Anal Chem 1995, 67:4565-4570.
3.
Caprioli RM, Farmer TB, Gile J: Molecular imaging of biological samples: localization of peptides and proteins using MALDITOF MS. Anal Chem 1997, 69:4751-4760. www.sciencedirect.com
Imaging thin tissue sections by MS Chaurand, Cornett and Caprioli 435
4.
5.
Stoeckli M, Chaurand P, Hallahan DE, Caprioli RM: Imaging mass spectrometry: a new technology for the analysis of protein expression in mammalian tissues. Nat Med 2001, 7:493-496. Todd PJ, Schhaaff TG, Chaurand P, Caprioli RM: Organic ion imaging of biological tissue with SIMS and MALDI. J Mass Spectrom 2001, 36:355-369.
6.
Chaurand P, Schwartz SA, Caprioli RM: Profiling and imaging proteins in tissue sections by mass spectrometry. Anal Chem 2004, 76:86A-93A. Review article summarizing tissue section imaging by MALDI-MS. 7.
Jackson SN, Wang HYJ, Woods AS: Direct profiling of lipid distribution in brain tissue using MALDI-TOF MS. Anal Chem 2005, 77:4523-4527.
8.
Chaurand P, Sanders ME, Jensen RA, Caprioli RM: Proteomics in diagnostic pathology: profiling and imaging proteins directly in tissue sections. Am J Pathol 2004, 165:1057-1068. Review article summarizing the clinical potential of protein profiles and images. 9.
Brunelle A, Touboul D, Piednoel H, Voisin V, De La Porte S, Tallarek E, Hagenhoff B, Halgand F, Laprevote O: Direct mapping on muscle tissues of duchenne myopathy biomarkers using both MALDI and cluster-SIMS imaging mass spectrometry. Mol Biol Cell 2004, 15:103A-104A.
10. Touboul D, Piednoel H, Voisin V, De La Porte S, Brunelle A, Halgand F, Laprevote O: Changes in phospholipid composition within the dystrophic muscle by matrix-assisted laser desorption/ionization mass spectrometry and mass spectrometry imaging. Eur J Mass Spectrom 2004, 10:657-664. 11. Stoeckli M, Staab D, Staufenbiel M, Wiederhold K-H, Signor L: Molecular imaging of amyloid beta peptides in mouse brain sections using mass spectrometry. Anal Biochem 2002, 311:33-39. 12. Pierson J, Norris JL, Aerni HR, Svenningsson P, Caprioli RM, Andren PE: Molecular profiling of experimental Parkinson’s disease: direct analysis of peptides and proteins on brain tissue sections by MALDI mass spectrometry. J Proteome Res 2004, 3:289-295. 13. Pierson J, Svenningsson P, Caprioli RM, Andren PE: Increased levels of ubiquitin in the 6-OHDA-lesioned striatum of rats. J Proteome Res 2005, 4:223-226. 14. Troendle FJ, Reddick CD, Yost RA: Detection of pharmaceutical compounds in tissue by matrix-assisted laser desorption/ ionization and laser desorption/chemical ionization tandem mass spectrometry with a quadrupole ion trap. J Am Soc Mass Spectrom 1999, 10:1315-1321. 15. Reyzer ML, Hsieh Y, Ng K, Korfmacher WA, Caprioli RM: Direct analysis of drug candidates in tissue by matrix-assisted laser desorption/ionization mass spectrometry. J Mass Spectrom 2003, 38:1081-1092. 16. Rohner TC, Staab D, Stoeckli M: MALDI mass spectrometric imaging of biological tissue sections. Mech Ageing Dev 2005, 126:177-185. 17. Wang HYJ, Jackson SN, McEuen J, Woods AS: Localization and analyses of small drug molecules in rat brain tissue sections. Anal Chem 2005, 77:6682-6686. 18. Reyzer ML, Caldwell RL, Dugger TC, Forbes JT, Ritter CA, Guix M, Arteaga CL, Caprioli RM: Early changes in protein expression detected by mass spectrometry predict tumor response to molecular therapeutics. Cancer Res 2004, 64:9093-9100. Key reference demonstrating the correlation between the presence of a drug in tumor tissue and its influence on the proteome monitored by imaging mass spectrometry. 19. Schwartz SA, Reyzer ML, Caprioli RM: Direct tissue analysis using matrix-assisted laser desorption/ionization mass spectrometry: practical aspects of sample preparation. J Mass Spectrom 2003, 38:699-708. www.sciencedirect.com
Methodology article detailing the steps necessary for the successful analysis of tissue specimens by MALDI-MS. 20. Kruse R, Sweedler JV: Spatial profiling invertebrate ganglia using MALDI MS. J Am Soc Mass Spectrom 2003, 14:752-759. 21. Fournier I, Day R, Salzet M: Direct analysis of neuropeptides by in situ MALDI-TOF mass spectrometry in the rat brain. Neuroendocrinol Lett 2003, 24:9-14. 22. Lemaire R, Tabet JC, Ducoroy P, Hendra JB, Salzet M, Fournier I: Solid ionic matrixes for direct tissue analysis and MALDI imaging. Anal Chem 2006, 78:809-819. First use of ionic matrices for profiling and imaging peptides and proteins from tissue sections by MALDI-MS. 23. Rujoi M, Estrada R, Yappert MC: In situ MALDI-TOF MS regional analysis of neutral phospholipids in lens tissue. Anal Chem 2004, 76:1657-1663. 24. Chaurand P, Caprioli RM: Direct profiling and imaging of peptides and proteins from mammalian cells and tissue sections by mass spectrometry. Electrophoresis 2002, 23:3125-3135. 25. Chaurand P, Schwartz SA, Billheimer D, Xu BJ, Crecelius A, Caprioli RM: Integrating histology and imaging mass spectrometry. Anal Chem 2004, 76:1145-1155. First use of MALDI-compatible tissue staining procedures and conductive glass slides as section support for profiling and imaging mass spectrometry. 26. Crecelius AC, Cornett DS, Caprioli RM, Williams B, Dawant BM, Bodenheimer B: Three-dimensional visualization of protein expression in mouse brain structures using imaging mass spectrometry. J Am Soc Mass Spectrom 2005, 16:1093-1099. 27. Yanagisawa K, Shyr Y, Xu BJ, Massion PP, Larsen PH, White BC, Roberts JR, Edgerton M, Gonzalez A, Nadaf S et al.: Proteomic patterns of tumour subsets in non-small-cell lung cancer. Lancet 2003, 362:433-439. First demonstration that protein profiles generated by MALDI-MS from lung cancer and control tissue sections can be correlated with patient diagnosis and outcome. 28. Schwartz SA, Weil RJ, Johnson MD, Toms SA, Caprioli RM: Protein profiling in brain tumors using mass spectrometry: feasibility of a new technique for the analysis of protein expression. Clin Cancer Res 2004, 10:981-987. 29. Schwartz SA, Weil RJ, Thompson RC, Shyr Y, Moore JH, Toms SA, Johnson MD, Caprioli RM: Proteomic-based prognosis of brain tumor patients using direct-tissue matrix-assisted laser desorption ionization mass spectrometry. Cancer Res 2005, 65:7674-7681. Demonstration that protein profiles generated by MALDI-MS from brain gliomas and control tissue sections can be correlated with patient diagnosis and outcome. 30. Chaurand P, Schwartz SA, Caprioli RM: Imaging mass spectrometry: a new tool to investigate the spatial organization of peptides and proteins in mammalian tissue sections. Curr Opin Chem Biol 2002, 6:676-681. 31. Aerni HR, Cornett DS, Caprioli RM: Automated acoustic matrix deposition for MALDI sample preparation. Anal Chem 2006, 78:827-834. Key reference showing that arrays of small matrix droplets can be automatically printed on tissue sections for MALDI imaging mass spectrometry. 32. Stoeckli M, Farmer TB, Caprioli RM: Automated mass spectrometry imaging with a matrix-assisted laser desorption ionization time-of-flight instrument. J Am Soc Mass Spectrom 1999, 10:67-71. 33. Spengler B, Hubert M: Scanning microprobe matrix-assisted laser desorption ionization (SMALDI) mass spectrometry: instrumentation for sub-micrometer resolved LDI and MALDI surface analysis. J Am Soc Mass Spectrom 2002, 13:735-748. 34. Chaurand P, Fouchecourt S, DaGue BB, Xu BJ, Reyzer ML, Orgebin-Crist MC, Caprioli RM: Profiling and imaging proteins in Current Opinion in Biotechnology 2006, 17:431–436
436 Protein technologies
the mouse epididymis by imaging mass spectrometry. Proteomics 2003, 3:2221-2239. 35. Luxembourg SL, Mize TH, McDonnell LA, Heeren RMA: High-spatial resolution mass spectrometric imaging of peptide and protein distributions on a surface. Anal Chem 2004, 76:5339-5344. Interesting instrumentation and methodology for imaging by MALDI-MS using a position-sensitive mass spectrometer.
Current Opinion in Biotechnology 2006, 17:431–436
36. Chaurand P, Schwartz SA, Caprioli RM: Assessing protein patterns in disease using imaging mass spectrometry. J Proteome Res 2004, 3:245-252. 37. Caprioli RM: Deciphering protein molecular signatures in cancer tissues to aid in diagnosis, prognosis, and therapy. Cancer Res 2005, 65:10642-10645. Perspective on the future role of profiling and imaging mass spectrometry in the clinical setting.
www.sciencedirect.com