JOURNAL OF MASS SPECTROMETRYJ. Mass Spectrom. 2003; 38: 699–708Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jms.505

SPECIAL FEATURE:PERSPECTIVE

Direct tissue analysis using matrix-assisted laserdesorption/ionization mass spectrometry: practicalaspects of sample preparation

Sarah A. Schwartz, Michelle L. Reyzer and Richard M. Caprioli∗

Mass Spectrometry Research Center, Vanderbilt University, Nashville, TN 37232, USA

Received 1 June 2003; Accepted 18 June 2003

Practical guidelines for the preparation of tissue sections for direct analysis by matrix-assisted laserdesorption/ionization (MALDI) mass spectrometry are presented. Techniques for proper sample handlingincluding tissue storage, sectioning and mounting are described. Emphasis is placed on optimizing matrixparameters such as the type of matrix molecule used, matrix concentration, and solvent composition.Several different techniques for matrix application are illustrated. Optimal instrument parameters and thenecessity for advanced data analysis approaches with regards to direct tissue analysis are also discussed.Copyright 2003 John Wiley & Sons, Ltd.

KEYWORDS: sample preparation; MALDI-MS; tissue analysis; imaging MS; proteins

INTRODUCTION

Matrix-assisted laser desorption/ionization mass spectrom-etry (MALDI-MS)1 has revolutionized the study of biologyand biochemistry by permitting sensitive, rapid, and molec-ularly specific analyses of peptides and proteins.2,3 Thistechnology provides information on molecular weights withhigh mass accuracy, protein modifications such as phospho-rylation and glycosylation, and allows for the identificationof proteins by peptide sequencing combined with proteindatabase searches.

MALDI-MS is often used for the analysis of relativelypure proteins obtained from a complex biological sample bya rather lengthy set of isolation and purification procedures.Although extremely powerful, the overall sample prepara-tion can be time-consuming, involving multiple steps thatmay include extraction of the peptides or proteins from abiological system of interest (cells, tissues, fluids, etc.), sep-aration and purification of the proteins (gel electrophoresis,HPLC), and concentration of the individual proteins. Sig-nificant losses of material are often encountered throughoutthese steps. Even so, tissue homogenates offer little informa-tion about protein spatial localization and expression levelswithin the tissue.

ŁCorrespondence to: Richard M. Caprioli, Vanderbilt University,Mass Spectrometry Research Center/824A RRB, 23rd Ave. S. @Pierce Ave., Nashville, TN 37232-6400, USA.E-mail: r.caprioli@vanderbilt.eduContract/grant sponsor: NIH/NIGMS.

Direct tissue analysis by MALDI-MS can be performed onintact tissue for the determination of the spatial distributionof compounds and their relative expression levels withoutthe need for molecularly specific exogenous compoundssuch as radiolabeled or immunochemical reagents.4 – 15 Thisprocess involves preparation procedures with minimalsample handling, which decreases analyte losses.

Over the past several years, investigators have demon-strated that peptide and protein signals can be effectivelydesorbed directly from cells and tissues using MALDI-MS.4 – 13 Cellular analysis has included work involving neu-rons from the freshwater snail, Lymnea stagnalis,13 and theanalysis of single crayfish neurosecretory cells for the expres-sion of two precursors of the hyperglycemic hormone.10 Areview of single-cell MALDI published in 2000 provides anoverview of the technique and summarizes much of thework that has been done primarily on cells using MALDI-MS.4 Direct analyses of peptides and proteins from tissuesinclude the detection of proteins from rat pituitary,5 ratpancreas,5 mouse brain,11 human brain tumor xenografts,11

and mouse prostate.14 In addition, small molecular weightpharmaceutical compounds have also been directly detectedin tissue.12,15 The detection of the drug paclitaxel in ratliver and human ovarian tumor xenograft tissue usingMALDI-MS/MS analysis in a quadrupole ion trap instru-ment has been reported.12 The anti-tumor drug candidateSCH 226374 was detected in mouse tumors via MALDI-MS/MS analysis in a hybrid quadrupole-time-of-flight massspectrometer.15

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The direct proteomic analysis of tissue sections byMALDI-MS is an emerging technique of great potential.Simple sample preparation procedures allow for fast andreliable tissue analysis, typically resulting in the detectionof many hundreds of ion signals in the mass range 2000 to70 000 Da. More importantly, these signals can be localizedto discrete tissue regions, thereby adding a new dimensionto the analysis. This paper outlines sample preparationand analysis protocols including practical aspects of tissuesectioning and matrix deposition, and general strategies fordirect tissue analysis by MALDI-MS.

EXPERIMENTAL METHODS

The MALDI matrices 2,5-dihydroxybenzoic acid (DHB) and˛-cyano-4-hydroxycinnamic acid (HCCA) were purchasedfrom Aldrich Chemical Co. (Milwaukee, WI, USA), andsinapinic acid (SA) from Sigma Chemical Co. (St. Louis, MO,USA).

MALDI-TOF mass spectra were acquired using a VoyagerDE-STR mass spectrometer (Applied Biosystems, Foster City,CA, USA) equipped with a nitrogen laser (337 nm). Datawere obtained in positive, linear mode, with an acceleratingvoltage of 25 kV, 95% grid voltage, 0.05% ion guide wirevoltage, a delay time of 150 ns, and a flight path length of2.0 m. In general, 250 laser shots were averaged for eachspectrum.

INITIAL TISSUE TREATMENT

Tissue preparation methods should be carefully followed inorder to maintain the integrity of the spatial arrangementof compounds. Mishandling or improperly storing tissuesamples in the early sample preparation steps may causedelocalization and degradation of the analytes. Considera-tion must be given to how the tissue is treated immediatelyafter surgical removal, how it is sectioned (including tem-perature, section thickness, and use of fixing and embeddingmedia), the method used to attach it to the MALDI targetplate, the type of target plate used, how the matrix is applied,and finally how the tissue is stored.

It is essential to surgically remove tissue samplescarefully so that the native shape of the tissue is retainedas much as possible. Immediately after removal, the tissuemay be loosely wrapped in aluminum foil and frozen inliquid nitrogen by gently lowering the tissue into the liquidnitrogen over a period of 30–60 s. This preserves the shape ofthe tissue and also protects the biological tissue componentsfrom degradation. (Rapid plunging of the tissue into theliquid nitrogen is not recommended since it often leads tocracking and brittle edges.) The foil acts to stabilize moremalleable tissues as well as to inhibit tissue adhesion to thesides of the liquid nitrogen dewar. Placing freshly excisedtissues in plastic tubes and then freezing them should beavoided, as the tissues may stick to the sides of the tubeor may take on the shape of the tube. Whole tissues mayremain frozen in a freezer at �80 °C for at least a year withno significant degradation.

Frozen tissues are cut into thin sections in a cryostat forsubsequent mounting onto MALDI plates. Optimal tissue

thickness is a compromise—thicker sections are easier tomanipulate, but take longer to dry and may not be electricallyconductive once in the mass spectrometer, resulting in lesserquality spectra. On the other hand, very thin sections aredifficult to manipulate and can tear easily. Thus, for MALDIanalysis, tissues are optimally sliced to a thickness in theorder of the diameter of a mammalian cell (¾10–20 µm), thusthe majority of the cells in the slice are cut open, exposingthe intracellular contents.

The use of cryostats to section frozen tissues allowsaccurate sectioning to be accomplished, while minimizingsample contamination. Tissue samples are attached to asample stage and sliced with a stainless steel microtomeblade. The sample stage is generally held between �5 °Cand -25 °C, depending on the tissue type. Fatty tissuestend to require lower temperatures to achieve high-qualitysections. Samples such as breast tissue or visceral fattissue can coagulate during sectioning at warmer cryostattemperatures, negatively affecting spatial information.

Traditional tissue sectioning for histological stainingrequires the use of an embedding medium, such as agar orOCT (optimal cutting temperature polymer), which stabilizesthe tissue and provides a smooth cutting surface. However,this is not recommended when using mass spectrometrybecause the presence of OCT suppresses ion formation inMALDI-MS analysis. For example, Fig. 1 shows the massspectra obtained from two slices of rat liver tissue. InFig. 1(A), OCT was used to adhere the liver slice to thecryostat stage by acting as an adhesive, but the cryostatblade never contacted the OCT as the tissue was sliced. Theresulting mass spectrum reveals many intense signals withinthe mass range shown of m/z 4500–10 500. In Fig. 1(B), theliver tissue was embedded in OCT. As the tissue was sliced,the blade cut through both the OCT and the liver tissue.The resulting liver slice thus had some OCT spread acrossits surface, and the mass spectrum obtained from the OCT-embedded liver tissue contains only about half of the signalspresent compared with the spectrum of the non-embeddedtissue.

Transfer of the tissue slice to a MALDI target plate canbe accomplished in several ways. The preferred methodinvolves a cold MALDI plate that is prepared by simplyplacing the plate in the cryostat chamber held at �15 °Cwhile sectioning. The tissue section is gently picked upusing forceps for thicker sections or an artist’s brush forthinner sections and positioned on the cold plate. The plateand tissue section are then quickly warmed together, andthe tissue is thus thaw-mounted onto the plate. With thismethod, there is no loss of water-soluble proteins since anyice crystals that have formed on the section transfer to theMALDI plate along with the section. The second methoduses a plate held at room temperature that is placed overthe frozen section and, as the section thaws, it adheres tothe MALDI plate. Often, however, a ‘residue’ of ice crystalsis left on the cryostat cutting surface. Sections transferredwith this method give poorer mass spectra, likely becauseof the loss of soluble proteins in the ice left behind. While itis possible that proteins may delocalize during the thawingprocedure, traditional biochemical assays using this method,

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Figure 1. Effect of OCT on MALDI signal from rat liver. (A) Optimal procedure where OCT is used to adhere the tissue to the samplestage but does not come into contact with the sliced tissue. The resulting spectrum shows many intense signals between m/z 4500and 10 500. (B) The tissue was embedded in OCT and attached to the sample stage. The resulting tissue slice is surrounded by OCTon the MALDI plate, and the resulting spectrum contains only about half of the signals as that in (A).

such as immunohistochemistry and autoradiography, showwell-defined protein localization.

It is important with both tissue transfer methods thatthe section is transferred to the MALDI plate with greatcare so that it retains its shape, eliminating complications ofdeformities, scratches, tears, and rolled-up edges. Folds inthe tissue section prevent the detection of some proteins fromsurfaces that are covered and give a non-flat surface that maynegatively affect the accuracy of mass measurements. Tearsor rips distort the tissue section, as well as create holes orgaps, which are not present in the native tissue. This not onlydistorts the protein profile, but also complicates comparisonof the analyzed section with a stained histological section.

One additional practical point can be made aboutthe composition of the MALDI plate. It is very usefulif the tissue can be differentiated from the surface ofthe plate when it is in the mass spectrometer in orderto position the tissue accurately and reproducibly. Apolished, gold-coated surface provides high contrast to thedull surface of the tissue, thus enhancing tissue outlinerecognition. Additionally, differences in cell morphologycan frequently be distinguished in sections on a gold surface.This property is useful for obtaining high-quality digitalphotographs of tissue sections. Stainless steel plates donot provide this advantage. Novel conductive glass slidesand MALDI-friendly staining protocols are being developedin this laboratory as alternatives to metal plates.16 Theseslides allow one tissue section to be first stained forhistological classification and then analyzed by MALDI-MS. This approach offers the advantage of being able todirectly analyze the stained section by MALDI, whereasmetal plates require collecting an adjacent section of tissueon a microscope slide for staining in order to correlatemorphological regions with the mass spectrometric analysis.

Other specific considerations may arise depending onthe application. For example, some drugs that have beenanalyzed for their distribution in tissue are light-sensitive.Therefore, once a tissue is cut and a section is on a MALDIplate, the drugs present in the tissue section are vulnerable tophotodegradation. To minimize this effect, the section shouldbe kept in the dark as much as possible before analysis.

MATRIX APPLICATION: GENERALCONSIDERATIONS

Once the initial tissue preparation steps have been com-pleted, matrix solution is deposited on the tissue surfaceprior to analysis. Choosing the best matrix and optimizingapplication parameters are necessary to obtain high-qualitymass spectra directly from tissue samples as well as main-taining the spatial integrity of the tissue surface. The choiceof matrix and matrix solvent conditions greatly affects ionproduction. While matrix selection and concentration aregenerally fixed for a given application, variables such assolvent composition can be fine-tuned to focus on specificmolecules in the tissue sample.

Matrix selection plays an important role in obtaininghigh-quality spectra. Sinapinic acid (3,5-dimethoxy-4-hydroxycinnamic acid, SA) is routinely used forhigher molecular weight proteins while ˛-cyano-4-hydroxycinnamic acid (HCCA) is a more common matrixfor lower molecular weight peptide species. Because of thewidely varying surface properties of tissue sections, thematrices used must effectively crystallize over all areas ofthe tissue in order to yield high-quality spectra. Evaluation ofthe three most common matrices on tissue, SA, HCCA, andDHB, demonstrates that SA yields the best combination ofcrystal coverage and signal quality (Fig. 2). Matrix droplets

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Figure 2. Matrix crystallization on tissue and resulting mass spectra. Solutions of three different matrices, SA, DHB, and HCCA,were made at 20 mg/ml in 50 : 50 acetonitrile/0.1% TFA in water. Each matrix (0.25 µl) was applied (2ð) to a section of mouse livertissue, and the spots were analyzed with a MALDI-TOF mass spectrometer. The resulting optical images and mass spectra areshown for (A) SA, (B) DHB, and (C) HCCA. As shown, both SA and HCCA form dense crystals on the tissue, while DHB does notcrystallize well. However, SA results in a superior quality mass spectrum, containing over 300 individual signals, goodsignal-to-noise, and a low baseline. HCCA results in an intermediate spectrum, with some protein signals, but relatively poorsignal-to-noise and a falling baseline. DHB produces only a few broad, low signals.

(250 nl, 20 mg/ml of each matrix in 50 : 50 acetonitrile/0.1%trifluoroacetic acid (TFA) in water) were deposited on thesurface of a mouse liver section and analyzed. As shown inthe figure, SA and HCCA effectively crystallize on the tissuesurface while DHB produces few or no visible crystals on thetissue. For both HCCA and DHB, recorded signals had lowresolution and low signal-to-noise. In contrast, SA produceda high-quality spectrum consisting of over 300 individual ionsignals. Thus, overall, SA serves as a good general matrix fortissue applications.

However, some improvements can be made to thecrystallization process on tissues. For example, while thesignal obtained from HCCA on native tissue is poor, thesignal obtained when HCCA is spotted on tissue that hasbeen washed in 70% ethanol is significantly improved. Asshown in Fig. 3, the spectral quality from a section of mouseliver tissue spotted with HCCA is poor prior to washing(Fig. 3(A)), but after washing (Fig. 3(B)) the signal is similarin the number of proteins extracted and overall signal qualityto a SA spot. For washing, the plate with the tissue section isimmersed in 70% ethanol for 30 s, removed, and immersed

a second time in a fresh 70% ethanol solution for 30 s. Theethanol wash step allows for the removal of salts and excesshemoglobin from the tissue surface. The overall result isimproved signal quality due to the enhanced crystallizationof HCCA on the tissue and decreased signal suppression.Because ethanol is a known tissue fixative, delocalization ofproteins is not expected to be significant and has not beenobserved to any measurable extent. However, any proteinssoluble in the aqueous ethanol may be removed during thewashing step. The washing step also marginally improvesthe quality of spectra obtained from sinapinic acid (Figs. 3(C)and 3(D)). No significant improvement was observed forDHB (data not shown).

Matrix concentration is also important in direct tissueanalysis affecting both the ultimate crystal coverage as well asthe mass spectral signal quality. Figure 4 demonstrates thathigher matrix concentrations result in higher quality spectra.Matrix solution droplets (0.25 µl) from 10 mg/ml, 20 mg/ml,and saturated (>30 mg/ml) SA in 50 : 50 acetonitrile/0.1%TFA were applied by micropipette to mouse liver sections.MS analysis performed on each droplet clearly shows that

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Figure 3. Washing tissue samples prior to matrix coating enhances MALDI signals. Mouse liver sections analyzed using HCCAmatrix demonstrate significantly enhanced ion signals following ethanol wash (B) when compared with an unwashed section (A), toppanel. Similar results are obtained when an unwashed tissue section spotted with SA matrix (C) is compared with a washed tissuesection (D), bottom panel.

a saturated matrix solution yields the highest number ofion signals with the best resolution compared with a 10 or20 mg/ml solution.

Many matrix/solvent combinations can be successfullyemployed. However, the goal of the matrix solution is toeffectively dissolve the analyte of interest; in this case,to extract a protein from its native environment on thetissue. Thus, solvent combinations will vary depending onthe molecule to be analyzed as well as the type of tissuesample being analyzed. A series of saturated SA solutions invarying organic solvent/water combinations were depositedon a mouse liver section and analyzed by MALDI-MSin order to show the effect of solvent composition onthe resulting mass spectra. Solvent combinations consistedof 30 : 70, 50 : 50, and 70 : 30 organic solvent/0.1% TFA indeionized water; organic solvents used include acetonitrile,ethanol, methanol, isopropanol and acetone. A comparisonof the spectra obtained highlights the variation in proteinsignals detected with different solvent systems (Fig. 5).Generally, the 75% and 25% organic solutions resultedin poorer quality spectra compared with a 50% organicsolution. Additionally, spectra obtained from the methanol-,isopropanol-, and acetone-based solutions yielded poorersignals compared with the acetonitrile- or ethanol-basedsolutions. Interestingly, while both solutions of 50 : 50acetonitrile/water and ethanol/water resulted in similar

profile patterns, they each exhibited a few protein signalsnot found in the other. Using 25 : 25 : 50 or 35 : 35 : 30acetonitrile/ethanol/0.1% TFA as the matrix solvent didnot result in a combined protein profile. Therefore, 50 : 50acetonitrile/water and ethanol/water are recommended asgood general solvents for tissue analysis since these twosolvent combinations have been found to give consistentlygood results on a variety of tissue samples. The use ofhigher organic solvents may be helpful in decreasing proteindelocalization if this should occur to a significant extent in aparticular application.

Similarly, the TFA concentration also affects the proteinprofile observed from direct tissue analysis. As noted above,as different solvent parameters affect the overall proteinprofile, changing the concentration of TFA also changes thespecific molecules measured. Figure 6 demonstrates that, asthe TFA concentration increases from 0 to 3% TFA, thetotal number of ion signals and the specific ions detectedvaries. Generally, we have found that a concentration rangeof 0.3–1% TFA maximizes the total number of proteinsanalyzed. However, in some cases, this concentration mayneed to be modified to obtain optimum performance.

METHODS FOR MATRIX APPLICATION

Two strategies are employed to apply matrix directly ontoa tissue surface: (1) depositing small droplets of matrix on

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Figure 4. Effect of matrix concentration on crystallization and the resulting mass spectra. Solutions of sinapinic acid (SA) weremade up in 50 : 50 acetonitrile/0.1% TFA in water at concentrations of (A) 10 mg/ml, (B) 20 mg/ml, and (C) saturated (>30 mg/ml).One spot (0.25 µl) was applied to a section of mouse liver tissue, and a mass spectrum was acquired. As shown, the greater theconcentration of matrix, the greater the crystal coverage, and the better the resulting mass spectrum (better signal-to-noise, moresignals detected).

specific morphological regions of the tissue and (2) coatingthe entire tissue with the matrix solution (Fig. 7). Thefirst approach results in low-resolution images, or profiles,whereby protein signals in a defined area of the tissueare obtained. This method allows for a relatively quickcharacterization of the protein content within a tissue sectionas well as facilitating a comparison between tissues (i.e.,disease vs. normal, treated vs. untreated, etc.). Matrixmay be deposited using either a low-volume automaticpipette or a syringe pump attached to a small capillary(50–100 µm i.d.). The placement of matrix droplets canbe guided by histological staining of an adjacent tissuesection. Small droplets incorporate proteins from a relativelylarge cell population. For example, deposition of a 100-nldroplet results in about a 900-µm diameter crystalline spot,incorporating proteins from about 1000 individual cells givena cell diameter of 30 µm. Although this type of profiling hasbeen performed reproducibly at matrix volume levels aslow as 5 nl, signal quality improves with increasing dropletvolume due to the greater amount of protein present andthe increased effectiveness of protein extraction due to thelonger drying time. MS analysis is then performed on eachindividual matrix droplet by summing 100 or more lasershots taken within the area of the droplet. A molecular profileis then generated for each spot on the tissue surface. Onemust assume that, within the area of each spot, proteins have

mixed and the spatial orientation, if any, is lost (althoughthis may not necessarily be the case).

High-resolution images can be obtained by homoge-neously coating the tissue surface with the matrix solutioneither as a continuous coating or as a high density of low-volume droplets. In this way the laser can sample the tissuein a raster, obtaining a spectrum at each point. An image ofa specific m/z signal can then be constructed that shows thespatial distribution of that protein within the tissue. Imageresolution is limited by two key parameters, crystal size andlaser diameter. Smaller crystal sizes are necessary for increas-ing imaging spatial resolution. Larger crystals require largerstep sizes between laser shots, while smaller crystals allowfor more crystals to be ablated in a single laser shot anda decreased laser step size. With crystal diameters smallerthan the laser beam, typically 50–150 µm depending on theinstrument, imaging resolution is generally limited to thelaser diameter.

Reproducible whole tissue coatings have been achievedusing a deactivated glass spray nebulizer to spray matrixdirectly onto the tissue surface. The primary goal isto maximize solubilization of proteins within the tissuein order to enhance co-crystallization of proteins andmatrix molecules as well as the final protein profile, whileminimizing protein delocalization. Increasing the length oftime proteins are exposed to large quantities of solvent also

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Figure 5. Solvent composition affects the tissue protein profile. Saturated sinapinic acid matrix in solvents of 50 : 50 organicsolvent/0.1% TFA in water as shown was deposited onto mouse liver tissue sections. The use of different solvent systems results indifferent protein profiles. Overall 50 : 50 ethanol/0.1% TFA resulted in the best protein profile for the mouse liver section, but 50 : 50acetonitrile/0.1% TFA in water has been shown to give better profiles for other organs (such as rat brain).

increases the chance the molecules can physically moveover the tissue surface. For full matrix coverage over theentire tissue sample, a cycle of matrix coatings is performedin which small volumes of matrix solution are depositedduring each cycle. The sample plate is held vertically about20–30 cm from the sprayer nozzle. As matrix is nebulizedfrom the sprayer, the device is moved parallel to the target toevenly apply matrix and just barely wet the sample surface.Deposition of a large quantity of solvent in any one regionof the tissue must be avoided. The sample is allowed to dryfor 1–2 min before the next coating cycle is performed. Anaverage of about ten cycles of coating and drying should beperformed to achieve an even, dense crystal field. Cyclingsmall volumes of matrix spray allows for a slow developmentof a dense matrix coat while minimizing the time thesample remains damp. Since different types of tissues canhave different surface properties, the number of coatingcycles can vary. For some samples, a more even coating isobtained when the final spray coating consists of only matrixsolvent. This serves to redissolve and recrystallize the matrix,increasing the incorporation of proteins in the crystals.

Other coating approaches have been evaluated in thislaboratory, including dragging a large matrix droplet acrossthe tissue surface,11,17 electrospray deposition, immersingthe tissue in a high-percentage organic solvent matrix solu-tion, and using robotics to uniformly deposit small matrixdroplets across the tissue surface. The large-droplet depo-sition method involves depositing a matrix droplet beside

the tissue under cold temperature conditions (–4 °C), drag-ging the solution across the surface with a glass rod, andallowing the sample to dry. This approach can result inexcellent protein signals and reasonable crystal coverage,but we have found this procedure to have poor reproducibil-ity. The electrospray technique requires a capillary deviceto travel over the tissue surface, depositing a fine spray ofmatrix solution over the sample. When applied carefully,this approach can yield a homogenous crystal coating thatcovers the entire tissue surface. However, in a practicalsense, we have had problems with reproducibly obtaininghigh-quality protein signals. Since the matrix is sprayed asvery small particles, crystals can easily form before reachingthe tissue surface due to the droplets drying in transit. Thissubstantially minimizes protein incorporation into the crys-tals and consequently decreases spectrum quality. Therefore,for an even crystal coat to be applied, several passes of theelectrospray device at optimum coating parameters may berequired. A dipping technique that involves soaking a tissuesection in a high-percentage organic solvent matrix solution(usually 20 mg/ml SA in 90% ethanol for 5–10 min) hasbeen successfully applied, although the procedure appearsto be very sensitive to conditions involving the drying pro-cess (humidity, temperature, etc.). Finally, a robot capable ofacoustic droplet ejection (RapidSpotter; Picoliter Inc., Sunny-vale, CA, USA) is being used to eject picoliter-sized matrixdroplets onto the surface of the tissue, effectively coatingthe section. This technique results in a crystal field similar

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Figure 6. TFA concentration affects the protein profile during direct tissue analysis. Sinapinic acid matrix (saturated in 50 : 50acetonitrile/water) with increasing concentrations of TFA was deposited onto mouse liver tissue sections as shown. The presence ofTFA increases the number of ions monitored, but TFA concentrations over 2% begin to inhibit specific ion signals.

(a) Imaging Tissues

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Figure 7. Direct tissue sample analysis. Sectioned tissues are thawed onto MALDI target plates and matrix was deposited by eithercoating the entire tissue section with matrix (A) or depositing matrix droplets onto the tissue surface (B). The resulting spectrademonstrate the complexity of protein expression from discrete tissue regions.

to electrosprayed surfaces, but droplet deposition allows formultiple droplet coats to be applied continuously. Dropletscan therefore remain wet longer while minimizing molecule

delocalization to within the droplet area. Figure 8(A) showsa robotically spotted section of mouse liver, where 20droplets were deposited consecutively onto the same spot at

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Figure 8. Robotically spotted mouse liver. (A) Saturated sinapinic acid matrix was deposited in an array onto a liver section. Eachcrystal spot is the result of 20 consecutive matrix depositions. The droplet diameter measures on average 130–150 µm.(B) MALDI-MS analysis of one matrix spot.

a frequency of 1 Hz to form an array of spots over the tissue.The resulting spectrum, shown in Fig. 8(B), is very similar toa spectrum obtained from a large, manual spot, indicatingthe feasibility of using this technique for precision spottingand imaging applications.

Choosing a method of matrix application is dependenton the overall goal of the experiment. Low-resolution tissueprofiling can be useful when the goal is to simply comparetwo regions of the same tissue or the same region of severalindividual tissue samples, such as determining the proteindifference between treated and untreated samples. Theresulting data set from this experimental approach is muchsmaller, 4–5 megabytes for 30 profiles. Many experimentsinvolving this approach can be analyzed without the aid ofcomplex computer programs and the entire procedure canbe performed quickly (less than 1 h). On the other hand, datacollected from imaging MS yield much more detailed (high-resolution) protein profiles across the tissue and data files areon the order of 1–2 gigabytes. High-resolution images maytake many hours to acquire, depending on the pixel densityrequired and instrument speed, and image data analysisrequires complex software viewing programs. Signal qualityis generally better in profiled samples when compared withimaged samples.

MASS SPECTROMETRIC ANALYSIS

Desorption of proteins from a complex sample such asa tissue section presents many challenges. Compoundsincluding salts containing NaC and KC, lipids, and other

proteins and biological compounds within the tissue sectioncan adversely affect the MS analysis. While ion suppressionand adduct formation can be expected at some level in allanalyses, methods for removing non-protein componentsusually compromise the spatial integrity of the surfaceand decrease sample throughput. In addition, instrumentparameters are difficult to optimize for detection acrossa large mass range, typically 70 000 Da, compromisingsensitivity and mass accuracy in some regions of this range.Nonetheless, high-quality and useful spectra can be obtainedby understanding the interplay of these issues and effectivelyoptimizing instrument parameters.

Ionization suppression, a common phenomenon in MSanalysis, occurs when one analyte is present in great excessover another and where one analyte is more efficientlyprotonated in the desorption process. For example, thisis commonly encountered in tissues with the ˛- and ˇ-chains of hemoglobin, proteins that can be present at highlevels. Typically, singly and doubly protonated forms ofboth hemoglobin chains are recorded. At high levels, thesesignals significantly compromise other protein signals in thespectrum. This phenomenon can generally be minimizedby limiting tissue contact with excessive amounts of bloodduring the surgical process as well as careful placementof matrix spots on the tissue so as to avoid reddish areascontaining excessive hemoglobin. At low levels, the presenceof hemoglobin chains can be advantageous because the ionsignals can be used as internal calibrants.

Optimizing the spectrum quality from a tissue samplealso requires tuning the instrument settings to maximize

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signal quality across the entire spectrum. Ideally, instrumentparameters should be optimized for individual, smaller massranges, especially when specific molecules are monitored ona tissue section in order to achieve the best signal resolutionand mass accuracy. Key among the parameters to vary in aTOF instrument are the delayed extraction parameters: gridvoltage and pulse delay. The grid voltage is a percentageof the accelerating voltage applied to a variable-voltage gridover the sample plate. The pulse delay is the time (in ns)after the laser ionizes the sample that the full acceleratingvoltage is applied. For any given mass range, there is a setof grid voltage and pulse delay values that are optimal,but they vary across the mass range. For example, optimalsettings for a low mass range (2000–10 000) consist of agrid voltage of 90–94% with a delay time of 50–150 ns. Ahigher mass range (20 000–100 000) typically requires a lowergrid voltage (87–92%) and a higher delay time (200–800 ns).Additionally, if a guide wire is in use, higher voltages aretypically required to achieve adequate sensitivity for highermass ions (>50 000). Increasing the laser intensity over thelevels typically used for more purified samples increases thetotal number of unique analytes detected. In addition, inthe profiling mode, the number of laser shots averaged perspectrum is typically between 250 and 750. This is less usefulin the high-resolution imaging mode because the crystal layeris usually less dense and high-quality signals can typicallyonly be obtained during the first 30–200 laser shots.

The matrix coating methods described thus far arebest for analyzing soluble proteins. Hydrophobic pro-teins, such as membrane-bound or transmembrane pro-teins, are not generally extracted and analyzed. Meth-ods for dissolving molecules in organic/water solutionsare ineffective at removing these molecules. Approachesapplying MS-’friendly’ detergents to the tissue surface intissue profiling experiments have significantly enhancedmembrane/lipophilic protein profiles. These detergentsinclude well-known MS detergents such as octyl ˇ-D-glucopyranoside (n-octyl glucoside) as well as cleavablesurfactants recently developed in this laboratory.18

CONCLUSIONS

Direct analysis of native tissue sections by mass spectrometryprovides a wealth of information. Protein localization andprotein–protein co-localization can be determined, as well ashow this changes in a compromised tissue. However, care intissue handling and preparation is required in order to extractthat information in a biologically meaningful way. Thetechniques discussed in this paper have all been successfullyapplied toward that goal, and, while no one procedure isoptimal for every type of analysis and tissue, the generaltrends and guidelines presented here should assist thoseapplying this methodology to their specific applications.

AcknowledgementsThe authors acknowledge NIH/NIGMS for funding and thank HansAerni for the RapidSpotter data. MLR acknowledges Philip Morris,Inc. for a post-doctoral research fellowship.

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