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Imaging Mass Spectrometry of IntactBiomolecules in Tissue Sections

Erin H. Seeley, Richard M. CaprioliMass Spectrometry Research Center, Department of Biochemistry,Vanderbilt University School of Medicine, Nashville, TN, USA

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O U T L I N E

Introduction 39

3

Matrix Application 394

Protein Analysis 396

Peptides and Protein Digests 397

Lipid Analysis 399

Drug Analysis 400

roteomic and Metabolomic Approaches to Biomarker Discoveryttp://dx.doi.org/10.1016/B978-0-12-394446-7.00025-X 393

3D Imaging 40

1

High-Speed Imaging 403

Conclusions and Perspectives 403

Acknowledgments 403

References 403

INTRODUCTION

Imaging mass spectrometry (IMS) technologyenables the in situ analysis of biomoleculesand pharmaceutical compounds directly fromthin tissue sections.1e3 Thin sections (typically5e20 mm) are taken from a block of tissue andcollected onto a target in a fashion similar tothat used for histological analysis. After collec-tion, sections are processed by one of severalmethods depending on the class of moleculesto be analyzed. These steps may include

dehydration, washing with organic solvents orbuffers, and matrix application. Mass spectraldata are then generated using an ionizationprobe passed over the tissue surface in a rasterpattern, where each ablated spot or pixel givesrise to an individual mass spectrum.

Traditional proteomic techniques for the anal-ysis of molecules from tissue specimens, such asLC-MS or 2D gel analysis, require that the tissuebe homogenized prior to analysis, eliminatingthe spatial localization of analytes. Immunohis-tochemistry allows for the localization of specific

Copyright � 2013 Elsevier Inc. All rights reserved.

25. IMAGING MASS SPECTROMETRY OF INTACT BIOMOLECULES IN TISSUE SECTIONS394

molecules within a tissue section but requiresthat the specific analyte of interest be known inadvance and that an antibody-based reagentagainst it must exist. Additionally, only a fewmolecules can be probed from a single tissuesection. Conversely, IMS is carried out withoutthe need for homogenization, allowing for theanalysis of hundreds to thousands of biomole-cules in their native locations within a singletissue specimen. This technology is excellent fordiscovery because no target-specific reagentssuch as antibodies are required.

Several surface ionization techniques can beused to generate data for imaging applications.One of the early approaches reported issecondary ion mass spectrometry (SIMS), inwhich a surface is bombarded with a primaryion beam, leading to desorption of moleculesand generation of secondary ions.4e6 Althoughthe SIMS approach allows for very high spatialresolution imaging (<1 mm), the high-energyprimary ion beam is destructive to surfacebiomolecules generated, effectively limiting thepractical mass range of analysis to less than1,000 Da. Desorption electrospray ionization(DESI) uses a capillary spray of solvent ontothe surface of the tissue to desorb ions froma tissue surface that can then be introducedinto a mass spectrometer through a second inletcapillary.7e9 Advantages of the DESI techniqueare that it can be used at ambient pressure andrequires minimal sample preparation. However,it is most effective in analyzing small moleculessuch as drugs and lipids and spatial resolutionis typically 100 mm or more. Liquid extractionsurface analysis (LESA) uses a small volume ofsolvent applied to an area of interest on a tissuesection using a pipet, which is then drawnback up into the tip.10 The extracted moleculesare separated by rapid liquid chromatographyand detected via mass spectrometry, often byselected reaction monitoring. The LESA tech-nique is limited by low spatial resolution; tar-geted areas are often on the order of 1 to 2 mmbut could be applicable to any type of molecule

that can be extracted from the tissue surface.Other lesser-used approaches include laser abla-tion electrospray ionization,11 matrix assistedlaser desorption electrospray ionization,12 andjet desorption electrospray ionization.13 Theselatter techniques will not be discussed in detailin this chapter and the reader is referred to recentreviews14,15 for more information.

In the present chapter, we will focus on theuse and application of matrix-assisted laserdesorption/ionization imaging mass spectrom-etry (MALDI IMS) as the most broadly appli-cable approach for the analysis of a widevariety of biomolecules. MALDI IMS has beenused for the analysis of proteins, peptides, lipids,drugs, and metabolites from tissue specimensranging in size from cell clusters16 to wholeanimal sections.17 In the MALDI approach,a matrix (typically a small organic molecule) isapplied to the surface of a tissue section ina solvent that aids in the extraction of analytes.The specific matrix and solvent used can beoptimized for different classes of molecules.15

A raster of the tissue surface by the laser inducesthe desorption and ionization of molecules fromthe tissue surface (Figure 1). Commercial instru-ments allow for achievable spatial resolutions ofapproximately 20 mm, and custom modificationsthrough use of specialized optics have allowedfor spatial resolutions of approximately 1 mm.18

As usual, the trade-off between spatial resolu-tion and sensitivity ultimately determinesthe actual resolution employed in a givenexperiment.

MATRIX APPLICATION

A variety of techniques can be used for theapplication of matrix to the tissue surface andthe method of choice depends on the imagingtask that is to be done. Wet extraction methodssuch as spotting19 and spraying20 result ina higher degree of analyte extraction, especiallyfor larger polar molecules such as proteins, but

FIGURE 1 Workflow for MALDIimaging mass spectrometry. Sectionsare taken from a tissue specimen andcollected onto a target plate ora microscope slide for histologicalstaining. Matrix is applied via spraycoating, sublimation, or robotic spot-ting. Spectra are collected in anordered array from the surface of thetissue section. Ion images can begenerated from any of the signals inthe mass spectra.

MATRIX APPLICATION 395

may cause large crystals to be formed, limitingsomewhat the attainable spatial resolution. Driermatrix application methods such as sublima-tion21,22 are less efficient at extracting largerbiomolecules but result in much smaller matrixcrystals on the sample surface, allowing higherspatial resolution to be achieved.

The simplest and most inexpensive approachis through the use of an airbrush sprayer to

manually apply matrix.23 This approach hasbeen used successfully for imaging applicationsbut leads to the production of moderately largecrystals (20e100 mm) and suffers from a lowerdegree of reproducibility between differentspray devices and individuals. Automated spraydevices such as the TM Sprayer from HTX Tech-nologies and the ImagePrep from Bruker Dalton-ics have been introduced that allow more

25. IMAGING MASS SPECTROMETRY OF INTACT BIOMOLECULES IN TISSUE SECTIONS396

homogeneous and reproducible coatings to beachieved. Spray coatings can be used forimaging any class of molecules at relativelyhigh spatial resolution; however, care must betaken with any spraying technique to ensurethat the surface is not made too wet, which canlead to delocalization of analytes.

Very high spatial resolution can be achievedthrough the use of sublimation to deposit matrixonto the tissue section. Powdered matrix isheated under vacuum and is redeposited ontoa cooler sample target that is suspended abovethe matrix. Crystals formed are typically lessthan 1 mm and the coating is very homogeneous.When conditions such as temperature, pressure,and time are carefully controlled, sublimatedcoatings are highly reproducible and are appli-cable for the analysis of most classes of lipids,as no solvent is needed for extraction. If largermolecules such as peptides and proteins are tobe analyzed, the use of a rehydration/recrys-tallization step is employed to help cocrystalizethese molecules with the matrix.24

Matrix and other reagents can also be appliedto the tissue through the use of robotic spotterssuch as acoustic spotters (Labcyte Portrait)19

and chemical inkjet printers.9 Although thesespotters cannot achieve the spatial resolution oftheuniform coating technologies describedprevi-ously (they are typically limited to ~150e200 mmspot size), they tend to have the highest reproduc-ibility of any of the coatings due to the tightcontrol of the exact volume of reagent appliedto a specific location on a surface. Spotting gener-ally leads to the most robust analyte signal due tothe ability to thoroughly wet small areas ofa tissue section for increased extraction withoutthe risk of delocalization that occurs with sprayor rehydration techniques. Because of the smallreservoir volume (hundreds of microliters) ofcommercial spotters, they may also be employedfor the application of relatively costly reagents tothe tissue section including enzymes for proteo-lytic digestion and internal standards for druganalysis and quantitation.

An additional benefit of robotic spotters is theability to deposit matrix or other reagents at veryspecific locations within a tissue section, allow-ing for an approach known as histology directedprofiling.25 In this type of experiment, twosections of a tissue specimen are collected, oneon a MALDI target and a serial section on a stan-dard microscope slide for histological staining.After a photomicrograph has been taken of thestained section, the image is annotated bya pathologist or biologist to indicate areas ofinterest for a given sample. The annotated imageis then merged with an image of the unstainedserial section using photo processing softwareand the coordinates of the annotations are deter-mined. These coordinates are transferred to therobotic spotter and matrix is applied only tothose specific locations. Typically 10 to 20 areasare targeted per cell type per tissue, with 10 ormore samples analyzed in a single experiment,greatly reducing the volume of data collected,increasing the throughput, and enabling morefacile statistical analysis. Histology directedprofiling can be thought of as a type of low-resolution imaging through correlation witha microscopy image. In this way, the spatiallocalization of analytes is preserved while gener-ating high-quality data.

PROTEIN ANALYSIS

Imaging mass spectrometry has been used forthe analysis of proteins in cancer tissue includ-ing breast,26e30 prostate,10,31,32 kidney,33 skin,34

colon,35 lung,36e40 ovarian,41 and gastric42,43 aswell as other diseases such as inflammatorybowel diseases,44 S. aureus infection,45 andsarcoidosis.46 The aim of most of these investiga-tions is the identification of proteins and proteinsignatures for improved diagnosis, prognosis,prediction of response to treatment, as well asdetermination of potential drug targets.

IMS has been used in the classification ofpapillary bladder tumors into high grade (HG)

PEPTIDES AND PROTEIN DIGESTS 397

and low grade (LG).47 These types of tumors hadpreviously been classified into three groupsdG1, G2, and G3daccording to histology, but in2004 the World Health Organization eliminatedthe middle grade group. Although high-gradeand low-grade tumors could be easily differenti-ated based on histological characteristics, theformerly classified grade 2 tumors provedmore challenging. To better differentiate thisgroup, a support vector machine algorithm wasgenerated from IMS data from 27 LG tumorsand 21 HG tumors that was then applied toa set of 31 G2 tumors. In the initial training set,cross-validation accuracies of 97.82% and96.54% were obtained for the LG and HGgroups, respectively. Classification of the G2samples designated 23 samples as LG and 8 asHG. Samples were blindly classified by two uro-pathologists and the results compared to the IMSclassifications. Of these, 78.3% of the LG and87.5% of the HG were correctly classified.Follow-up analyses of the five misclassified LGsamples showed that three of these patients pro-gressed to have HG tumors within five years.The one misclassified HG sample showed notumor recurrence within five years of follow-up.

IMS has recently been applied to a study ofgastric cancer biopsies for proteins that correlatedwith patient outcome.42 A cohort of 63 sampleswere used for a training set, 47 with good prog-nosis and 16 with poor prognosis, to determineproteins that could be correlated with outcome.All samples were subjected to MALDI IMS andthen regions of interest corresponding to tumorwere extracted and a subset of these spectraused for statistical analysis. Analyses werecarried out using ClinProTools� (Bruker) andthe Statistical Analysis of Microarrays plugin forMicrosoft Excel�. A signature of sevenproteinsdm/z 3445, m/z 6278, m/z 8406, m/z8453, m/z 10098, m/z 11353, and m/z 11613dwasfound that correlated with a nonfavorable effecton patient survival and could not be definitivelyattributed to any other patient or tumor charac-teristics. Three of these proteinsdm/z 3445, m/z

8406, and m/z 10098dwere identified as humanneutrophil peptide-1, cysteine-rich intestinalprotein-1 (CRIP1), and S100A6, respectively.These three proteins were then used to validatean independent set of 118 samples by immuno-histochemistry. Of the three proteins, CRIP1showed the strongest correlation with patientsurvival. Additionally, a support vector machinealgorithmwasgeneratedusing the sevenproteinsthat resulted in 98% classification accuracy.

PEPTIDES AND PROTEIN DIGESTS

Endogenous or enzymatically producedpeptides can be analyzed directly from tissuesections. The latter technique has allowed forthe analysis of formalin-fixed, paraffin-embedded biopsies,48 opening up a vast bankof tissues for IMS analysis as the majority of clin-ical samples are preserved and stored in thismanner. This analysis is accomplished by firstsubjecting tissue sections to deparaffinizationusing xylene and graded alcohols beforeapplying heat-induced antigen retrieval,commonly performed in histology laboratories.Trypsin (or another enzyme) is then applied tothe surface of the tissue by either spraying orspotting before application of matrix. Peptidescan be analyzed from these microdigestion spotsand serve as sequence tags for the parentproteins, thus allowing for analysis of highermolecular weight proteins than those thatare typically accessible by traditional proteinimaging.49 Additionally in many cases,peptidesdeither endogenous or enzymaticallyproduceddcan be sequenced and identifieddirectly from tissue sections through tandemmass spectrometry without further isolation orpurification.

We have recently shown the application of thecombination of histology-directed profiling andtissue tryptic digestion in the study of Spitzoidskin lesions.50 Spitz nevi (SN) are benign skinlesions that often occur on the face or lower leg

25. IMAGING MASS SPECTROMETRY OF INTACT BIOMOLECULES IN TISSUE SECTIONS398

of young children, whereas Spitzoid melanomas(SM) are malignant tumors with Spitzoidfeatures. These two types of lesions can at timesbe difficult to differentiate, and treatments forthe two disease types are quite different. Toprovide a better molecular diagnoses, weprofiled a total of 114 Spitzoid lesions. Areas oftumor and surrounding stroma (dermis) weretargeted from each sample when sufficient tissuewas available (Figure 2). These were subdividedinto 26 SN and 25 SM in a training set and 30 SNand 29 SM in an independent validation set.After analysis of tryptic peptides from the tumorin the training set, a genetic algorithm modelwas generated using ClinProTools, consistingof five peptides that could correctly diagnoseall samples in the training set. This algorithm

FIGURE 2 Analysis of Spitzoid lesions. (A) Spitzoid melanomtumor (blue) and dermis (yellow) and targeted for on-tissue tryspectra from tumor regions from Spitzoid melanoma (red) andmarked with an asterisk.

was then applied to the independent validationset with 29 out of 30 SN and 26 out of 29 SMcorrectly classified. Two of the peptides thatmade up the signature were identified by MS/MS directly from the tissue sections. A peptideat m/z 976.5 was identified as originating fromactin and one at m/z 1428.6 as being fromvimentin.

In addition to traditional biopsies, tissuemicroarrays (TMA) have been analyzed usingthese techniques allowing for the analysis ofa hundred or more samples in a single experi-ment. This was first demonstrated by Grose-close et al.51 in the analysis of a lungcarcinoma TMA comprised of 112 cores from50 different patients. The patient diagnosescould be subdivided into adenocarcinoma (21),

a (top) and Spitz nevi (bottom) were annotated for areas ofptic digestion and mass spectrometry. (B) Portion of averageSpitz nevi (green). Peptides that are part of the classifier are

LIPID ANALYSIS 399

squamous cell carcinoma (21), bronchioloalveo-lar carcinoma (4), metastatic colon cancer (2),carcinoid biopsy (1), and plasma cell granuloma(1), with a focus on adenocarcinoma and squa-mous cell carcinoma for clinical analysis. Subse-quent to on-tissue tryptic digestion and IMS,statistical analyses were carried out anda support vector machine algorithm made upof 73 peaks was generated that could accuratelyclassify all of the cancerous cores into theirdesignated subtypes. It was also found fromthis analysis that there was a subset of squa-mous cell carcinoma cores that could be differ-entiated from the others based on theexpression of a peptide identified as beingfrom cytokeratin 5. TMA analysis by IMS hasalso been demonstrated in pancreatic,52

gastric,53 and prostate54 cancers.Endogenous peptide level variations were

shown in a rat model of Parkinson’s disease(PD).55 PDwas induced in rats through theunilat-eral injection of 6-OHDA-HCl into the substantianigra with saline administered to control animalsfollowed by treatment with L-DOPA to alleviatePD symptoms and induce levels of dyskinesia.Coronal sections of the ventral midbrain contain-ing the substantia nigra were coated with DHBusing a chemical inkjet printer and imaged. Twopeptides in particular, dynorphin B and alpha-neoendorphin (aNeo), were found to be signifi-cantly elevated in the lesioned substantia nigrain the animals with high dyskinesia as comparedto animals with low dyskinesia and controls. Inparticular, these peptides showed considerablyhigher levels (1.75 fold higher for dynorphin Band 2 fold higher for aNeo) in the lateral portionof the lesioned sustantia nigra as compared tothe contralateral side. Additionally, the peptidemetabolites Leu-Enk-Arg, Leu-Enk-Arg-Arg,and aNeo (1-7) showed similar trends in expres-sion of being higher in the lateral portion ofthe substantia nigra in the lesioned side of thebrain as compared to intact but did not showa strong correlation with dyskinesia. Leu-Enk-Arg showed opposite trends with dynorphin B

with respect to spatial localization indicatingthat it is being produced by the metabolism ofdynorphin B. Other peptides, such as dynorphinA (1-8), dynorphin A (1-17), and substance Pshowed no treatment induced changes.

LIPID ANALYSIS

Lipids are an emerging area of interest tothe IMS community as they have been associatedwith a variety of biological and disease processesincluding signal transduction,56 kinase path-ways,57 cancer,58,59 Alzheimer’s disease,60 andembryo implantation,22 as well as being highlyexpressed in every organ of the body, especiallythe brain.61 Sample preparation protocols havehelped to improve ionization of lipids whiledecreasing ambiguity due to coordination withmultiple cations. Addition of lithium to thematrix solution has been used to drive lipids totheir lithiated form enhancing sensitivity aswell as improving fragmentation efficiency.62

Washing tissue with ammonium formate priorto matrix application and mass spectral analysishas proven to enhance lipid signals particularlyin negative ion mode.63

Menger et al. have recently demonstrated therole that lipids play in myocardial infarction.64

A rat model of infarction was generated bysurgically ligating the left anterior descendingcoronary artery. The rats were sacrificed andthe hearts dissected 24 hours later. Transversesections were collected from the infarctedheart containing the damaged area. Areas ofdamage were confirmed by staining with2,3,5-triphenyltetrazolium chloride (TTC) asinfarcted tissue lacks the dehydrogenase thatconverts TTC to formazan. Samples were coatedwith DHB containing alkali metals to help drivethe ionization of lipids to a single species. MSand MS/MS imaging were then carried outusing a Thermo Scientific LTQ XL in positiveion mode. Infarcted areas could be clearly differ-entiated from normal areas based on the absence

25. IMAGING MASS SPECTROMETRY OF INTACT BIOMOLECULES IN TISSUE SECTIONS400

of creatine and the increased presence of lyso-phospholipids in the damaged tissue. Lysophos-pholipids LPC 16:0 [M þH]þ (m/z 496), LPC 16:0[M þ Na]þ (m/z 518), LPC 18:0 [M þ H]þ orunidentified LPE (m/z 524), LCP 18:1 [M þNa]þ or LPC 20:4 [M þ H]þ (m/z 544) and LPC18:0 [M þ Na]þ (m/z 546) were found to be atsignificantly greater levels in the infarcted tissuethan in the surrounding unaffected tissue.Conversely, intact phospholipids PC (18:0/20:4) [M þ Na]þ (m/z 832) and PC (18:0/20:4)[M þ K]þ (m/z 848) were found to be in highabundance in the normal tissue but absentfrom the infarcted tissue (Figure 3). The presenceof increased levels of lysophospholipids alongwith decreased levels of intact lipids indicatedincreased activity of phospholipase A2, whichhydrolyzes the SN-2 acyl bond of intact phos-pholipids, within the infarcted tissue area. Thisfinding is in accordance with previous reports65

that phospholipase A2 acts on lipids containing

(A)

(C)

FIGURE 3 MS images for the[M þ K]þ of LPE 18:0 at m/z 520 (A),the [M þ Na]þ of LPC 18:0 at m/z 546(B), the [M þ Na]þ of LPC 16:0 at m/z518 (C), and the [M þ K]þ of PC(18:0/20:4) at m/z 848 (D) from a heartfollowing [left anterior descending]coronary artery ligation. All imageswere generated by plotting the ionintensity divided by the TIC. (Re-printed with permission from reference#64. Copyright 2012 American ChemicalSociety.)

arachidonic acid in an effort to protect againstischemic cell death.

DRUG ANALYSIS

Imagingmass spectrometryhasbeenemployedin the pharmaceutical industry for determinationof the spatial distribution and quantitation ofdrugs and theirmetabolites in tissue sections.66e68

Traditional drug analyses have been carried outusing quantitative whole body autoradiographythat allows for the visualization of radiolabeledcompounds throughout thin sections of dosedanimals.69 However, this technology shows onlythe location of the label and does not identify thecompound (parent drug or metabolite). Alterna-tively, LC-MS-based approaches can easily distin-guish differentmetabolites but spatial localizationis lostdue to thehomogenizationof the tissuepriorto analysis. IMS is ideal for this purpose because it

(D)

(B)

3D IMAGING 401

allows for the distinction between drugs andmetaboliteswhilemaintaining the spatial localiza-tion of these compounds.

IMS of drugs and other small molecules has theadded challenge in that there is redundancy ofnominal masses observed in the lower molecularweight part of the spectrum. Often severalcompounds within a tissue section can have thesame nominal mass as the molecule of interestand may not be able to be resolved by lower massresolution analyzers such as quadrupole andtime-of-flight-based analyzers. Three differentapproaches can be taken to circumvent this issue.The first is to use a much higher resolution massanalyzer such as a Fourier transform ion cyclotronresonance (FT-ICR) instrument that can allow forthe distinction of compounds separated by frac-tions of one mass unit and confidently validatethe identity of a compound by accurate mass.68,70

A second approach is to carry out the imaging inanMS/MSmode by following a specific transitionfrom the parent mass to a fragment ion of thecompound.17,71 A third approach is to use ionmobility mass spectrometry to separate moleculesof interest from other isobaric compounds basedon their gas phase conformations and capture crosssectional areas.11,72

Castellino et al. have shown the applicabilityof the FT-ICR approach in the imaging of severaldrugs and metabolites in animal tissue.68 Organswere collected from dogs dosed with lapatiniband subjected to imaging using a Bruker solariX12T FT-ICR mass spectrometer. Through use ofthis high-resolution instrument, investigatorswere able to distinguish two different metabo-lites, GW006 and M2, which differ in mass by0.013 amu and display different spatial distribu-tions within the liver of the dosed dog. This workhas led to the identification of a total of 21different metabolites in dog liver sections.68

Alternatively, selected reaction monitoringthrough the application of MS/MS imaging canbe used in the accurate determination of the spatiallocalization and quantitation of drug compoundsin tissue sections. Goodwin et al. have employed

this approach in the analysis of positron emissiontomography (PET) tracers in the brains andkidneys of mice.73 Two compounds, 3,5-dichloro-N-[(2S)-1-ethylpyrrolidin-2-yl]methyl-2-hydroxy-6-methoxybenzamide (raclopride) or 7-cholor-3-methyl-1-phenyl-1,2,4,5-tetrahydro-3-benzazepin-8-ol (SCH 23390) were administered by injectionat different doses through the tail veins of rats.Animals were sacrificed at 1, 5, or 30 minutespost dose and the brains and kidneys dissectedand snap frozen. Midpoint sections of the organswere collected and CHCA matrix was depositedby dry coating, which allowed for desorptionand ionization of both compounds without thedelocalization that can occur with wet, solvent-based matrix application. Both compoundswere imaged as intact parent masses along withMS/MS imaging. Raclopride was imaged bymonitoring the transition m/z 347 to m/z 129 andm/z 111 while SCH 23390 was imaged by moni-toring the transition m/z 288 to m/z 179. Imagesof parent compounds collected on both qTOFand FT-ICR instruments showed excellent corre-lation with MS/MS images of the compoundsconfirming their localization and identities(Figure 4). Raclopride was found to be at highestlevels in the brain at 1 minute post dose with an80% drop in concentration by 30 minutes postdose as evidenced by decreased signal intensity.Similar results were obtained in the kidney. Thismethod was also used to quantitate raclopridein the tissue sections by use of a serial dilutionof the compound on control tissue section toa generated calibration curve based on signalresponse. Raclopride was determined to be ata concentration of 60 nM at 1 minute post 7.5mg/kg dose and a concentration of 15 nM at 1minute post 2 mg/kg dose.

3D IMAGING

An exciting application of imaging massspectrometry is the ability to generate three-dimensional volumes of biomolecules within an

FIGURE 4 IMS abundance distributions obtained on a range of MALDI mass spectrometers for rat brain tissue sections (14mm thick) coated with a solvent free dry matrix (CHCA). Raclopride brain tissue sections (i.v. 2.5 mg/kg) dose tissues (A)analyzed by MALDI q-TOF at a spatial resolution of 100 mm. Abundance displayed on a heat-map scale. (B) Analyzed byMALDI FT ICR at a spatial resolution of 100 mm. (C) Pseudo-SRM images produced by mapping the distribution of the MSMSfragmentation masses for raclopride m/z 347.0 to 129.1 (C.i) and 111.9 (C.ii) at a spatial resolution of 200 mm performed ona MALDI q-TOF. SCH 23390 brain tissue sections (iv, 5 mg/kg) (D) analyzed by MALDI q-TOF at a spatial resolution of 100mm. Abundance displayed on a heat-map scale. (E) analyzed by MALDI TOF at a spatial resolution of 200 mm. (F) Pseudo-SRM image produced by mapping the distribution of the MSMS fragmentation masses for SCH 23390 m/z 289.1 to 179.0abundance as heat-map scale (F.i) and monochrome red scale (F.ii) at a spatial resolution of 200 mm performed on a MALDI q-TOF. (Reprinted with permission from reference #73. Copyright 2011 American Chemical Society.)

25. IMAGING MASS SPECTROMETRY OF INTACT BIOMOLECULES IN TISSUE SECTIONS402

organ or animal oftenwith co-registration to otherimaging modalities. This approach has been usedto create 3Dvolumes of the corpus callosum74 andthe substantia nigra within the rodent brain.75 Co-registration of IMS with magnetic resonanceimaging (MRI) was shown in a xenograph modelof a human glioma in a rat brain.76 Proteomicdata from imaged brain sections could be corre-lated with data from noninvasive MR data.

Applications of IMS involving 3D imagingrequire that many sections must be registered toeach other in order to construct a molecularvolume. Thus, it is necessary to minimizeany physical distortion of the samples during

sectioning of the tissue.70,77 It is also importantthat all sample preparation and data collectionparameters are kept constant throughout theexperiment to minimize nonbiological variability.

Recently we have shown the co-registration ofIMS and MRI in a murine model of staphylo-coccus infection and treatment with linezolid.77

Animals were infected retro-orbitally and theinfection allowed to progress for 96 hours beforetreatment with 0.2 mg/mL of linezolid for 96hours. The animals were splinted to preventmovement and subjected to MRI measurementsof the abdomen. Large abscesses were observedin the kidneys of untreated animals as

REFERENCES 403

previously described45 with smaller abscessesfound in the treated animals. Animals werethen sacrificed, snap frozen, and sectioned ina whole body cryomacrotome. Prior to eachsection being cut, a photograph of the blockfacewas taken. About 40 sections were collected peranimal in the region encompassing the kidneysand subjected to protein IMS. Three-dimensionalvolumes were generated of proteins that local-ized to the abscesses and to normal kidney struc-tures and were then co-registered through theuse of the optical blockface intermediary to theMRI volumes. Calgranulin A showed localiza-tion to the abscesses in the IMS volume and cor-responded to the observed abscesses in the MRIvolume. A second protein fragment, a-globinresidues 2-47, was found to localize to the cortexof the kidney. This study clearly showed theability of IMS to monitor the infection processand treatment through multimodality imagingusing MRI and IMS.

HIGH-SPEED IMAGING

One of the major challenges when movingto higher spatial resolutions and higher datavolumes as in the case of 3D imaging is thetime necessary to complete the imagingprocess. The use of high-repetition-rate lasers12

and continuous raster sample stages are highlybeneficial in achieving high-speed imaging.This has been recently shown in imagesacquired using the new instrument fromSimulTOF� (Virgin Instruments, Inc.).78 Thismass spectrometer couples a 5 kHz laser repe-tition rate with continuous raster. Pixel dimen-sions are determined by the number of shotsper pixel, the stage speed, and the repetitionrate of the laser. By operating the laser at 3kHz, a stage speed of 5 mm/s, and acquiring60 shots per pixel, an image of a sagittal ratbrain section (185 mm2) consisting of approxi-mately 19,000 pixels was acquired in under10 minutes. Such technological advances are

imperative as IMS moves to higher numbersof samples and higher spatial resolution.

CONCLUSIONS ANDPERSPECTIVES

Imaging mass spectrometry has shown wideapplicability in the study of biological systemsallowing for the in situ analysis of many classesof biomolecules. Proteins, peptides, lipids, anddrugs/metabolites have all been successfullyimaged at high spatial resolution with relativequantitation, providing valuable insight into theirinvolvement in biological and disease processes.Although relative quantitation has been clearlydemonstrated, processes necessary to achieveabsolute quantitation are just beginning. IMS hasenabled investigation of biological processes andhas provided insights into better diagnostics,outcomes, and treatment response, especially inareas where traditional histology has not beeneffective. As the technology and sample prepara-tion methods advance, IMS is becoming easier touse in a wide variety of applications in the biolog-ical andmedicalfields.Themolecular specificityofthis imaging technology brings a very powerfuland enabling tool to these disciplines.

AcknowledgmentsThe authors acknowledge funding from NIH/NIGMS 8P41GM103391-02 (formerly NIH/NCRR 8P41 RR031461-02),NIH/NIGMS 5R01 GM058008-13, and the DOD grantW81XWH-05-1-0179.

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