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DOI: 10.1080/01926230590881862 2005; 33; 92 Toxicol Pathol

Pierre Chaurand, Sarah A. Schwartz, Michelle L. Reyzer and Richard M. Caprioli Imaging Mass Spectrometry: Principles and Potentials

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Toxicologic Pathology, 33:92–101, 2005Copyright C© by the Society of Toxicologic PathologyISSN: 0192-6233 print / 1533-1601 onlineDOI: 10.1080/01926230590881862

Imaging Mass Spectrometry: Principles and Potentials

PIERRECHAURAND, SARAH A. SCHWARTZ, MICHELLE L. REYZER, AND RICHARD M. CAPRIOLI

Mass Spectrometry Research Center and Department of Biochemistry, Vanderbilt University Medical Center, Nashville,Tennessee 37212, USA

ABSTRACT

Direct tissue profiling and imaging mass spectrometry (MS) allow for detailed mapping of the complex protein pattern across a tissue sample.Utilization of these tools provides spatial information across a tissue section for target protein expression and can be used to correlate changes inexpression levels with specific disease states or drug response. Protein patterns can be directly correlated to known histological regions within thetissue, allowing for the direct monitoring of proteins specific for morphological regions within a tissue sample. Profiling and imaging MS have beenused to characterize multiple tissues, including human gliomas and lung cancers, as well as tumor response to specific therapeutics, suggesting theuse of proteomic information in assessing disease progression as well as predicting patient response to specific treatments. This article discussesboththe technology and methods involved in analyzing proteins directly from tissue samples as well as several MS applications, including profiling humantumors, characterizing protein differences between tumor grades, and monitoring protein changes due to drug therapy.

Keywords. Mass spectrometry; tissue; protein; profiling; imaging; cancer.

INTRODUCTIONIn recent years, mass spectrometry has become an in-

dispensable tool for proteomic studies (Aebersold andGoodlett, 2001; Godovac-Zimmermann and Brown, 2001;Lahm and Langen, 2000; McDonald and Yates, 2000;Pandey and Mann, 2000; Roepstorff, 1997; Russell andEdmondson, 1997). Desorption and ionization techniquessuchasmatrix-assisted laser desorption ionizationmassspec-trometry (MALDI MS) (Hillenkamp et al., 1991; Karas andHillenkamp, 1988) and electrospray ionization mass spec-trometry (ESI MS) (Fenn et al., 1989) have literally revolu-tionized our ability to analyze proteins. These improvementsoffer levels of sensitivity and mass accuracy never beforeachieved for the detection, identification and structural char-acterization of proteins. It is now possible to routinely mea-sure molecular weights above 200 kDa as well as obtain lowparts per million mass measurement accuracies for the de-termination of peptides and proteins. Protein identificationhas been greatly facilitated because of the rapid expansionof protein and gene databases. Modern mass spectrometerscan now rapidly map and fragment peptides that result fromprotease digestion in order to obtain sequence informationand identify proteins.MALDI MS is an ideal tool to investigate complex pro-

tein mixtures. It utilizes a matrix, a small acidic aromaticmolecule that absorbs energy at the wavelength of the irra-diating laser. The analyte molecule is mixed with the matrixin a ratio of typically 1/5000, deposited on a target plate andallowed to dry. During the drying process, matrix-analyte co-crystals form. These crystals are then submitted to very short

Address correspondence to: Richard Caprioli, Mass Spectrometry Re-search Center, 9160 MRB III, Vanderbilt University, Nashville, Tennessee37232-8575, USA; e-mail: [email protected]: MALDI MS: matrix-assisted laser desorption ionization

mass spectrometry; ESI MS: electrospray ionization mass spectrometry;IMS: imaging mass spectrometry; m/z: mass-to-charge; OCT: OptimumCutting Temperature; RP-HPLC: reverse phase high pressure liquid chro-matography; CAD: collision activated dissociation.

laser pulses (typically UV laser light), resulting in the des-orption and ionization of the analyte molecule. Mostly intactprotonated molecular ions are formed ([M+H]+, where M isthe molecular weight of the analyte molecule). The mass-to-charge (m/z) of the ion is typically measured in a time-of-flight mass analyzer (Cotter, 1999) (Figure 1).There are several technologies available to analyze pro-

teins in a tissue specimen. To date, the most commonly usedtechnology is the separation and visualization of proteinsby 2-dimensional (2-D) gel electrophoresis and subsequentidentification by mass spectrometry and database search-ing (Lahm and Langen, 2000; Godovac-Zimmermann andBrown, 2001). One of the drawbacks of the 2-D gel technol-ogy is that sample preparation removes the direct relationshipbetween morphological tissue regions and a specific protein.One solution to this problem is to purify cells from thin tis-sue sections by laser capture microdissection prior to proteinextraction. Although such an approach has been successful(Curran et al., 2000), the extraction of a sufficient quantity ofmaterial is very labor intensive and requires a large amountof microdissected cells.One of the recent applications of MALDI MS is its use to

profile and image proteins directly from thin tissue sections(Todd et al., 2001; Chaurand and Caprioli, 2002; Chaurandet al., 2002, 2004b). MALDI imaging mass spectrometry(IMS) is a new technology that allows for simultaneousmap-ping of hundreds of peptides and proteins present in thintissue sections with a lateral resolution of about 30–50µm.Matrix is first uniformly deposited over the surface of thesection, utilizing procedures optimized to minimize proteinmigration. Proteins are then desorbed from discrete spotsor pixels upon irradiation of the sample in an ordered ar-ray or raster of the surface. Each pixel thus is keyed to afull mass spectrum consisting of signals from protonatedspecies of molecules desorbed from that tissue region. Aplot of the intensity of any one signal produces a map ofthe relative amount of that compound over the entire im-aged surface. This technology provides an extremely power-ful discovery tool for the investigation of biological processes

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Vol. 33, No. 1, 2005 IMAGING MASS SPECTROMETRY STUDIES 93

FIGURE 1.—Principle of time-of-flight mass spectrometry. Ions formed by laser irradiation in the source area are accelerated by a positive (or negative) potentialdifference applied between the sample plate and the extracting electrode(s). Ions of the same nominal charge acquire the same kinetic energy; however, their respectivevelocities will depend on their mass. Ions are then separated in time based on their velocities in a field free time-of-flight tube and are finally detected by an iondetector. The time each ion takes to travel through the mass spectrometer is precisely measured. After instrument calibration, the ion flight times are converted tomass. Effectively, time-of-flight mass spectrometers measure the mass-to-charge (m/z) ratios of ions.

because the identities of proteins observed do not need tobe known in advance. IMS under various forms has alreadybeen successfully used to further characterize the expres-sion of proteins and other organic biological compounds innumerous normal and diseased tissues. For example, pro-tein organization in mouse colon (Chaurand et al., 1999),brain (Stoeckli et al., 2001; Todd et al., 2001; Chaurandet al., 2002, 2004b) and epididymis (Chaurand et al., 2003) aswell as phospholipid organization in mammalian lens tissue(Rujoi et al., 2004) has been studied. Variations in proteinexpression have been investigated in the cases of Parkinson’s(Pierson et al., 2004) and Alzheimer’s (Stoeckli et al., 2002)diseases. Several forms of cancers have also been inves-tigated including gliomas (Stoeckli et al., 2001; Chaurandet al., 2004b; Schwartz et al., 2004), breast cancer (Palmer-Toy et al., 2000; Xu et al., 2002), prostate cancer (Masumoriet al., 2001), colon cancer (Chaurand et al., 2001a) and lungcancer (Bhattacharya et al., 2003; Yanagisawa et al., 2003).In this later study (Yanagisawa et al., 2003), protein patternshave been shown to be predictive of diagnosis and prognosis.Methodologies aimed at detecting and mapping pharmaceu-tical compounds by direct MALDI MS analysis of sectionsfrom dosed tissues have also been described (Troendle et al.,1999; Reyzer et al., 2003).We review here the basic methodologies for tissue sample

handling and preparation for analysis by MALDI MS usingvarious mouse and human cancer tissue samples and discusscurrent and future potentials of the methodology for basicresearch and in clinical applications.

GENERATINGPROTEINPROFILES FROMTHINTISSUE SECTIONS

A schematic diagram of the general experimental proce-dure is given in Figure 2 (Chaurand et al., 2004b). Afterdissection, the tissue sample should be loosely wrapped inaluminum foil, immediately snap-frozen in liquid nitrogenand stored at−80◦C until analyzed. Thin frozen sections

are typically cut at−15◦C using a cryostat (the optimum cut-ting temperaturemaybe tissuedependent) and thaw-mountedonto a conductive target plate (Schwartz et al., 2003). The tis-sue sample is maintained with the desired orientation on thecutting block using a medium such as OCT (Optimum Cut-ting Temperature) polymer. However, it is important to notethat OCT is only used to mount the tissue on the cryostatblock and should not come into contact with the surface ofthe tissue to be sectioned. The surface of the tissue should beleft available for sectioning free from the polymer. To aligntissue features andmolecular images, 2 sections are typicallyused, one is stained (typically with hematoxylin and eosin)for optical evaluation and the second is investigated by IMS.Although this approach is robust, misalignment between the2 sections is of concern especially for very small tissue sam-ples such as needle biopsies. One of the latest tissue analysisprotocols developed utilizes optically transparent glass slidesas target plates (which have a thin conductive coating on thesurface) together with MALDI MS friendly tissue stainingprotocols (Chaurand et al., 2004a). This makes possible themicroscopicevaluationofa tissuesectionbyapathologist fol-lowed by the molecular imaging of the same section by IMS.In general, there are two basic modes of data acquisition:

profiling and imaging (Figure 2). In the profiling experiment,one is interested in comparing protein patterns from adiscrete number of spots or areas. Although there is no spotlimitation, typically this is done for 5–20 regions across agiven tissue section. Matrix is applied, as discrete droplets(spots) to the regions of interest, and the sample is placedinto the MS source. We have found sinapinic acid (saturatedin 50/50/0.1—acetonitrile/H2O/trifluoroacetic acid) to bethe matrix of choice for the analysis of proteins (Schwartzet al., 2003). Typically, 200 to 500 nL droplets of matrixare deposited using an automatic pipette covering an areaof 2–4 mm2. Smaller drop sizes (5–100 nL) can be appliedwith a fine capillary attached to a Hamilton type syringecontaining the matrix. In this case, matrix deposition isgenerally performed under low to medium magnification



94 CHAURAND ET AL. TOXICOLOGICPATHOLOGY

FIGURE 2.—Scheme outlining the different steps involved for profiling and imaging mass spectrometry of mammalian tissue samples. See text for details. Fromreference (Chaurand et al., 2004b).

to precisely deposit the matrix droplets at the desired tissuecoordinates. The laser beam irradiates each sample spot andion signals from 200–1000 consecutive shots are averagedacross the droplet surface generating a mass spectrum. Uponanalysis, the resulting mass spectra typically yield from 300to in some cases over 1000 signals, of various intensitiesover three to four orders of magnitude, in am/z range from2000 up to, in some cases, overm/z 200,000 (Chaurandand Caprioli, 2002). However, because of the inefficiency ofMALDI time-of-flight mass spectrometers to resolve (Bahr

et al., 1997) and detect higher molecular weight compounds(Brunelle et al., 1997; Westmacott et al., 2000), most signalsdetected are belowm/z 30,000. One may also presume thatthe most intense signals come essentially from soluble andabundant protein species.In the imaging experiment (Figure 2), the goal is to dis-

play a detailed molecular image of an entire tissue sectionor a specific subregion. In this case, matrix needs to behomogeneously deposited across the section without gen-erating any major lateral protein migration. In an attempt




to “fix” the proteins in a section, the sections mounted onthe target plate may be soaked in an ethanol fixing solutioncontaining matrix (sinapinic acid, 20 mg/mL in 90/10/0.1—ethanol/H2O/trifluoroacetic acid) for 5–10 minutes and al-lowed to dry at room temperature. The fixing procedurealso tends to seed matrix on the sections. Sections arethen coated with matrix solution (sinapinic acid, 20 mg/mLin 50/50/0.1—acetonitrile/H2O/trifluoroacetic acid) using apneumatic handheld Venturi glass sprayer. At this stage, spe-cial care needs to be taken not to “overwet” the section. Onaverage, 10 spray cycles are necessary to achieve a thin, rela-tively homogeneous, matrix coating that covers the entire tis-sue section. The coating proceduremay bemonitored under amicroscope to assess crystal size and density (Schwartz et al.,2003). A second coating approach currently developed in ourlaboratory consists of matrix deposition using an automatedspotter (Rapidspotter TM, Labcyte Inc., Sunnyvale, CA),generating a Cartesian microdroplet array over the surface ofthe section (Aerni et al., 2003, 2004). Although this tissue-coating approach does not currently permit imaging resolu-tion below 200µm, it has the advantage of limiting proteinmigration within the surface covered by each microdroplet.In an imaging experiment, thousands of spots can define

the array with each spot associated with a full mass spec-trum. Maximum resolution depends primarily on the dimen-sions of the ionizing laser beam. With commercially avail-able instruments, beam dimensions of about 25–100µm indiameter are attainable with minimum efforts (Caprioli et al.,1997). Specialized instrument control software is used to seta data acquisition grid that defines a discrete Cartesian pat-tern across the sample surface (Stoeckli et al., 1999, 2002).This pattern has a fixed center to center distance betweenspots, typically ranging from 25 to 250µmdepending on theimaging resolution required. The mass spectrometric data isthen acquired utilizing this grid pattern with a predeterminednumber of laser shots per grid coordinate. The signal inten-sity for a selectedm/z value at every acquisition coordinateis then integrated and a 2-dimensional ion density map, orimage, is reconstructed. An image can be generated for eachof the mass signals detected throughout the section. From asingle acquisition, several hundred images, each at a specificmolecular weight, can be reconstructed. Data acquisition andprocessing (image reconstruction) is done with specializedsoftware (Stoeckli et al., 2002).All of the samples presented herewere cut at−15◦C in 10–

12µmsections using a Leica CM 3050 S cryostat (LeicaMi-crosystems AG, Welzlar, Germany). Sections were analyzedusing sinapinic acid as matrix. Mass spectrometric data wereacquired in the linear mode under optimized delayed extrac-tion conditions using a Voyager DE-STR MALDI time-of-flight mass spectrometer (Applied Biosystems, Framingham,MA).

PROFILING AND IMAGING MAMMALIAN TISSUESECTIONSExamples of protein profiles obtainedbyMALDIMS from

various human cancer tissue biopsies are presented in Fig-ure 3. Tissues were cut in 12µm sections and processed asdescribed above. Overlaid in Figure 3 are three typical pro-files obtained from a grade IV glioma (blue trace), a colorec-tal adenocarcinoma (red trace) and a breast adenocarcinoma(green trace). Signals were detected throughout the studied

m/z range (from 3,000 to 70,000) with the large majority ofthese belowm/z30,000. A close inspection of these profilesrevealed that, although some signalswere common to 2 of thecancer forms, very few signals are common to all 3 forms ofcancer. Pattern recognition studies could therefore determineprotein signals specific for each tissue studied, illustrating thehigh specificity of the profiles for a given tissue sample.Preliminary investigations of human tumor biopsies by

MALDIMS demonstrate that proteomic patterns can be usedto distinguish cancerous tissue from normal tissue as wellas subclassify cancers by histological grade (Yanagisawaet al., 2003; Schwartz et al., 2004). In these studies, prospec-tively collected, snap-frozen normal and tumor specimensfrom multiple patients were examined using MALDI MS.Peptide and protein expressions were compared and the pat-terns assessed through hierarchical cluster analysis. In thestudy from Schwartz et al., the mass spectral patterns couldreliably distinguish gliomas from nontumor brain tissue aswell as subclassify grade IV gliomas from grades II andIII. Figure 4 presents the simultaneous analysis by imagingmass spectrometry of two 12µm sections obtained from agrade II (low-grade) and a grade IV (high-grade) resectedhuman glioma biopsy (Figure 4b, 4a, respectively). The sec-tions were coated with matrix using the automated spotter,and the images were acquired with a lateral resolution of250µm. Figure 4c presents survey protein profiles obtainedfrom the low-grade (red trace) and high-grade glioma (bluetrace) samples. In them/z range displayed, numerous signalswere found to be expressed with different intensities in boththe low-grade and the high-grade biopsies (highlighted bystars). These differences have been used to subclassify lowgrade from high grade gliomas (Schwartz et al., 2004). Fig-ure 4d–h presents 5 ion density maps, or images, acquired fordifferentmolecular weight signalswhich displayed strong in-tensity differences between the low-grade and the high-gradetumor. The mass signals found to statistically discriminatebetween normal and tumor and the different grades of can-cer are currently being identified. Biomarker identificationis performed by well-established methods that consist of ex-traction of the proteins from the tissue followed by proteinseparation (RP-HPLC, anion exchange, and size exclusion).After screening byMALDIMS, the HPLC fractions contain-ing the targeted molecular weight markers are digested withtrypsin and the resulting peptides mapped and sequenced bymass spectrometry. The proteins are identified by interro-gating gene or protein databases with the experimentally re-covered peptide mass maps and sequences (Chaurand et al.,2001b, 2003; Stoeckli et al., 2001).Basedonsignal (or protein) expressionmeasuredat precise

coordinates across the sections using high resolution IMS,changes in cellular morphology may be distinguished and insome cases, the cellular origins for the different protein sig-nals can be determined. This later point is demonstrated forthe signal atm/z 10836 in the previous analysis, identifiedas S100β protein (Swiss-Prot accession Number P04271),which was found increased by about a factor of 4 in the high-grade sample (Figure 5a). Figure 5b–c presents the corre-spondingmassspectrometric iondensitymapsobtainedwhenintegrating thesignal for theS100β proteinatm/z10836.Theimages clearly showstrongerS100β protein expression in thehigh-grade tumor with respect to the low-grade tumor. Based




FIGURE 3.—MALDI MS protein profiles obtained after analysis of 12µm tissue sections from human breast adenocarcinoma, colorectal adenocarcinoma andgrade IV glioma. Alpha and beta hemoglobins are labeledα andβ, respectively.




FIGURE4.—IMS analysis of 12µm low-grade (LG) and high-grade (HG) human gliomas. (a, b) Photomicrographs of the sections thaw-mounted on the MALDIsample plate prior tomatrix deposition. (c) Survey protein profiles obtained after homogeneousmatrix deposition. Stars indicate signals for whichsignificant intensityvariations were observed between the 2 profiles. d, h) Ion density maps obtained at differentm/z values with an imaging resolution of 250µm. The ion density mapsare depicted as grey scale images with white representing the highest protein concentration and black the lowest.




FIGURE5.—Localization of the S100β protein in human low-grade and high-grade glioma biopsies by imaging mass spectrometry and immunohistochemistry. (a)Partial survey MALDI MS protein profiles obtained from low-grade (LG, red trace) and high-grade (HG, blue trace) 12µmglioma sections. The signal atm/z10836was identified as the S100β protein. S100β ion intensity maps obtained from (b) high-grade and (c) low-grade glioma sections. Highmagnification photomicrographsobtained from (d) high-grade and (e) low-grade glioma sections after immunostaining for the S100B protein.




on signal expression across the high grade glioma section(Figure 5b), stronger intensity signals for S100β were corre-lated to regions with higher concentrations of astrocytes. Inparallel, S100β protein expression levels between low-gradeand high-grade gliomas were also investigated by immuno-histochemistry. Figure 5d–e displays magnified photomicro-graphs of high-grade and low-grade glioma tissue sections(cut from the same biopsies investigated by IMS) after im-munoreaction (epifluorescence microscopy). The photomi-crographs show a number of astrocytes in the high-grade tu-morwith pronouncedS100β immunoreactivity in the cytosol(arrowhead) and weak immunopositivity in the oligodendro-cytes in the low-grade tumor. Similar IMS and immunos-taining patterns have been seen when comparing grade IVastrocytomas with grade II astrocytomas.Imaging mass spectrometry can also be used to measure

the precise localization of drugs andmetabolites in tissue sec-tions (Reyzer et al., 2003; Troendle et al., 1999). One of thedifficultiesof imaging lowermolecularweight compoundsbyMALDI mass spectrometry comes from significant interfer-ence and overlap with signals from endogenous compoundsin the tissue or originating from various matrix cluster ionstypically present in them/z range up to about 2,000. In orderto increase selectivity as well as sensitivity, collisionally acti-vated dissociation (CAD) is employed (Reyzer et al., 2003).The general strategy involves mass selection of the intactdrug ion, fragmentation of the ion by CAD, and detectionof a unique fragment ion. Imaging of drugs on tissue is thenaccomplished in an analogous fashion to imaging of proteinson tissue. In this case, the intensity of the drug specific frag-ment ion is plotted as a function of pixel coordinate. Similarsoftware is required to control the x/y movement of the sam-ple stage, to identify the area to be imaged, and to define theimaging resolution.Imaging drugs by mass spectrometry offers significant ad-

vantages over traditional imaging techniques (such as au-toradiography and fluorescence spectroscopy). Because in-tact drugs may be desorbed directly from tissue surfaces, noadditional syntheses are required, thus allowing this type oflocalization analysis to occur earlier in the drug discoveryprocess with less cost. In addition, any confounding pharma-cological effects due to a bulky label are eliminated, as are theenvironmental issues associated with radioactivity. Finally,the molecular specificity of mass spectrometry allows the in-tact drug to be distinguished from itsmetabolites that differ inmass. The resulting images generated by mass spectrometryare thus specific for the intact drug, while the independentlocalization of metabolites may also be determined.In parallel to drug localization, variations of the proteome

induced by the drugs may be investigated as a function ofdosage or time to determine their efficacy. This strategy maybe extremely useful to assess at very early time points posi-tive (or negative) reactions to therapy. The efficacy of an ex-perimental inhibitor drug against human melanoma was ex-amined via MALDI mass spectrometry. The drug was orallyadministered tomice bearing humanmelanoma xenograft tu-mors at low and high physiological doses (16-fold increase).This compound inhibits one of the steps of the ras genepathway and has been shown to decrease tumor volume atthe doses used here. The mice were dosed for 19 days andthe tumors were removed 12 hr after the last dose. Multiple

mass spectra from several sections from each sample wereacquired and averaged. These are shown in Figure 6. Manysignals showed differences in intensities between the 2 mea-surements, indicating changes in the proteome as a responseto dosage in the drug treatment. For example, several regionsof the spectrum have been expanded to show signals such asm/z 4738 andm/z 8452 which were significantly reducedfrom low dose to high dose treatment. Other signals such asm/z 8720 show an inverted trend, with a significant increasein expression after high-dose treatment. These data suggestthat changes in multiple protein signals can be used to indi-cate which tumors will be responsive to drug therapy at insome cases, an early stage.

PERSPECTIVESImaging mass spectrometry is a new technology that is

currently undergoing further development to make it moreroutinely accessible to users. Imaging time depends on sev-eral instrumental parameters, namely the laser repetition rate,spot-to-spot sample repositioninganddataprocessing. Laserswith repetition rates at or above 1 kilohertz and improvedelectronics will considerably reduce acquisition times fromhours to minutes. Acquisition algorithms capable of record-ing high-throughput data and specially targeted data miningtools are also being developed. Imaging resolution, currentlyin the 50µm range for tissue level analysis, may be increasedto 1–5µm for applications requiring subcellular analyses.Such developments are ongoing in our laboratory and else-where (Spengler and Hubert, 2002). Efforts to improve sam-ple preparation and matrix coating procedures are also beingundertaken to provide protocols thatmore easily achieve highsensitivity and high resolution images.The potential of such a molecular imaging technology is

considerable. The fundamental contributions of the technol-ogy are rapidly providing molecular weight specific profilesand images at relatively high resolution and sensitivity. Theseprovide important information in the investigation of cellu-lar processes in both health and disease. Imaging MS is ofextraordinary benefit as a discovery tool because one doesnot need to know in advance the specific proteins that havechanged in a comparative study. Furthermore, the cellularorigins and relative concentrations of the markers across thesection can be assessed. Once a marker of interest is iden-tified, its precise (subcellular) location, concentration, regu-lation and function may be investigated, to help understanddisease progression at the molecular level. Although currentimaging MS technology does not allow analysis of individ-ual cells, we anticipate that new developments will allow thisapplication in the near future.Clinically, IMS can provide a molecular assessment of tu-

mor progression and treatment obtained from biopsies, withthe potential to identify tumor subpopulations and predictpatient survival that is not evident based on the cellular phe-notype determined histologically. Further, assessment of theefficacy of drug treatment through comparative proteomics isfeasible. The information obtained by IMS significantly aug-ments, but does not replace, existing molecular diagnostictools. Together, these tools promise to promote new discov-eries in biology and medicine.




FIGURE 6.—Average MALDI MS protein profiles obtained after analysis of 12µm tissue sections from human melanoma xenograft tumors implanted in miceshowing signal variations induced by a 19-day drug treatment at low- and high-dose (16-fold increase).

ACKNOWLEDGMENTS

Theauthorswould like to thank the followingpersons fromVanderbilt University (Nashville, TN): Dr. Malin Andersson,Dr. Marta Guix, Dr. Jiaqing Li, and Hans-Rudolf Aerni fortheir help in generating some of the data presented here.The authors also thank MDS Inc. (Odense, Denmark) for theS100β protein identification. The authors acknowledge fund-ing by theNational Institutes of Health (grantGM58008–05)and the National Cancer Institute (grant CA 86243–02).

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