Integrating Histology and Imaging Mass Spectrometry - American

Integrating Histology and Imaging Mass. Spectrometry. Pierre Chaurand,† Sarah A. Schwartz,† Dean Billheimer,‡ Baogang J. Xu,† Anna Crecelius,â...
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Anal. Chem. 2004, 76, 1145-1155

Integrating Histology and Imaging Mass Spectrometry Pierre Chaurand,† Sarah A. Schwartz,† Dean Billheimer,‡ Baogang J. Xu,† Anna Crecelius,† and Richard M. Caprioli*,†

Mass Spectrometry Research Center and Department of Biochemistry, Department of Biostatistics, Vanderbilt University Medical Center, Nashville, Tennessee 37232-6400

MALDI (matrix-assisted laser desorption/ionization) imaging mass spectrometry (IMS) is a new technology that generates molecular profiles and two-dimensional ion density maps of peptide and protein signals directly from the surface of thin tissue sections. This allows specific information to be obtained on the relative abundance and spatial distribution of proteins. One important aspect of this is the opportunity to correlate these specific ion images with histological features observed by optical microscopy. To facilitate this, we have developed protocols that allow MALDI mass spectrometry imaging and optical microscopy to be performed on the same section. Key components of these protocols involve the use of conductive glass slides as sample support for the tissue sections and MS-friendly tissue staining protocols. We show the effectiveness of these with protein standards and with several types of tissue sections. Although stain-specific intensity variations occur, the overall protein pattern and spectrum quality remain consistent between stained and control tissue samples. Furthermore, imaging mass spectrometry experiments performed on stained sections showed good image quality with minimal delocalization of proteins resulting from the staining protocols. MALDI imaging mass spectrometry (IMS) generates profiles and two-dimensional ion density maps of molecules, primarily peptides and proteins, directly from the surface of thin tissue sections1-4 showing the relative abundance and spatial distribution of these molecules. The technique makes use of thin sections cut from fresh frozen using a crystat that are thaw-mounted on target plates, and MALDI matrix is subsequently deposited on the sections. Discrete droplets of matrix can be deposited at various coordinates on the section (profiling). Spectra acquired from these spots typically display well over 500 signals, most of which are in a molecular weight range below 50 kDa. In specific cases, signals * To whom correspondence should be addressed. Phone: (615) 322 4336. Fax: (615) 343 8372. E-mail: [email protected]. † Mass Spectrometry Research Center and Department of Biochemistry. ‡ Department of Biostatistics. (1) Stoeckli, M.; Chaurand, P.; Hallahan, D. E.; Caprioli, R. M. Nat. Med. 2001, 7, 493-496. (2) Todd, P. J.; Schhaaff, T. G.; Chaurand, P.; Caprioli, R. M. J. Mass Spectrom. 2001, 36, 355-369. (3) Chaurand, P.; Caprioli, R. M. Electrophoresis 2002, 23, 3125-3135. (4) Chaurand, P.; Schwartz, S. A.; Caprioli, R. M. Curr. Opin. Chem. Biol. 2002, 6, 676-681. 10.1021/ac0351264 CCC: $27.50 Published on Web 01/09/2004

© 2004 American Chemical Society

with molecular weights well above 200 kDa have been observed.3 When matrix is deposited over the section in a homogeneous manner, it is possible to perform high-resolution imaging of the section. In this mode, mass spectrometric data is acquired over the area of the section using a discrete Cartesian coordinate pattern with a fixed center-to-center distance between spots. Typically, a 25-200-µm separation is used, depending on the image resolution required. A mass spectrum is acquired at each coordinate, and from the m/z values recorded, two-dimensional ion density maps, or images, are constructed. An image can be generated for each of the protein mass signals detected throughout the section. From a single acquisition, hundreds of specific ion images can be displayed. Profiling and imaging mass spectrometry have already proven to be valuable tools to investigate both normal and diseased tissues. This includes studies of the spatial distribution of proteins in xenographs of human gliomas;1 both normal and cancerous mouse prostate;5 both healthy and cancerous mouse colon tissue;6 neuropeptides in the rat brain;7 human lung tumor biopsies;8 and very recently, the mouse epididymis.9 Other studies have combined laser capture microdissection of cells from human breast ductal carcinoma in situ with analysis by mass spectrometry.10-12 One of the important aspects of the investigation of disease tissue is the comparison of histological features obtained from stained sections using light microscopy with molecular images obtained by mass spectrometry. Previously, this was accomplished using two separate adjacent sections, one for histology and one for mass spectrometry.7,8 Often visual registration between both sections was difficult because of differences in morphology or because of the physical changes induced by preparation proce(5) Masumori, N.; Thomas, T. Z.; Chaurand, P.; Case, T.; Paul, M.; Kasper, S.; Caprioli, R. M.; Tsukamoto, T.; Shappell, S. B.; Matusik, R. J. Cancer Res. 2001, 61, 2239-2249. (6) Chaurand, P.; DaGue, B. B.; Pearsall, R. S.; Threadgill, D. W.; Caprioli, R. M. Proteomics 2001, 1, 1320-1326. (7) Fournier, I.; Day, R.; Salzet, M. Neuroendocrinol. Lett. 2003, 24, 9-14. (8) Yanagisawa, K.; Shyr, Y.; Xu, B. J.; Massion, P. P.; Larsen, P. H.; White, B. C.; Roberts, J. R.; Edgerton, M.; Gonzalez, A.; Nadaf, S.; Moore, J. H.; Caprioli, R. M.; Carbone, D. P. Lancet 2003, 362, 433-439. (9) Chaurand, P.; Fouchecourt, S.; DaGue, B. B.; Xu, B. J.; Reyzer, M. L.; Orgebin-Crist, M. C.; Caprioli, R. M. Proteomics 2003, 3, 2221-2239. (10) Palmer-Toy, D. E.; Sarracino, D. A.; Sgroi, D.; LeVangie, R.; Leopold, P. E. Clin. Chem. 2000, 46, 1513-1516. (11) Xu, B. J.; Caprioli, R. M.; Sanders, M. E.; Jensen, R. A. J. Am. Soc. Mass Spectrom. 2002, 13, 1292-1297. (12) Bhattacharya, S. H.; Gal, A. A.; Murray, K. K. J. Proteome Res. 2003, 2, 95-98.

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dures. Similarly, it was difficult to locate many tissue features, which may not always be visible when sections are mounted on different opaque media. Thus, it became imperative to devise protocols that would allow histology and MS analyses on the same section. Histology is normally performed on stained thin tissue sections mounted on glass slides. Numerous dyes, staining different cellular components, are available and regularly used by pathologists for diagnostic purposes.13 One of the commonly used staining procedures employs hematoxylin and eosin (H&E). Mass spectrometric analysis of H&E-stained laser-captured cells and tissues has been recently performed, giving some molecular information.10-12 However, with this particular staining procedure, one or both of the dyes directly interfere with either the proteins present in the section or with the MALDI process. We have found that the quality of the mass spectra from H&E-stained sections is significantly compromised relative to that obtained from unstained sections.11,14 To perform both an optical evaluation and a protein analysis by MALDI time-of-flight MS, it is imperative that tissue sections be mounted on optically transparent and noninsulating sample plates. In time-of-flight mass analyzers, this latter point is particularly important because any significant local transient charging of the surface will induce variations of the accelerating potential field, resulting in measurable time-of-flight shifts for all of the ions present in the spectrum. Bulk or optically transparent glass and quartz have been used as target surfaces for MALDI-MS15-19 and as reactive surfaces for the affinity capture of proteins.20,21 Recently, atmospheric pressure MALDI was reported for samples prepared on indium-tin oxide (ITO)-coated conductive glass slides.22 The current paper explores the potential and advantages of protocols developed to allow direct analysis of a given mammalian tissue section by both microscopy and IMS. We present comparative results for the MALDI-TOF-MS analysis of peptides and proteins from tissue sections mounted on metallic surfaces and conductive (ITO-coated) glass slides. Several tissue-staining protocols are described that are compatible with histology and mass spectrometry performed on thin sections and laser captured cells. MATERIALS AND METHODS Indium-tin oxide (ITO)-coated glass slides were purchased from Delta Technologies Ltd. (Stillwater, MN). Unpolished 1.1mm-thick glass was coated with a thin ITO layer ∼160 to 240 Å thick and cut into 45 × 45 mm2 pieces, dimensions compatible (13) Horobin, R. W. Understanding Histochemistry: Selection, Evaluation and Design of Biological Stains; Ellis Horwood Ltd: Chichester, 1988. (14) Todd, P. J.; McMahon, J. M.; Short, R. T.; McCandlish, C. A. Anal. Chem. 1997, 69, 9, A529-A535. (15) Ehring, H.; Costa, C.; Demirev, P. A.; Sundqvist, B. U. R. Rapid Commun. Mass Spectrom. 1996, 10, 821-824. (16) Schurenberg, M.; Schulz, T.; Dreisewered, K.; Hillenkamp, F. Rapid Commun. Mass Spectrom. 1996, 10, 1873-1880. (17) Lennon, J. D.; Glish, G. L. Anal. Chem. 1997, 69, 2525-2529. (18) Preisler, J.; Foret, F.; Karger, B. L. Anal. Chem. 1998, 70, 5278-5287. (19) Perez, J.; Petzold, C. J.; Watkins, M. A.; Vaughn, W. E.; Kenttamaa, H. I. J. Am. Soc. Mass Spectrom. 1999, 10, 1105-1110. (20) Afonso, C.; Fenselau, C. Anal. Chem. 2003, 75, 694-697. (21) Koopmann, J. O.; Blackburn, J. Rapid Commun. Mass Spectrom. 2003, 17, 455-462. (22) Galicia, M. C.; Vertes, A.; Callahan, J. H. Anal. Chem. 2002, 74, 18911895.

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with the Applied Biosystems Inc. (Framingham, MA) disposable MALDI plate holder. The dimension of the glass target and ITO coating thickness give a surface resistance of ∼70 to 100 Ω. The thin ITO film permits ∼85% visible light (500 < λ < 900 nm) transmission through the slide, allowing clear microscopic tissue section examination. Mass spectrometric analyses were performed in the linear mode at +25 kV of accelerating potential on an Applied Biosystems Inc. (Framingham, MA) Voyager DE-STR time-of-flight mass spectrometer under optimized delayed extraction conditions for all of the investigated molecules. This mass spectrometer is equipped with a 337-nm N2 laser capable of operating at repetition rates of 3 or 20 Hz. For IMS experiments, an iris positioned in front of the laser beam was partially closed to define on the surface of the target a laser spot size of roughly 50 µm in diameter. Ion image acquisition was performed by home-built software (MALDI mass spectrometry imaging tool, MMSIT23,24), which interfaced with the Applied Biosystems instrument controller and acquisition software. The MMSIT software controls data acquisition over a predetermined area and reconstructs ion density maps by measuring the signal intensities of the ions monitored. If desired, images for every mass signal observed within a scanned area can be obtained. To test the potential of ITO-coated conductive glass slides as a sample support for MALDI samples in a time-of-flight mass spectrometer, different peptide and protein standards as well as different matrixes were used. Solvents, matrixes, and standard peptides and proteins were purchased from Sigma (Saint Louis, MO). Two different MALDI samples were used. The first sample was obtained by mixing on the target plate 500 nL of angiotensin II (MWmono 1045.54, at a concentration of 1 pmol/µL) with 500 nL of R-cyano-4-hydroxycinnamic acid as matrix (CHCA, prepared at a concentration of 10 mg/mL in 50:50 acetonitrile/0.1% trifluoroacetic acid by volume). The second sample was prepared by mixing on the target plates 500 nL of a protein mixture containing porcine insulin (MWav 5777.6, at a concentration of 1.34 pmol/ µL), horse cytochrome c (MW 12 360.1, at a concentration of 2.06 pmol/µL) and horse skeletal muscle apomyoglobine (MW 16 951.6, at a concentration of 2.04 pmol/µL) with 500 nL of sinapinic acid as matrix (SA, prepared at a concentration of 20 mg/mL in 50:50:0.1 acetonitrile/H2O/trifluoroacetic acid by volume). Tissue sectioning and staining was performed at the Vanderbilt Tissue Acquisition Core Laboratory. Thin tissue sections (10-12 µm thick) were cut with a Leica Jung cryostat (Leica Microsystems AG, Welzlar, Germany) at -15 °C from fresh snap-frozen mouse liver and human brain glioma samples. The glass and metallic target plates were maintained at -15 °C within the cryostat chamber. The different frozen sections were then deposited on the cold target plates and thaw-mounted by simply warming the plate.25 Prior to staining, macroscopic drying was achieved by placing the plates in a desiccator for up to 30 min. Table 1 lists the different stains tested and the corresponding staining protocols. In general, several hundred microliters of dye prepared in (23) Stoeckli, M.; Farmer, T. B.; Caprioli, R. M. J. Am. Soc. Mass Spectrom. 1999, 10, 67-71. (24) Stoeckli, M.; Staab, D.; Staufenbiel, M.; Wiederhold, K.-H.; Signor, L. Anal. Biochem. 2002, 311, 33-39. (25) Schwartz, S. A.; Reyzer, M. L.; Caprioli, R. M. J. Mass Spectrom. 2003, 38, 699-708.

Table 1. List, Manufacturer, and Preparation Protocols of the Stains stain

protocol

stain time (s)

Toluidine Blue (Electron Microscopy Sciences, Catalogue No. 22050)

5% Toluidine Blue in 1% aqueous borax (hydrated sodium borate, Na2B4O7‚10H2O, Fisher Scientific, ACS grade). Dissolve borax in warm distilled water, add Toluidine Blue, stir overnight, filter before use. 0.1% Nuclear Fast Red in 5% aqueous aluminum sulfate (Al2(SO4)3). Ready to use. 0.15% in 70% ethanol (AAPER alcohol, absolute 200 proof). Dissolve Methylene blue in ethanol, stir overnight, filter before use. 0.02% aqueous Methylene Blue in 0.02% aqueous potassium carbonate (K2CO3, Merck KGaA, ACS grade). Dissolve potassium carbonate in distilled water, add Methylene Blue, stir overnight, filter before use. 0.5% aqueous (deionized water). Stir overnight on low heat, filter solution, add 2 drops of glacial acetic acid to a 100-mL solution, filter before use.

5-10

Nuclear Fast Red (Newcomer Supply, Catalogue No. 1255A) Methylene Blue (Sigma, Catalogue No. MB-1) Terry’s Polychrome

Cresyl Violet (Sigma, Catalogue No. C1791)

either water or ethanol-based solvents were directly deposited on the sections using a Pasteur pipet and allowed to react for the time indicated. Excess stain was removed by plunging the plates for 15 s in two successive Petri dishes, containing 70 and 100% ethanol, respectively. Similar destaining/fixing procedures have been found compatible with MALDI-MS analysis11 without any indication of protein migration across the section. The sections were allowed to dry in a desiccator for up to 30 min. Photomicrographs of the sections were obtained under magnification using an Olympus BX 50 microscope (Olympus America Inc., Melville, NY) equipped with a digital camera. Matrix (sinapinic acid at 20 mg/mL in a mixture of 50:50:0.1 acetonitrile/H2O/trifluoroacetic acid by volume) was either deposited in discrete droplets or spray-coated on the various tissue sections. For droplet deposition, two 200-nL drops of matrix were successively deposited on the section at the same coordinates using an automatic pipet and allowed to dry. The first droplet was allowed to almost dry before the second droplet was deposited. For spray coating, a continuous matrix coating is applied over the entire surface of the section as previously described.3,4,25 This protocol has been optimized to favor the formation of small matrix crystals (∼20 to 40 µm in length) on the surface of the sections while minimizing protein migration across the section. To achieve this, dry powder matrix was initially brushed over the tissue section.26 Excess matrix was gently blown off using clean air, generating a homogeneous layer. Matrix solution was then sprayed onto the sections using a commercially available glass venturi nebulizer. This process must be done with care, using multiple spray cycles of a few seconds each. Large droplets of matrix solution on the tissue sections must be avoided. Complete coverage of the section surface (better than 95%) is achieved by overlaying 10 spray cycles with a 1 min drying time between cycles. Crystal formation and coverage can be followed using a microscope. Laser capture microdissection (LCM) of hepatic cells from control and stained mouse liver tissue was performed using the PixCell II LCM system (Arcturus, Mountain View, CA). Fivemicrometer liver sections were cut and mounted on uncoated glass slides and immediately dehydrated using successive ethanol (26) Aerni, H. R.; Erskine, A. R.; Reyzer, M. L.; Lee, D.; Cornett, D. S.; Caprioli, R. M., Proceedings of the 51st ASMS Conference on Mass Spectrometry and Allied Topics, Montreal, Quebec, Canada, June 8-12, 2003.

45 5-10 30

30

Figure 1. MALDI-TOF-MS signals acquired under delayed extraction conditions from porcine insulin prepared in SA on a nonconductive glass slide. For this experiment, the laser repetition rate was adjusted to 3 Hz. The dotted line trace presents a signal resulting from a single laser shot, and the trace in solid line was obtained averaging the signals from 20 successive shots.

washes. The control sections were dehydrated according to the following procedure: 30 s in 70% ethanol, 1 min in 95% ethanol, 1 min in 100% ethanol (twice), and 2.5 min in xylene (twice). The sections were then allowed to dry for 2 min at room pressure and temperature. The Methylene Blue and Cresyl Violet stained sections were first processed according to the protocol described in Table 1, then dehydrated according to the following procedure: 1 min in 95% ethanol, 1 min in 100% ethanol (twice), and 2.5 min in xylene (twice). The sections were then allowed to dry for 2 min at room pressure and temperature. For each section, ∼400 cells were captured on a CapSure thermoplastic film LCM cap (Arcturus, Mountain View, CA) (see Figure 9a-c). After LCM, the thermoplastic film was removed from the LCM cap using forceps and placed on the MALDI plate using conductive doublesided tape. A finely pulled glass capillary was employed to precisely deposit the matrix solution (sinapinic acid, prepared as mentioned above) only on the captured cells under microscope visualization. EXPERIMENTAL RESULTS (1) Fundamentals. (a) Using Conductive Glass as a Target Support for MALDI-TOF-MS. To assess the effectiveness of the Analytical Chemistry, Vol. 76, No. 4, February 15, 2004

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Figure 2. MALDI-MS protein profiles obtained from 12-µm mouse liver sections mounted on (a) a gold-coated stainless steel plate, and (b) an ITO-coated conductive glass slide. Both sections were spotted with SA at roughly the same coordinates.

ITO-coated glass slide as a target plate for MALDI-TOF-MS performed under delayed extraction conditions, peptide and protein samples were prepared on both ITO-coated glass and goldcoated metallic target plates. As a control experiment, standards were also prepared on a noncoated (nonconductive) microscope glass slide. The spectrum presented in Figure 1 displays positive ion signals from porcine insulin prepared in SA on a nonconductive glass slide. The laser repetition rate was set at 3 Hz. The dotted trace is the signal obtained from a single laser shot while the solid trace was obtained by averaging signals from 20 successive shots. Between the first and the last laser shots, a shift of the signal of up to 100 ns toward shorter times-of-flight was observed, leading to a signal resolution M/∆M (full width at half-maximum definition, fwhm) of no better than 400. For insulin, on the basis of the accelerating potential, instrumental dimensions and delayed extraction parameters, this time shift represents a mass shift of more than 10 amu. Similar mass-shifting behavior was also observed when the same sample was investigated under constant field extraction. Under these same experimental conditions, when analysis was performed after a delay of 5 min between the time the potential was applied and the time of analysis, the signal was found shifted toward lower flight times for the first laser shot. All of these behaviors are consistent with a progressive accumulation of positive charges due to the applied electric field on the sample plate. This charging effect results in an increase in potential difference with respect to the first extracting electrode and, therefore, a decrease in flight time. 1148

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MALDI-TOF-MS signal quality between gold-coated metallic or ITO-coated glass slides was also systematically investigated under optimized laser energy and delayed extraction conditions. Analysis performed on several protein standards (angiotensin, insulin, cytochrome c and apomyoglobin) prepared on both types of sample plate demonstrated no significant difference in signal quality in terms of intensity and resolution (data not shown). For angiotensin II, an identical isotopic resolution (fwhm) of ∼3900 was obtained with both types of target plates. For insulin, cytochrome c, and apomyoglobin, similar apparent resolutions (fwhm) around 1000 were observed with both types of target plates. MALDI TOF-MS signal quality from tissue sections either mounted on gold-coated metallic or ITO-coated glass target plates were also investigated. Figure 2 presents two MALDI-MS protein profiles in the m/z range from 3000 to 35 000 obtained from 12µm serial mouse liver sections mounted on both types of plates with matrix (SA) deposited at similar coordinates on both sections. Acquisition was performed by averaging 750 laser shots randomly collected across each matrix droplet. The same experimental parameters were used on both target surfaces. No major differences between the two mass spectra were observed when comparing the relative intensities and resolutions of the signals detected. With an instrumental configuration having a maximum laser repetition rate of 20 Hz, scanning thin tissue sections for image acquisition usually requires several hours. To assess the longterm electrical stability of the conductive glass slides over this

Figure 3. Imaging of angiotensin II distribution within a MALDI spot prepared in CHCA on a coated glass slide. (a) Photomicrograph of the spot under 40× magnification. Lines of higher matrix crystal density, resulting from contact of the pipet tip with the target plate during sample preparation, were observed. Image acquisition was performed with resolution of 50 µm. (b) ion density maps obtained for the first isotope signal of angiotensin observed at m/z 1046.54. (c) MALDI-MS spectra of angiotensin II acquired from the droplet. The dotted line trace was obtained by averaging 250 laser shots randomly acquired across the sample surface. The solid line trace was generated by averaging all of the mass spectra acquired across the surface of the sample during the course of image acquisition. The baselines of the spectra are shifted in intensity for clarity.

period of time, images of peptide distributions were acquired over time. Angiotensin II was prepared in CHCA on a coated glass slide. Figure 3a displays a photomicrograph of the spot under 40× magnification. Lines of higher matrix crystal density, resulting from contact of the pipet tip with the target plate during sample preparation, were observed. Image acquisition was performed by averaging 20 laser shots per spectrum with a laser repetition rate of 20 Hz (1 s per data point) and a resolution of 50 µm. Considering the dimensions of the spot (∼4.0 × 4.8 mm2), this represents a pixel array of 80 × 96 ) 7680 data points (or spectra) acquired over a time period of ∼2 h. Figure 3b displays the ion density maps obtained for the first isotope signal of angiotensin observed at m/z 1046.54. Higher intensity signals were generated from the sample area with higher crystal densities. Figure 3c presents the overlay of two signals from angiotensin II acquired from the droplet in Figure 3a. The dotted trace was obtained by averaging 250 laser shots randomly acquired across the sample surface. The solid trace was generated by averaging all of the mass spectra acquired across the surface of the sample (∼6000 spectra, representing 120 000 laser shots). The baselines of the spectra are shifted in intensity for clarity. In both cases, on the basis of the chemical formula for angiotensin II (C50H71N13O12), the experimental relative isotopic intensity distributions were found to be within a few percent of the calculated distribution. No significant shift in mass or loss of resolution was observed

between the two spectra, indicating that for laser repetition rates up to 20 Hz, excellent electrical stability can be maintained across the conductive glass slides for extended periods of time. (b) Developing Tissue Staining Protocols. The incorporation of a MALDI-friendly tissue staining procedure with ITO-coated glass slides allows for the direct selection of morphological regions of interest for MS analysis. To this end, several tissue-staining procedures were developed that are compatible with both MALDI mass spectrometry and histology. All of the dyes tested here are nuclear stains that bind DNA and, therefore, should have minimal chemical interference with MALDI-MS analyses of proteins. Furthermore, visualization of the cell nucleus is of prime importance in cancer diagnostic where nucleus shape and size is used for cell identification as well as the degree of cell differentiation for tumor stage or grade assessment. Five different staining protocols are listed in Table 1. Methylene Blue was prepared either in ethanol-based (Methylene Blue) or water-based (Terry’s Polychrome) solutions. All of the other dyes were prepared in water-based solutions. Since all of the tested dyes are also soluble in ethanol, a common ethanol-based destaining procedure aimed at removing excess dye from the sections was optimized. Similar ethanol-based solutions have been successfully used to dehydrate thin tissue sections prior to MALDI-MS analysis.11 To monitor the effects of the different solvents used in the staining protocols on the MALDI-MS protein response, several control experiments Analytical Chemistry, Vol. 76, No. 4, February 15, 2004

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Figure 4. MALDI-MS protein profiles generated from three serial 10-µm human brain glioma tissue sections: (a) unprocessed section; (b) a section successively rinsed in 70 and 100% ethanol; and (c) a section first washed for 30 s in deionized water, then successively rinsed in 70 and 100% ethanol. All of the sections were spotted with SA at approximately the same coordinates.

Figure 5. High-magnification (×400) photomicrographs of unstained (control) and stained 10-µm serial human glioma tissue sections. In the upper right corner of each image, the lower left edge of the matrix sample can be seen.

were performed. Figure 4 presents three protein profiles generated from serial 10-µm-thick tissue sections from a human brain glioma: (4a) unprocessed section, (4b) a section successively rinsed in 70% and 100% ethanol, and (4c) a section first washed 1150

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for 30 s in deionized water, then successively rinsed in 70 and 100% ethanol. Matrix (SA) was deposited on all of the sections at approximately the same coordinates. No significant distortions in the protein profiles were observed after the water/ethanol and

Figure 6. MALDI-MS protein profiles acquired from unstained (Ct: control section rinsed in 70 and 100% ethanol) and stained grade 4 human glioma tissue sections. TP, Terry’s Polychrome; TB, Toluidine Blue; NFR, Nuclear Fast Red; CV, Cresyl Violet; MB, Methylene Blue.

ethanol rinsing steps with respect to the profile obtained from the unprocessed section. Similar results were also obtained from serial mouse liver sections (data not shown). Figure 5 presents six high-magnification (×400) photomicrographs of unstained (control) and stained 10-µm serial grade 4 human glioma tissue sections. In the upper right corner of each image, the lower left edge of the matrix sample can be seen. All of the sections were spotted twice at the same tissue coordinate with 200 nL of sinapinic acid matrix solution. For all of the tested protocols, staining of the cell nuclei was observed. Staining with Nuclear Fast Red did not give as much contrast between the nucleus and cytoplasm when compared to the other dyes. Furthermore, with Nuclear Fast Red, consistent staining reproducibility proved harder to obtain. For Methylene Blue and Toluidine Blue, some light cytoplasmic staining was also observed. Changes in tissue preparation such as how samples are frozen or stored and for how long a section is stained may drastically affect the staining quality. The specific stain or type of solvent used, stain concentration, and age of solution or solvents may also change the staining quality. Additionally, different types of tissues will stain differently. Therefore, staining protocols may need to be optimized for specific applications. (2) Applications. (a) MS Protein Profiling. Using the above staining protocols, general regions of interest within a tissue sample, such as a tumor site surrounded by nontumor tissue, can be selected for profiling MS analysis. Figure 6 presents MALDIMS protein profiles acquired from unstained (control section rinsed in 70 and 100% ethanol) and stained grade 4 human glioma tissue sections. Overall, good quality protein profiles were obtained

after staining over the entire mass range investigated. Furthermore, the general protein pattern, intensities, and resolutions were found equivalent to those obtained from the unstained section. Depending on the staining protocol, however, the intensities of some signals were affected. For this sample, the profiles obtained after staining with Terry’s Polychrome and Toluidine Blue displayed the strongest variations with respect to the control trace. This is particularly evident for m/z 4937 and m/z 4965 (corresponding to the Thymosins Beta 10 and Beta 4, respectively1) and for m/z 15 127 and m/z 15 868 (corresponding to R- and β-hemoglobins, respectively), which in most instances were found suppressed. After staining with Toluidine Blue, however, stronger intensity signals were detected in the m/z range from 40 000 to 50 000. This was not the case after staining with Terry’s polychrome, when no significant signals above m/z 30 000 were observed. Staining with Nuclear Fast Red suppressed signals associated with various histones5 (m/z 11 306 and 11 348 corresponding to two different acetylated forms of the histone H4, m/z 13 784 corresponding to histone H2.B, m/z 14 007 corresponding to histone H2.A, and m/z ∼15 335 corresponding to histone H3). For this sample, the profiles recovered after staining with Cresyl Violet and Methylene Blue showed the least variation with respect to the control profile. When taking into consideration both the quality of the staining and the quality of the mass spectrometric results, Methylene Blue and Cresyl Violet were preferred over the other stains. In particular, staining with Cresyl Violet offered high nuclear/cytoplasm contrast necessary for adequate histological analysis of cancerous tissue. Analytical Chemistry, Vol. 76, No. 4, February 15, 2004

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Figure 7. Deviation from the control (unstained) of mean (n ) 20) MALDI-MS protein profiles obtained after staining with Methylene Blue, Cresyl Violet, and Terry’s Polychrome (solid black lines). Point-wise 99.9% confidence intervals for the difference from control are also shown (dotted lines). To aid trend interpretation across the m/z range, smoothed mean differences (red lines) are also presented. See text for details.

A more systematic quantitative study of the variations introduced in the profiles by different staining procedures was performed. For this analysis, we chose to use mouse liver because of its relative homogeneity over a fairly large surface area and range of depth. Forty successive serial 12-µm sections were cut from one liver lobe. To average variations within the tissue as a function of depth, consecutive sections were successively used as control (rinsed but not stained), stained with Methylene Blue, stained with Cresyl Violet, or stained with Terry’s Polychrome as described in the Materials and Methods Section. Two distinct matrix droplets (sinapinic acid) were deposited approximately at the same coordinates on each section throughout the set. Instrumental acquisition parameters (delayed extraction settings and laser energy) were kept constant throughout the experiment. Protein profiles were acquired from each spot by averaging 500 consecutive laser shots. We therefore obtained 20 profiles, reflecting the protein expression of mouse liver for the control and each of the chosen staining protocols. Prior to the statistical 1152 Analytical Chemistry, Vol. 76, No. 4, February 15, 2004

analysis, each spectrum was internally calibrated (cytochrome c oxidase VIIC at m/z 5444.4, and β-hemoglobin at m/z 15,617.9) and baseline-corrected using an in-house written algorithm. No intensity normalization was performed. After baseline correction, spectral intensity values were squareroot-transformed to reduce the mean-variance dependence, because in raw spectra, observations with high mean values tend to have larger variance than observations with low mean values. For each m/z value between 3000 and 25 000, a linear model (analysis of variance) was fit to the transformed intensity values to estimate the mean intensity for each stain (as well as control) and to compute an estimate of residual variance. Subsequently, a pointwise 99.9% confidence interval was computed for the difference in means between each stain and control (Methylene Blue minus control, Cresyl Violet minus control, and Terry’s Polychrome minus control, respectively). This high confidence level was selected to accommodate the large number of intervals computed while acknowledging the strong short-range positive correlation

Figure 8. IMS analysis of a 12-µm coronal mouse brain section. (a) Photomicrograph of a Cresyl Violet-stained section showing different anatomic brain substructures. (b) MALDI-MS protein profile obtained after homogeneous matrix deposition averaging all of the individual spectra acquired from the section. (c-g) Ion density maps obtained at different m/z values with an imaging resolution of 100 µm. The ion density maps are depicted as pseudocolor images with white representing the highest protein concentration and black the lowest.

between intensities at adjacent m/z values. Excursions beyond these confidence limits are intended to indicate m/z regions where individual stain means differ from control. For graphical display, mean differences and confidence limits were smoothed using a smoothing spline with the smoothing penalty chosen by crossvalidation.27 Finally, for each stain, the deviations from control were smoothed using supersmoother.28,29 This is a nonlinear, variable span smoother that uses cross-validation to adapt to local changes in the curvature of the underlying function. It is used here to indicate the general trend of each stain’s mean intensity with respect to control. Figure 7 presents the difference in means between each stain and control. A positive signal at a particular m/z value indicates that it is on average more intense after staining with respect to the control, whereas a negative signal indicates that it is on average less intense after staining with respect to the control. Also displayed in Figure 7 are the deviations from the control profile within a 99.9% confidence interval range (dotted lines). After staining with Methylene Blue, in the m/z presented, except for a very limited set of signals, the mean deviation from the control always falls within the control’s confidence interval. This indicates a high degree or similarity with the control profile. The general trend of the effects of the staining as a function of molecular weight can be visualized by plotting a supersmoothed curve of the mean deviation. In the case of Methylene Blue, the super(27) Green, P. J.; Silverman, B. W. Nonparametric Regression and Generalized Linear Models; Chapman and Hall: London, 1994. (28) Friedman, J. H. A variable span scatterplot smoother. Laboratory for Computational Statistics; Stanford University Technical Report No. 5, 1984. (29) Hastie, T. R.; Tibshirani, R. J. Generalized Additive Models; Chapman and Hall: London, 1990.

smoothed curve follows the zero y axis with high fidelity across the entire m/z range. After staining with Cresyl Violet and Terry’s Polychrome, between 20 and 30 signals strongly deviated from the confidence interval of the control profile, some by more than 100-fold. Signals that display a decreasing intensity are, however, in most cases still detected. The data indicate that above m/z ∼12 000, on average, Cresyl Violet staining results in a suppression of mean intensity. The variations of mean deviation from the control as a function of m/z detected after Cresyl Violet staining suggest a negative deviation above m/z ∼12 000. This trend is better visualized when considering the behavior of the supersmoothed curve of the mean deviation. After staining with Terry’s Polychrome, the trend of the supersmoothed curve indicates that signals below m/z ∼12 000 are better detected with stronger intensities. Above m/z ∼12 000, the supersmoothed curve equally deviates from the zero y axis between positive and negative values. One may also note that the behaviors of the mean deviations from the control and supersmoothed curves observed after staining with Cresyl Violet and Terry’s Polychrome show similar trends, which are fairly different than the ones observed for Methylene Blue. It is possible that the differences observed after staining may, in fact, be the results of the nature of the solvents in which the dyes are prepared. Cresyl Violet and Terry’s Polychrome are prepared in water-based solvents, whereas Methylene Blue is prepared in an ethanol-based solvent (see Table 1). (b) MS Imaging. For smaller tissue regions or a higher resolution analysis, tissue sections can also be analyzed by imaging mass spectrometry. This analysis yields information about the protein pattern trends within a tissue sample. Critical to this analysis is the correlation between the protein changes within a Analytical Chemistry, Vol. 76, No. 4, February 15, 2004

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Figure 9. (a-c) LCM of Cresyl Violet-stained mouse hepatic cells. MALDI-MS protein profiles obtained from microdissected mouse hepatic cells. Ct, control; MB, stained with Methylene Blue; and CV, stained with Cresyl Violet.

tissue to the visual cellular changes across the sample. Using these staining procedures, a visual analysis of a tissue section prior to matrix coating and image acquisition can be linked to the resulting protein analysis, aiding the identification of morphologically linked protein changes. To demonstrate this, MS images of mouse brain sections were obtained after staining with Cresyl Violet. An array of 104 × 77 ) 8008 data points over the section was created with a lateral resolution of 100 µm, averaging 20 laser shots per point. Figure 8a presents a photomicrograph of a 10-µm-thick section mounted on a conductive glass slide and stained with Cresyl Violet prior to matrix spray deposition. Several tissue features, such as the cerebral cortex, the corpus callosum and the striatum can be easily distinguished. Figure 8b presents the MALDI-MS protein profile obtained when averaging all of the individual mass spectra recorded over the section during image acquisition (representing about three-quarters of the data points) in the m/z range between 2000 and 30 000. All of the individual spectra were baselinecorrected. Figure 8c-g presents five ion density maps obtained for different m/z values. These signals, in particular, were found to be highly localized within specific brain structures. For example, m/z 7339 was found exclusively in the striatum, whereas m/z 7844 was found exclusively in the corpus callosum. Similar images have previously been obtained from unstained sections.1,2,4 In general, for the signals detected from the section after staining with Cresyl Violet, it is important to note that no significant delocalization was observed at the image resolution used. (c) Laser Capture Microdissection. Laser capture microdissection (LCM) can be utilized to select specific cells from an intact 1154

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tissue section,30 and adequate staining becomes an important step for accurate cell selection. Analysis of the proteins from the dissected cells can be performed using multiple approaches, including analyzing extracts by 2-D gel electrophoresis,31,32 as well as applying a matrix solution to the surface of the cells, allowing direct access to the protein content. MALDI-MS analysis of lasercaptured cells has been successfully demonstrated and used to study normal and cancerous human breast epithelial cells10-12 as well as various cell types from the mouse epididymis.9 Our interest has focused on analyzing the proteins within the intact cells, which minimizes the protein extraction and concentration steps. LCM requires a drastic dehydration of the tissue sections to permit the capture process (see Material and Methods), including several ethanol dehydration steps and an additional rinse in xylene. Since many of these stains are soluble in ethanol or xylene, it is important to ascertain if fully dehydrated sections remain stained and can be used in LCM of cells and subsequent MALDI-MS analyses. Consecutive 5-µm mouse liver sections were cut and stained with Cresyl Violet or Methylene Blue. Some Cresyl Violet LCM-compatible staining protocols have been reported elsewhere33 and on the Internet (www.ambion.com, www.arctur.com). (30) Simone, N. L.; Bonner, R. F.; Gillespie, J. W.; Emmert-Buck, M. R.; Liotta, L. A. Trends Genet. 1998, 14, 272-276. (31) Banks, R. E.; Dunn, M. J.; Forbes, M. A.; Stanley, A.; Pappin, D.; Naven, T.; Gough, M.; Harnden, P.; Selby, P. J. Electrophoresis 1999, 20, 689-700. (32) Curran, S.; Lawrie, L.; McLeod, H. L.; Fothergill, J. E.; Cash, P.; Murray, G. I. J. Pathol. 2000, 192, 14A-14A. (33) Luo, L.; Salunga, R. C.; Guo, H. Q.; Bittner, A.; Joy, K. C.; Galindo, J. E.; Xiao, H. N.; Rogers, K. E.; Wan, J. S.; Jackson, M. R.; Erlander, M. G. Nat. Med. 1999, 5, 117-122.

An unstained control section was also processed. About 400 hepatic cells were microdissected from each section, spotted with matrix, and analyzed. For both Cresyl Violet and Methylene Blue, the dehydration steps did not further destain the sections. Figure 9a-c presents photomicrographs of the Cresyl Violet stained section during the different LCM steps. Figure 9 also presents the protein profiles obtained after MALDI-MS analysis of the unstained (Ct) and Methylene Blue (MB)- and Cresyl Violet (CV)stained microdissected cells in the m/z range from 3000 to 30 000. Overall, quality spectra in terms of resolution and signal intensity were obtained from all of the microdissected cells and were found in good agreement with those obtained after direct section analysis (Figure 2). The profiles obtained from Methylene Blue-stained cells were found remarkably similar to those obtained from control cells over the entire studied m/z range. The profiles acquired from Cresyl Violet-stained cells showed some significant signal intensity differences from the control profiles. For example, the signals at m/z 9911, 14 287, and 29 281, which were detected with strong intensity in the control cells, were detected with much lower intensities after Cresyl Violet staining. As a general trend, stronger intensity signals were obtained after Cresyl Violet staining in the m/z range below ∼12 000. However, signals in the m/z range above to ∼18 000 were better detected from the control or Methylene Blue stained cells. These trends are in good agreements with those obtained after direct MALDI-MS analysis of liver sections. CONCLUSIONS We have demonstrated the compatibility of several wellestablished tissue staining protocols with MALDI-MS analyses, merging new molecular technology and time-honored pathology procedures. Comparisons have shown that the use of ITO-coated slides results in accurate, reproducible mass assignments as well as comparable signal resolution for both standards and tissue sections over several hours of analysis time. These glass slides have been used as a support for stained tissue sections, allowing direct visual analysis of the nuclei prior to MS analysis. Using these staining protocols, MS analysis can be focused on specific morphological regions of interest. This procedure avoids the constraints consistent with aligning consecutive stained sections with an original analyzed sample, such as general uncertainty in

alignment accuracy as well as the inability to actively select small regions or specific cells of interest, with minimal effects to the overall protein pattern. The staining protocols described here are compatible with MALDI-MS and generally result in quality nuclearspecific staining. Comparative analyses have demonstrated that, although stain-specific intensity variations can occur, the overall protein pattern remains consistent between stained tissue sections and control samples. Additionally, the majority of the stains presented here demonstrate a clear distinction between the stained nuclei and the surrounding cytoplasm. This distinction is necessary for many histopathological classifications, including identifying regions of tumor development or distinguishing between different cell types. Using these techniques, we have demonstrated that reliable histopathological and MS analysis can be performed on a variety of tissue samples. Direct tissue analysis, including stain-guided region-specific profiling and high-resolution imaging can be performed. These techniques can also be used in combination with LCM to allow the capture and direct analysis of specific cells of interest. Utilizing the ITO-coated plates as well as the staining protocols presented here further improves the accuracy of MS direct-tissue analysis and overall data reliability. A direct comparison between the sample analyzed and the cellular morphology allows for a molecular analysis of the determined pathology and should lead to the discovery of cell-specific biomarkers.8 ACKNOWLEDGMENT The authors acknowledge Pamela Adams and Anthony Frazier (Human Tissue Acquisition Core Laboratory, Vanderbilt University), Kimberly Johnson (Vanderbilt-Ingram Cancer Center), and Annette Erskine (Mass Spectrometry Research Center, Vanderbilt University) for their valuable help with tissue staining. The authors also acknowledge the Robert J. Kleberg, Jr. and Helen C. Kleberg Foundation, the National Institutes of Health (Grant GM 5800805) and National Cancer Institute (Grant CA 86243-02) for financial support.

Received for review September 25, 2003. Accepted December 1, 2003. AC0351264

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