instruments

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Chapter4

Imaging Mass Spectrometry:Sample Preparation,Instrumentation, andApplications

Kamlesh Shrivas†,‡ andMitsutoshi Setou†

Contents 1. Introduction 1462. Ionization Methods for Imaging Mass Spectrometry 147

2.1. Desorption Electrospray Ionization 1472.2. Secondary Imaging Mass Spectrometry 1492.3. Laser Ablation Electrospray Ionization 1492.4. Matrix-Assisted Laser Desorption/Ionization 149

3. MALDI Imaging 1503.1. Sample Handling 1513.2. Choice of Matrix 1553.3. Application of Matrix Solution 159

4. Instrumentation 1614.1. Quadrupole Mass Analyzer 1614.2. Time-of-Flight Mass Analyzer 1614.3. Sector-Type Mass Analyzer 1634.4. Ion Trap Mass Analyzer 1634.5. Orbitrap Mass Analyzer 1644.6. Ion Cyclotron Resonance Mass Analyzer 164

5. IMS Measurements 1656. Data Analysis 1657. Applications of IMS for Direct Analysis of Tissue 166

7.1. IMS for Lipidomics 1667.2. IMS for Proteomics 175

† Department of Cell Biology and Anatomy, Hamamatsu University School of Medicine, 1-20-1 Handayama,Higashi-Ku, Hamamatsu, Shizuoka 431-3192, Japan

‡ Department of Chemistry, Guru Ghasidas University, Bilaspur-495009, CG, India

Advances in Imaging and Electron Physics, Volume 171, ISSN 1076-5670, DOI: 10.1016/B978-0-12-394297-5.00004-0.Copyright c© 2012 Elsevier Inc. All rights reserved.

145

http://dx.doi.org/10.1016/B978-0-12-394297-5.00004-0


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146 Kamlesh Shrivas and Mitsutoshi Setou

7.3. IMS for Pharmacokinetic Studies 1777.4. IMS for Metabolomics 180

8. Summary 182Acknowledgments 183References 184

1. INTRODUCTION

The ability to visualize the molecular distribution in biological materialsuch as tissue samples has helped scientists to provide a better under-standing of the principles of life. The study of biomolecule distribution inorgans and its alterations with disease remains one of the most challeng-ing and intriguing scientific issues of recent times. Various techniques areused in laboratories around the world to visualize molecular systems—techniques such as magnetic resonance imaging (MRI) technology (Hurdand Freeman, 1989) and positron electron tomography (PET) (Ametameyet al., 2008). The Nobel Prize–winning MRI and PET technologies areknown as noninvasive techniques for medical diagnosis. Nuclear mag-netic resonance spectroscopy (NMRS) is also helpful for imaging andidentification of biomolcules in tissue sample (Hiltunen et al., 2002). Thelimitations of these techniques are the relatively poor resolution, sensitiv-ity, and requirement of labeling of molecules for detection (in the case ofthe PET method).

Imaging mass spectrometry (IMS) was introduced for spatial distribu-tion analysis of biomolecules without the need for extraction, purification,separation, or labeling of biological samples. Recent developments inmolecular imaging have created new opportunities to perform moleculardiagnostic and therapeutic procedures. The technique can be exploited tovisualize cellular and molecular processes that occur in two-dimensional(2D) or three-dimensional (3D) fashion without perturbing the structureof the system (Caprioli et al., 1997; Setou et al., 2010).

Mass spectrometry (MS) is a technique based on the measurementof the charged ions in an electric or magnetic field. Generally, a massspectrometer contains three distinct parts: (1) an ion source producingions from sample molecules; (2) a mass analyzer separating the dif-ferent molecules with respect to their mass-to-charge ratios (m/z), and(3) a detector, registering the ion m/z and the intensity at which theions were detected. Data are collected and visualized in a mass spec-trum where the different m/z ratios are displayed as a function of theirsignal intensity (Gross, 2004). MS is a great scientific tool because of thewide range of molecules that can be accurately detected and identified:large organic compounds and biomolecules of low molecular weight.


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Imaging Mass Spectrometry 147

In the beginning, mass spectrometric analysis was limited to samplesthat had undergone excessive preparation procedures, such as purifi-cation, separation, and concentration steps. These procedures not onlyjeopardize sample integrity, but also lead to the complete loss of any spa-tial distribution information. MS instruments are equipped with differentionization methods, including electron ionization and chemical ioniza-tion (Fales et al., 1972), fast atomic bombardment (Morris et al., 1981),electrospray ionization (ESI) (Fenn et al., 1989), and matrix-assisted laserdesorption/ionization (MALDI) (Karas et al., 1985) for the analysis of awide range of organic and bio-organic molecules. The introduction of the“soft” ionization sources such as ESI and MALDI transfigured MS, as itoffered the capability to analyze large intact biomolecules.

At present, IMS is a well-recognized technique for profiling the dis-tribution of biomolecules in tissue sample at micrometer to nanometerresolution (Caprioli et al., 1997; Goodwin et al., 2008; McDonnell andHeeren, 2007; Pol et al., 2010; Shimma et al., 2008). Data acquisition is per-formed through scanning a tissue section with a laser, thereby obtainingone mass spectrum for every pixel. The main principle of IMS is basedon desorption and ionization of biomolecules from the surface of thetissue sample. There are currently four important desorption/ionizationmethods: desorption electrospray ionization (DESI) (Takats et al., 2004),secondary ion mass spectrometry (SIMS) (Benninghoven, 1973), MALDI(Tanaka et al., 1988) and laser ablation electrospray ionization (LAESI)(Nemes and Vertes, 2007).

2. IONIZATIONMETHODS FOR IMAGING MASSSPECTROMETRY

2.1. Desorption Electrospray Ionization

DESI was introduced by R.G. Cooks in 2004. In DESI, the molecules areionized at atmospheric pressure without the use of any organic matrix(Dill et al., 2009) in a combination of ESI and desorption ionization (DI).The charged droplets of solvent generated during the electrospray stageare used to ionize molecules from the surface of the sample and the ionsproduced thereby are directed into an atmospheric inlet of the MS. Thecomponents and use of DESI in IMS are presented in Figure 1a. The spatialresolution obtained by this method is 0.3–0.5 mm, which is a low resolu-tion of tissue sample in IMS studies. DESI has been successfully appliedto IMS for the identification of lipids, drug metabolites, and antifungalmolecules in seaweeds (Dill et al., 2009; Lane et al., 2009; Wiseman et al.,2008).


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(a)

(b)

(c)

Spray droplets

Solvent

N2

Secondarydroplets

Sample

Inlet of massspectrometer

Primary ions

Sample



UV or IR laser

Laserattenuator

Analyte/matrixmixture

Secondaryions

FIGURE 1 Desorption-ionization techniques used in mass spectrometry imaging.(a) Desorption electrospray ionization (DESI). (b) Secondary ion mass spectrometry(SIMS). (c) Matrix-assisted laser desorption ionization (MALDI). UV, ultraviolet;IR, infrared. Reprinted from Pol et al. (2010) with permission from Springer.


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2.2. Secondary Imaging Mass Spectrometry

SIMS is a sophisticated technique that uses ion beams from metal ionssuch as Ar+, Ga+, and In+ (here denoted primary ion beams) to producesecondary ions from molecules on the surface of a sample. Ionizationis performed in high vacuum to avoid a collision with surrounding gasmolecules, and the primary ion beams can be focused down to 50 nmon the sample surface, with the resolution depending on the current andcharge state of the ions. SIMS coupled with time-of-flight (TOF-SIMS) is asuperior tool for high-spatial, submicron resolution (<10 nm). Thus SIMScan be applied for the differentiation of biomolecules that are presentall the way down to the cellular level. However, fragmentation of largermolecules on the sample surface is observed when strong laser energyis applied for the primary ion beam. Hence, SIMS is primarily applica-ble for the analysis of small molecules (<1000 Da) (Heeren et al., 2006;Slaveykova et al., 2009). Figure 1b shows the process of SIMS ioniza-tion of molecules from the sample surface. SIMS has been applied forimaging of samples such as single cells, embryos, brain, cocaine, and cin-namoylcocaine in coca (Colliver et al., 1997; Jones et al., 2007; Wu et al.,2007). The fragmentation of molecules in SIMS can be overcome throughthe treatment of an organic MALDI matrix; this approach is known asmatrix-enhanced (ME)-SIMS (Altelaar et al., 2007).

2.3. Laser Ablation Electrospray Ionization

LAESI was developed by Nemes and Vertes (2007) and is a method for MSanalysis of tissue samples without sample preparation under atmosphericpressure (Nemes and Vertes, 2007). Laser ablation from a mid-infrared(mid-IR) laser is combined with a secondary ESI process. The spatial res-olution for tissue samples using LAESI technique is better than DESI andcan be used for imaging of biomolecules from the surface of tissue sam-ple at a lateral resolution of <200 µm. The technique has been applied forimaging and identification of plants, tissues, cell pellets, and even singlecells (Nemes et al., 2010; Shrestha et al., 2010; Sripadi et al., 2010). Recentlyit has also been used in 3D imaging of molecules from the sample (Nemeset al., 2009).

2.4. Matrix-Assisted Laser Desorption/Ionization

MALDI was introduced as a soft ionization technique that causes littleor no fragmentation of the target molecules, allowing for the analysisof molecules at several hundred kilodaltons (i.e., high m/z values). Thisallows for mass spectrometric analysis of a wide range of molecules suchas amino acids, peptides and proteins, carbohydrates, and nucleic acidsand drugs and has proven to be one of the most powerful MS technologies


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to date. In traditional MALDI, an organic matrix is mixed with the sam-ple on the target plate and irradiated by a ultraviolet or IR light generatedby a pulsed and focused laser. The matrix absorbs the light at the wave-length of the laser, leading to a soft desorption/ionization of the intactcompounds of interest (Gross, 2004; Karas et al., 1985; Tanaka et al., 1988).Figure 1c illustrates the MALDI mechanism.

3. MALDI IMAGING

By scanning a sample surface with the MALDI matrix/laser setup andregistering individual mass spectra for each pixel, a 2D ion density mapcan be reconstructed using appropriate software. Direct MALDI-IMS anal-ysis of clinical samples offers a unique approach to reveal the spatialexpression of biomolecules linked with pathological disease and otherclinical information. MALDI imaging is also suitable as a biomarkerdiscovery tool by comparing the relative quantities and/or spatial dis-tribution patterns of molecules in pathological and normal samples. Thelocalization and abundance of biomarkers identified in tissue sectionsare used to understand disease progression at a molecular level. Themain advantages of a direct biomarker analysis using MALDI imagingare that it provides spatial distribution patterns and is free from extrac-tion, purification, or separation steps, hence avoiding procedures that areboth time-consuming and jeopardize sample integrity (Chaurand et al.,2006; Hayasaka et al., 2010; Herring et al., 2007; Schwartz et al., 2003;Sugiura et al., 2009). With the currently available imaging software pac-kages, we can construct multiplexed imaging maps of selected biomole-cules within tissue sections. The laser energy is used in a raster scanpattern to ionize the molecules, which are present as discrete spots orpixel. For each pixel the full mass spectrum is represented. The data acqui-sition time for IMS was shortened by the introduction of N2 (337-nm) orneodymium-doped yttrium aluminum garnet (Nd:YAG) (355-nm) laserswith repetition rates of 200–1000 Hz with pulse lengths of 3 ns. The laserspot size of MALDI-MS is decreased from 100–150 to 20 µm, renderinghigher spatial resolution of biomolecules on the tissue surface. Further,a higher spatial resolution can be attained with a MALDI instrumentequipped with a highly focused laser. Chaurand et al. (2007) used a laserbeam at 7 µm, which is in the order of the diameter of a single cell todetect protein ions. However, a decrease in sensitivity is observed whileincreasing the resolution in this manner. Figure 2 shows an example ofMALDI-IMS analysis of protein from tissue section.

In addition to increased sample integrity, the great advantage of IMSis that it allows the construction of numerous ion images of moleculesdetected in a single run. This technique does not require previous


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Slice frozen tissue oncryostat (~12 µm thick)

Thaw slice ontoMALDI plate,allow to dry

Highdensitydropletarray

Applymatrix

Acquiremass spectra

Molecularprofiles

Molecularimages

Laser

Laser

Profiling

Lowdensitydropletarray

Imaging

FIGURE 2 Scheme presenting the protein profiling and imaging analytical strategiesfrom thin tissue sections. Reprinted from Chaurand (2006) with permission fromAmerican Chemical Society.

labeling with fluorescent probes or radioactive isotopes. MS analyses maybe performed for imaging of biomolecules at low concentrations; thedetection of 500 attomol has been reported in a single cell (Northen et al.,2007). Another advantage when using MS is the specific identification ofmolecules; tandem MS is used to identify compounds for which no previ-ous knowledge is required. For this, two MS analyzers are used: one forthe selection of the ion of interest before fragmentation, and the second isused for the analysis of fragmented masses. Thus the use of MS is rapid,sensitive, and free from complicated sample procedures for the analysisof unknown biological tissue samples.

3.1. Sample Handling

Sampling handling is a very important concern for imaging and identifi-cation of biomolecules in tissue samples. Consideration must be given tothe storage of the tissue sample after surgical removal from the human oranimal body to prevent ex vivo degradation and alteration processes. Thesectioning, washing, and staining of tissue, the choice of matrix, and itsapplication on the tissue section are other parameters to optimize in orderto obtain better-quality data.


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3.1.1. Storage of Samples

Tissue storage is the most important part of the protocol for IMS studiesto maintain the integrity of both the molecular composition and the spa-tial localization of analytes. When sampling is performed through surgicalremoval of tissue, molecular processes such as protein degradation con-tinue in the ex vivo state. These processes should be halted immediately,either through freezing in liquid nitrogen or heat stabilization (Schwartzet al., 2003). Chaurand et al. (2008) reported a long preservation methodof tissue samples with ethanol for generating high-quality histologicalsections that enable high-quality images of biomolecules in tissue sam-ple. Previously archived samples, on the other hand, are often fixed withparaformaldehyde and embedded in paraffin. Due to the cross-linkingbetween molecules caused by this preservation method, special methodsfor specific tissue digestion have been developed (Wisztorski et al., 2010).

3.1.2. Sectioning of Tissue

The next important part of imaging experiments is the sectioning of tis-sue sample into thin slices and the subsequent mounting of these tissueslices onto an appropriate target. Before tissue sectioning, the frozen tissuesamples are transferred from the −80◦C freezer to the cryostat chamberat −20◦C for 30 minutes to thermally equilibrate the tissue. The tissueis usually embedded on an optimal cutting temperature (OCT) poly-mer, which supports easy handling and precise microtoming of sections.However, the use of OCT compounds causes a suppression of MALDIanalyte signals in MS and should, if possible, be avoided (Schwartz et al.,2003). Figure 3 shows the mass spectra of rat liver with suppression ofMALDI-MS signals when OCT is used as a supporting material.

The use of gelatin is an alternative method for embedding the tissuesample where the mass spectrum is free from background signals com-pared with the use of OCT (Chen et al., 2009). The embedded tissue isfixed on a sample stage and the temperature is maintained between 5◦Cand−25◦C. The optimal temperature is set depending on the type of tissueto be analyzed and is followed by slicing of tissue with a steel micro-tome blade. For MALDI-IMS, the tissue sections are usually 5–20-µm thick(Chaurand et al., 2006; Schwartz et al., 2003).

The next step is the proper transfer of the sliced tissue section onto anelectrically conductive steel plate or a glass slide. Thicker sections of tis-sue are more suitable when transferring them to the target plate becausethinner sections break more easily. The first method of tissue transfer isperformed by simply placing the plate in the cryostat chamber kept at−15◦C while sectioning. An artist’s brush is used to pick up the tissuesection and gently place it on the cold plate, followed by gentle warming


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(a)

(b)

OCT OCTTissue slice

4500

Inte

nsity

Inte

nsity

6000 7500m/z

9000 10500

FIGURE 3 Effect of optimal cutting temperature (OCT) on MALDI signal from rat liver.(a) Optimal procedure where OCT is used to adhere the tissue to the sample stage butdoes not come into contact with the sliced tissue. The resulting spectrum shows manyintense signals between m/z 4500 and 10500. (b) The tissue was embedded in OCT andattached to the sample stage. The resulting tissue slice is surrounded by OCT on theMALDI plate, and the resulting spectrum contains of only about half of the signals asin (a). Reprinted from Schwartz et al. (2003) with permission from John Wiley and Sons.

of the plate by touching the backside of the plate with a fingertip. Thetissue is thereby thaw-mounted on the target plate. In the second method,the plate is kept at room temperature and placed over the sliced frozensection, and the tissue is thereby simply thawed on the target plate.Great care should be taken with both methods to retain the shape of thetissue. Obviously, folding or stretching caused during the sectioning oftissue section may affect the molecule distribution analysis and preventsdetection of some of the molecules from the tissue surface.

3.1.3. Washing Tissue Sections

A tissue sample is generally washed to remove contaminants such astissue-embedding media as well as lipids or biological salts that may affectthe profiling and identification of peptides and proteins in MALDI-MSanalysis. Washing a tissue section with 70% ethanol can remove salts, fol-lowed by a 90%–100% ethanol wash to dehydrate and fixate the tissue(Lemaire et al. 2006b; Schwartz et al., 2003). Lemaire et al. demonstrated aprocedure for washing a tissue section with five different organic solvents(chloroform, xylene, toluene, hexane, and acetone) for the identificationof proteins in tissue samples and repeated the procedure with freshsolvents. The detection of protein signals is increased when the tissue


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FIGURE 4 Average MALDI-IMS protein profiles directly acquired from serial mouseliver tissue sections not washed or washed with different solvent systems. (a) Full massrange; (b)–(e) selected mass signals showing specific behaviors for the different washes.Reprinted from Seeley et al. (2008) with permission from Springer.

sections are washed with organic solvent compared with untreated sam-ples (Lemaire et al., 2006b). Seeley et al. (2008) reported a new washingprocedure to enhance protein detection in terms of both the number ofobserved peaks and the signal intensity. They demonstrated that the useof 12 different washing solvents established the most effective condi-tion for direct protein analysis from the surface of tissue section. Theyalso obtained a high detection sensitivity of protein signals, matrix crys-tal formations, and histological integrity of the tissues by washing with70% isopropanol for 30 seconds followed by a 90% isopropanol wash for30 seconds. Figure 4 shows the MALDI-IMS results for protein detection inmouse liver tissue sections after washing with different organic solvents.

3.1.4. Histological Staining of the Section

Histological staining of the tissue section is necessary to interpret the ionimages obtained from the IMS results with the tissue section used in theexperiments. The optical image obtained by the microscope is also used tosuperimpose the images acquired by IMS analysis to see the localizationof molecules in tissue section. Hematoxylin-eosin (H&E) staining is a verypopular histological method for MALDI-IMS results (Walch et al., 2008).In IMS, two serial sections are sliced from tissue; one is used for imaging

AcetoneChloroformEthanolWaterHexaneIsopropanolAcetic acidMethanolt-MBETolueneXyleneNo wash

2000015000Mass-to-charge (m/z)

100005000

2000

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(a)

Pars distalis Pars intermedia Pars neuralis

m/z 9685

m/z 6651

m/z 2897

1 mm m/z 6651 m/z 2897 m/z 9685 1 mm

a.u.12108642

3000 4000 5000 6000 7000 8000 9000 10000m/z

(b) (c)

(f)

(d) (e)

FIGURE 5 MALDI-IMS of a tissue section of rat pituitary gland. (a) Optical microscopicimage of an H&E-stained tissues section. The staining was done after the MALDImeasurement of the tissue section. (b)–(d) Visualized selected m/z species representingfeatures to pars distalis (m/z 6,651; green), pars intermedia (m/z 2,897; red), and parsneuralis (m/z 9,685; yellow). (e) Merge of (a–d). (f) MALDI-TOF-MS spectra obtainedfrom this case from pars distalis (green), pars intermedia (red), and pars neuralis (yellow)showing the molecular differences between the histological regions. Reprinted fromWalch et al. (2008) with permission from Springer.

and another section is cut for histological staining. They can then be super-imposed on each other and provide an absolute value of the moleculardistribution (Figure 5). Recently a new approach for tissue section stain-ing after the MALDI measurement has been reported. The results obtainedfrom IMS analysis were correlated with the H&E staining of the tissuesection (Schwamborn et al., 2007).

3.2. Choice of Matrix

The choice of a suitable matrix for MALDI-IMS analysis depends onthe mass range and analyte of interest. The main function of the matrixis to absorb laser energy from the source and transfer it to the analyte(Dreisewerd, 2003). The matrix thus ensures that efficient desorptionand ionization occur and protects the tissue section from the disrup-tive energy of the laser. Sinapinic acid is generally used for the analy-sis of higher-molecular-weight proteins, and α-cyano-4-hydroxycinnamicacid (CHCA) is used for lower-molecular-weight molecules such aspeptides (Schwartz et al., 2003). 2,4-dihydroxybenzoic acid (DHB) and2,6-dihydroxyacetophenone (DHA) are generally used for analysis of


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TABLE 1 Commonly used MALDI matrices for imaging of biomolecules in tissuesamples

Matrix Applications References

2,5-Dihydroxybenzoicacid (DHB)

Lipids, Sugars,peptides, nucleotides,glycopeptides,glycoproteins, andsmall proteins

Fournier et al. (2003);Herring et al. (2007);Tholey and Heinzle(2006)

α-Cyano-4-hydroxycinnamic acid(CHCA)

Peptides, smallproteins andglycopeptides

Schwartz et al. (2003);Tholey and Heinzle(2006)

2,6-Dihydroxyacetophenone(DHA)

Phospholipids Jackson et al. (2005);Seeley et al. (2008);Tholey and Heinzle(2006)

2,4,6-Trihydroxyacetophenone(THAP)

Lipids Stuebiger and Belgacem(2007)

p-nitroaniline (PNA) Phospholipids Estrada and Yappert(2004); Rujoi et al. (2004)

2-mercaptobenzothiazole(MBT)

Phospholipids Astigarraga et al. (2008)

Sinapinic acid (SA) Peptides and largeproteins

Schwartz et al. (2003)

CHCA/aniline, ionicmatrix

Peptides Lemaire et al. (2006b)

CHCA/n-butylamine,ionic matrix

Phospholipids Shrivas et al. (2010)

phospholipids (Herring et al., 2007; Seeley et al., 2008). A great variety ofmatrices are used for the analysis of biomolecules, some of which are listedin Table 1.

3.2.1. Ionic Matrices for IMS

Ionic matrices (IMs) constitute a new class of organic matrices reportedfor the analysis of a number of different molecules in MALDI-MS.IMs are good for MALDI-MS imaging studies due to the fact that theprocess solubulizes several analytes, has vacuum stability, and formshomogenous crystals with analyte molecules. IMs have been used toobtain enhanced sensitivity and good reproducibility in the analysis


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of biomolecules (Armstrong et al., 2001; Laremore et al., 2007). IMssuch as 2,5-dihdroxybenzoic acid butylamine (DHBB) and α-cyno-4-hydroxycinnamic acid butyl amine (CHCAB) render good crystal forma-tion, signal intensity, and reproducibility compared with conventionalmatrices such as DHB and CHCA (Shrivas et al., 2010). The results are

(a) (b)

(c) (d)

(e) DHB

703

731

760

786

(f) CHCA (g) DHBB (h) CHCAB

100%

0%

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× 1

03

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010 2 3 4 5 6

703731760786

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703731760786

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703731760786

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(o) (p)

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703731760786

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703

500 800

FIGURE 6 The crystal formation of (a) DHB, (b) CHCA, (c) DHBB, and (d) CHCABmatrixes with phospholipids on to a MALDI target plate. The pictures were taken with anUltraflex II TOF/TOF. The scale bar (white color line) is 100 µm; images (e) to (h) showthe ion image of phospholipids reconstructed obtained by using (e) DHB, (f) CHCA,(g) DHBB, and (h) CHCAB matrix at m/z 703, 731, 760, and 786. Images (i) to (l) showthe signal enhancement: 3- to 7-fold enhancement of signal intensity when DHBA IM(image i) is used as a matrix compared with DHB matrix (image j) and 50- to 100-foldimprovement of signal intensity using CHCAB IM (image k) compared with CHCA matrix(image l). Graphs (m) to (p) show the six replicate analyses of samples with± relativestandard deviation, % by using (m) DHB:± 20.5–40.8%, (n) CHCA:± 29.5–45.8%,(o) DHBB:± 14.5–21.8%, and (p) CHCAB:± 7.5–10.0%. Reprinted from Shrivas et al. (2010)with permission from American Chemical Society.


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shown in Figure 6. Direct tissue analyses of peptides in rat brain tissuesections using IMs improved the ionization efficiency and increased thesignal intensity of ion images of molecules compared with the conventionalmatrix (Lemaire et al., 2006a). IMs were also used for imaging and identifi-cation of gangliosides in mouse brain (Chana et al., 2009). DHB and CHCABIMs in MALDI-IMS were also used for analysis of mouse liver and cerebel-lum tissues to identify the different species of lipids; results with CHCABwere better than with conventional matrices (DHB and CHCA).

3.2.2. Nanoparticles as Matrices for IMS

In addition, nanoparticles (NPs) can be used as a matrix instead of organicmatrices for the analysis of low-molecular-weight molecules (<500 Da).One problem with the organic matrix ions is that they themselves producean intense peak in the mass spectrum and hence suppress detection of theanalyte of interest, which then obviously decreases the sensitivity of themethod. To circumvent this disadvantage, nanomaterials and inorganiccompounds have been introduced. The Tanaka and Sunner groups investi-gated the application of cobalt powder (NPs) and graphite microparticles,respectively, suspended in glycerol to analyze proteins and/or peptidesin MALDI-MS analyses. The use of NPs as a matrix in MALDI-MS allowsfor efficient absorption of laser energy as well as efficient subsequentdesorption and ionization of molecules from the sample surface (Sunneret al., 1995; Tanaka et al., 1988). Desorption/ionization on porous sili-con (DIOS) is another matrix-free method that is produced by etchingof the silicon surface. Small molecules can be efficiently ionized usingDIOS as an effective surface (Wei et al., 1993). Today nanomaterial surfacesare also applied for the direct analysis of tissue samples in MALDI-IMS.Northen’s group introduced a new nanostructure surface for imagingof biomolecules in tissue samples known as ionization nanostructure-initiator mass spectrometry (NIMS) (Northen et al., 2007). Several othersample preparation procedures, such as graphite-assisted laser desorp-tion/ionization (GALDI) (Cha and Yeung, 2007), nano-assisted laserdesorption/ionization (NALDI) (Vidova et al., 2010), and DIOS have beenproposed for imaging of biomolecules in tissue samples. Taira et al. (2008)developed another matrix-free method called nanoparticle-assisted laserdesorption/ionization imaging mass spectrometry (Nano-PALDI-IMS)that can be used to visualize peptides, phospholipids, and metabolitesin tissue sections. Recently silver (Hayasaka et al., 2010) and gold (Goto-Inoue et al., 2010a) NPs were applied for imaging and identification offatty acids and glycosphingolipids, respectively, an analysis that could bedifficult to perform by conventional MALDI-MS using DHB as a matrix.Figure 7 demonstrates imaging and identification of fatty acids frommouse liver sections using silver NPs as a matrix (Hayasaka et al., 2010).More recently, TiO2 NPs were applied for the analysis of low-molecular


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(a) (c)

4035302520

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u.)

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On tissue with AgNps

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1510

5

200 220 240 260 280 300 320

On tissue with DHB

340 m/z

(b)

4035302520

Sig

nal i

nten

sity

(a.

u.)

15105

200 220 240 260 280 300 320

Only AgNPs

340 m/z

(d)

4035302520

Sig

nal i

nten

sity

(a.

u.)

1510

5

200 220 240 260 280 300 320

Only DHB

340 m/z

Opticalimage

(e)

AgNPs

100%

0%

DHB

m/z 255.4(16:0)

m/z 279.4(18:2)

m/z 281.5(18:1)

m/z 283.4(18:0)

m/z 301.2(20:5)

m/z 303.3(20:4)

FIGURE 7 Identification of fatty acids from mouse liver sections in nano-PALDI-IMS.The serial sections were sliced to a thickness of 10 µm. Silver nanoparticles (NPs) or DHBmatrix solution was sprayed on the surface of the mouse liver sections, respectively.Their sections were measured with a scan pitch of 100 µm by nano-PALDI-IMS analysis innegative-ion mode. The mass spectra were obtained from the sections sprayed withsilver NPs (a) on tissue section, (b) only silver NPs or DHB matrix, (c) on tissue section,and (d) only DHB solution. The peaks used to reconstruct the ion image are indicated byarrows. (e) In the analysis using silver NPs and DHB, the ion signals at m/z 255.4 (16:0),279.4 (18:2), 281.5 (18:1), 283.4 (18:0), 301.2 (20:5), and 303.3 (20:4) were detected. The scalebars are 500 µm. Reprinted from Hayasaka et al. (2010) with permission from Springer.

weight-biomolecules in mouse brain without observing any NP-relatedpeaks. More individual signals and higher intensity were obtained whenTiO2 NPs were used as a matrix compared with a DHB matrix (Shrivaset al., 2011). Thus we can conclude that the use of a nanomaterial surfaceis efficient and effective for desorption and ionization of molecules; theprocess yields images with higher resolution.

3.3. Application of Matrix Solution

The deposition of matrix solution on the surface of a tissue section isanother important step in obtaining homogeneity, reproducibility, andgood resolution of the biomolecule. The matrix solution consists of three


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components—organic solvent, matrix, and trifluoroacetic acid (TFA).Crystal formation is affected by the concentration of matrix and the ratioof organic solvent to water; organic solvent is used to dissolve the solidmatrix and extract the molecules from the tissue section. This extrac-tion is followed by crystal formation on the surface of the tissue section.The addition of TFA provides free protons for effective ionization of theanalytes, and typically, singly charged [M+H]+ ions are formed. A num-ber of devices are useful for the deposition of matrix solution on thesurface of tissue sections—for example, chemical inkjet printer spotter(Baluya et al., 2007), robotic spotting depositors (Aerni et al., 2006), electro-spray depositors (Altelaar et al., 2007), and airbrush sprayers (Hayasakaet al., 2009). The sublimation (Hankin et al., 2007) and stainless steel sieve(Puolitaival et al., 2008) methods have demonstrated good signal intensityand sample reproducibility. Figure 8 shows a thin layer chromatography(TLC) sprayer (image a), sublimation apparatus (b), air brush sprayer (c),and a chemical inkjet printer (d) used for matrix deposition. The goal ofthese matrix deposition approaches is to improve the homogeneity of thesample surface and enhance the signal intensity for the identification ofbiomolecules compared with direct deposition of the matrix.

(b)(a)

(d)(c)

MALDIplate with

tissue slice

FIGURE 8 Apparatus used to deposit matrix on the tissue section. (a) Thin-layerchromatography sprayer, (b) sublimation apparatus, (c) air brush sprayer, and (d) chemicalinkjet printer. Reprinted from Hankin et al. (2007) with permission from Springer.


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4. INSTRUMENTATION

4.1. Quadrupole Mass Analyzer

A quadrupole mass analyzer is made from four parallel rods maintainedat fixed direct current (DC) with an alternating radiofrequency (RF). Withthis setup molecular ions formed at the source pass through the middleof the quadrupoles in the electric field region and the ions of a specificm/z have a stable trajectory path and may pass all the way to the detec-tor, while the remaining ions collide with the electrodes and never reachthe detector (Gross, 2004). Using a continuous and controlled manner tochange the frequency and potential, the quadropole transmits moleculesat certain m/z values. Figure 9a shows a diagram of quadrupole mass ana-lyzer. The sensitivity of the instrument can be enhanced by increasing thenumber of qaudupoles from two to three (triple quadrupole) in series. Intriple-quadrupole analyzers, the first (Q1) and third (Q3) quadrupoles actas filters, and the second (Q2) quadrupole functions as a collision cell. Thethird (Q3) quadrupole is worked at normal RF/DC or in the linear iontrap (LIT) mode (Douglas et al., 2005). Hopfgartner et al. (2009) demon-strated the fast imaging of complete rat sections using MALDI coupledwith a triple-quadrupole LIT where the precursor ion mode can be usedto monitor the presence of the parent drug in the tissue section.

4.2. Time-of-Flight Mass Analyzer

The TOF-MS analyzer has become valuable for direct analysis of biomole-cules from tissue samples. In TOF-MS, the different masses of ions areseparated based on their differences in travel time through a drift region.The lighter ions produced from the source travel faster at the end of thedrift region compared with heavier ions in the tube (see Figure 9b). How-ever, TOF-MS has disadvantages in mass accuracy, resolving power, andits inability to perform tandem MS experiments (Goto-Inoue et al., 2011;Gross, 2004). This drawback has been overcome by the introduction of anorthogonal geometry (oTOF)-MS analyzer to extract pulsed ions from acontinuous ion beam. Huang et al. (2011) investigated the use of oTOF-MSfor imaging and simultaneous detection of metal and nonmetal elementsin tissue section with spatial resolution of 50 µm.

Ion mobility (IM) spectrometry can also be coupled with the TOF-MSsystem for direct analysis of tissue samples. The instrument has oTOF-MSand is equipped with an IM spectrometer located between the quadrupoleand the TOF-MS analyzer. The IM spectrometer separates ions based ontheir IM (i.e., their shape) and TOF-MS separates ions according to theirm/z ratio in the MS (Verbeck et al., 2002). Separation of structurally similar


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(d)

(e)

(f)

Entranceslit Quadrupole

rods

(a)

Exitslit

Ringelectrode

Exitendcap

electrode

Entranceendcap

electrode

Detectorelectrode

Detectorelectrode

Centralelectrode

(b)

Flighttube Linear

detector

Reflectordetector

Reflector(lon mirror)

(c)

Magnet

Flighttube

Exitslit

Superconductivemagnet

Excitationplates

Trappingplates

Detectorplates

FIGURE 9 Schematic description of six mass analyzers used in mass spectrometers.(a) Quadrupole, (b) time-of-flight, (c) magnetic sector, (d) ion trap, (e) orbitrap, (f) ioncyclotron resonance. Reprinted from Pol et al. (2010) with permission fromSpringer.

ions and ions of the same charge state is thus possible through their differ-ent mobility in the IM spectrometer. The combined techniques of IM andTOF-MS were used for imaging and identification of digested proteins.IM separates isobaric ions that cannot be distinguished by MALDI-TOFalone, providing mass- and time-selected ion images of biomolecules intissue samples (Stauber et al., 2010).


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In addition, the combination of a quadrupole (Q) mass analyzer witha TOF-MS is called a Q-TOF-MS system and is used for structural analysiswith tandem MS. The localization of a xenobiotic substance in skin hasbeen reported by applying a Q-TOF-MS (Bunch et al., 2004). Anotherapproach for imaging and identification of molecules is the combinationof two TOF mass analyzers; this hybrid is called TOF/TOF. First, TOF-MSseparates precursor ions using a velocity filter; second, TOF-MS analyzesthe fragment ions (Gross, 2004). MALDI-TOF/TOF is a simple, rapid, andsensitive technique for MALDI imaging of biomolecules in tissue sections(Hayasaka et al., 2010; Sugiura et al., 2009).

4.3. Sector-Type Mass Analyzer

The sector mass spectrometer consists of large electromagnetic (“B”sector) and electrostatic focusing devices (“E” sector) that, dependingon the different manufacturers’ use, differ in their geometries (Cottrelland Greathead, 1986). The motion of the ions in the trajectory pathwaydepends on the strength of electric and magnetic field where each ion(m/z) travels with different speeds (see Figure 9c). Magnetic sectors areused for high-resolution elemental imaging and identification of samplesin combination with dynamic SIMS. The magnetic sector and several mov-able detectors allow a simultaneous detection of several elements or smallmolecules (within a narrow mass range) with higher sensitivity. Slodzianet al. (1992) used a SIMS coupled with a magnetic sector double-focusingmass spectrometer for simultaneous imaging of several elements in tissuesample.

4.4. Ion Trap Mass Analyzer

A quadrupole ion trap (QIT or 3D-IT) operates in a 3D quadrupole fieldmaintained at constant DC and RF fields to trap the moving ions of m/zrange. A QIT consists of three hyperbolic-shaped electrodes: the cen-tral ring electrode and two adjacent end cap electrodes (entrance andexit) (see Figure 9d). A 3D-IT is a small, relatively inexpensive instru-ment for sensitive analysis; it can also be used for MSn analysis ofmolecules in the tissue samples (Gross, 2004; Hopfgartner et al., 2004).Shimma et al. (2008) reported their use of a MALDI-QIT–TOF-MS instru-ment for imaging of phospholipids, glycolipids, and tryptic-digestedproteins. MS analyses were performed to confirm their presence. Recentlya mass microscope coupled with a high-resolution atmospheric pressure-laser desorption/ionization (AP-MALDI) and QIT-TOF was used forimaging and identification of volatile substances in ginger (Harada et al.,2009). This instrument allows researchers to precisely determine the


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specific tissue section prior to IMS and has spatial resolution (10 µm)higher than the naked eye.

In a linear quadrupole ion trap (LIT) or 2D traps (2D-IT), the ions aretrapped in a 2D quadrupole field and then pass axially. The 2D-IT iontrap produces reasonable mass accuracy, mass resolution, and sensitivity(Schwartz et al., 2002). LIT has a better ion storage capacity and a highertrapping efficiency compared with 3D-IT. However, the disadvantage ofLIT is the relatively narrow mass range of small molecule analysis. Garrettet al. (2007) described a new MALDI-LIT-MS for imaging of tissue sam-ples and also used for MSn analyses to confirm the molecules. Enomotoet al. (2011) demonstrated the visualization of phosphatidylcholine (PC),lysophosphatidylcholine, and sphingomyelin in mouse tongue using LTQ(linear trap quadrupole)-MALDI-IMS (Enomoto et al., 2011).

4.5. Orbitrap Mass Analyzer

In an orbitrap mass analyzer, the ions are rotated around a central elec-trode by applying an appropriate voltage between the outer and centralelectrodes. Hence, the ions of a specific m/z ratio cycle in rings thatoscillate around the central spindle and then pass through the detec-tor (Makarov et al., 2006). Figure 9e shows the overview of the orbi-trap mass analyzer. LTQ-Orbitrap has been used to analyze compoundswith high resolving power and excellent mass accuracy that apprecia-bly decrease false-positive peptide identifications in the sample (Adachiet al., 2006; Makarov et al., 2006). Verhaert et al. (2010) demonstratedthe use of LTQ-orbitrap for imaging of neuropeptides in neural tissuesamples. In addition, it has also been used for identification and sequenc-ing of neuoropeptides from neural tissue using MALDI-MS with an iontrap–orbitrap hybrid instrument. Landgraf et al. (2009) showed the highresolution and accurate measurement of ion images of lipids in spinalcord using MALDI-LIT–orbitrap-MS. Manicke et al. (2010) demonstratedimaging of lipids in rat brain tissue section with a high-resolving powerinstrument of DESI-LTQ–orbitrap-MS.

4.6. Ion Cyclotron Resonance Mass Analyzer

In an ion cyclotron resonance (ICR)-MS analyzer, the ions of a particularm/z ratio are isolated based on the cyclotron frequency of the ions in aconstant magnetic field. The oscillation of ions in ICR induces an alter-nating current that is equivalent to their m/z ratios. Figure 9f shows theschematic for an ICR analyzer. Fourier transforms (FT)-ICR-MS contin-ues to deliver the highest mass-resolving power and mass measurementaccuracy (Gross, 2004). The combination of MALDI-TOF-MS with theFT-ICR-MS technique is useful for high–spatial resolution analysis and


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identification of unknown biomolecules in tissue samples. Thus the highmass resolution of the FT-ICR-MS can be used to analyze compoundsthat cannot be distinguished with lower–mass resolution mass spectrom-eters (Taban et al., 2007; Wang et al., 2011). MALDI-FT-ICR has also beenreported for IMS analysis of drugs and metabolites in tissue. The accuratemass measurement can be performed using FT-ICR-MS, which provideda molecular specificity for the ion images obtained from tissue sampleanalysis (Cornett et al., 2008).

5. IMS MEASUREMENTS

MALDI-IMS experiments can be performed after the deposition of matrixon the tissue section and using different types of MS instruments as dis-cussed above. The setting of the laser energy, detector gain, and randomwalk function are optimized in order to obtain better signal intensity of thetarget molecules during the IMS analysis. Either a particular region ofthe tissue or the entire tissue section is selected for analysis, depending onthe particular interest. At present, the commercially available instrumentscan perform IMS analyses with the highest spatial resolution of ∼25 µm(Goto-Inoue et al., 2009a). Recently we developed a mass microscope thatcan move a sample stage by 1 µm, and the finest size of the laser diame-ter is approximately 10 µm (Harada et al., 2009). The measurement time ofIMS experiments depends on the number data spots, the frequency of thelaser, and the number of shots per spot.

6. DATA ANALYSIS

Due to the large (gigabytes) size of the dataset, high-capacity visualizationsoftware is required to visualize the ion image and distribution pattern ofbiomolecules in tissue samples. New computing methods are required forboth rapid, accurate data acquisition and the interpretation of the IMSanalysis results. Therefore, in addition to instrumental improvements,data acquisition and software development have been important for theproduction of reliable data. The databases used are based on algorithmsthat perform analysis through statistical evaluation of observed andtheoretical spectra of bimolecules. BioMap (http://www.maldi-msi.org,Novartis, Basel, Switzerland) and flexImaging (http://www.bdal.com,Bruker Daltonics GmbH, Bremen, Germany) imaging software are usedto identify biomolecules in various sample types. The intensity of thedifferent color images obtained by both software packages can relatethe distribution of biomolecules in the tissue section. These softwarepackages also help in understanding the localization of biomolecules at

http://www.maldi-msi.org

http://www.bdal.com


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particular regions of interest (ROIs) for mass spectral comparison andother statistical analysis.

BioMap imaging software can be used for different instruments suchas PET, nuclear magnetic resonance (NMR), computed tomography (CT),and near-infrared fluorescence (NIRF) as the result of multiple modi-fications. Interactive data language Virtual Machine (Research Systems,Boulder, CO) is required in the system to process the data obtained fromIMS analysis. BioMap software can also be used for baseline correction,spatial filtering, and averaging of spectra for presentation of the IMSresults.

The flexImaging software is used for the acquisition and evaluationof MALDI-TOF imaging results. The mass peaks (at m/z) obtained in themass spectrum are normalized to total ion current and then the peakintensity is taken into account to study the molecules distribution onthe tissue section. The imaging MS experiments are performed by col-lecting spectra at a resolution of 50 to 400 µm in the same m/z range asabove. Principal component analysis (PCA) is an unsupervised statisti-cal method used to identify groups of closely correlated variables; forMS imaging datasets these variables are spatial coordinates and mass.This approach also reduces the multidimensional datasets to the lowerdimensions (Chou, 1975). PCA analysis is performed using ClinProTools2.1 software (Bruker Daltonics). Zaima et al. (2009) performed a PCA forscreening of metabolites in the fatty liver. Several differences were foundin identifying the metabolites in fatty and normal liver tissue samples.PCA was also used in proteomics studies (Deininger et al., 2008; Djidjaet al., 2010; Yao et al., 2008).

7. APPLICATIONS OF IMS FOR DIRECT ANALYSIS OF TISSUE

7.1. IMS for Lipidomics

Lipids are the main constituents of cell membranes; the major functions oflipids are transportation of ions and signals across the cell membrane. Var-ious types of lipids, such as glycerophospholipids (GPLs), sphingolipids,sterol lipids, saccharolipids, waxes, and fat-soluble vitamins are found inbiological systems. Membranes act as barriers to separate compartmentswithin eukaryotic cells and to separate all cells from their surroundings(Brown, 2007; Fahy et al., 2009; Lee et al., 2003). The identification andquantification of lipids in tissue sample can help in understanding severalbiosynthetic and metabolic pathways that govern human diseases, suchas insulin-resistant diabetes, Alzheimer’s disease, schizophrenia, cancer,atherosclerosis, and infectious diseases (Oresic et al., 2008). Thus the analy-sis of lipids in tissue samples is a very important issue. High-performanceliquid chromatography (HPLC) (McCluer et al., 1986), TLC (Touchstone,


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1995), and MS have been used to analyze lipids in tissue samples. How-ever, the sample preparation procedures in chromatographic techniquesare lengthy and the localization of biomolecules in tissue sample surfacecannot be established. Therefore, different IMS systems are successfullyused for imaging of lipids. The analysis of glycerophospholipids, sph-ingolipids, and neutral lipids is discussed in detail in the followingsections.

7.1.1. Glycerophospholipids

GPLs are glycerol-based phospholipids and essential components of cellmembranes. They act as second messengers involved in cell prolifera-tion, apoptosis, and metabolism. The determination of GPL content intissue samples is useful for finding potential biomarkers for diseases suchas atherosclerosis or rheumatism (Fuchs et al., 2005; Schmitz and Rueb-saamen, 2010). Altered levels of lipids are found in many pathologicalconditions such as Alzheimer’s disease (Han et al., 2001, 2002), Down syn-drome (Murphy et al., 2000), diabetes (Han et al., 2007), and Niemann–Pickdisease (He et al., 2002). Figure 10 illustrates the basic structures of com-mon classes of GPLs such as PC, phosphatidylethanolamine (PE), phos-phatidylinositol (PI), and phosphatidylserine (PS) (Jackson and Woods,2009). PC is easily ionized due to its charged quaternary ammonium headgroup and has thus become a popular lipid species to investigate (Pulferand Murphy, 2003). The ionized molecules observed in the mass spectrumare usually either protonated [M+H]+, sodiated [M+Na]+, or pottasiated[M+K]+ in positive-ion mode. Phospholipids such as PE, PS, PA, PG, andPI may generate negative ions due to the presence of the phosphodiestermoiety and display molecular anions [M-H]− (Fuchs et al., 2010). Theaddition of potassium acetate (Sugiura et al., 2009) or LiCl (Jackson et al.,

Phosphatidylcholine

Phosphatidylinositol

Phosphatidylethanolamine

Phosphatidylserine

R1

R2

OP

OHOH

OHO

HOHO

HO

OOH

O

O

O

R1

R2

H2NO P

C

O

OHH

HHO

O

OO

O

O

O

R1

R2

POH

HH2N

O

O OO

O

O

O

O

O

−ONO O R1

R2OH

P

O

O

+

FIGURE 10 Structure of glycerophospholipids. Reprinted from Jackson and Woods(2009) with permission from Elsevier Science.


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2005) to the matrix solution has also been reported for effective ionizationof molecules in tissue samples.

The selection of MALDI matrices is an important issue. For MALDI-IMS, the matrix should have good vacuum stability and homogenouscrystal formation containing analyte molecules. Various matrices havebeen reported for the identification and characterization of lipids inMALDI-MS, including DHB (Petkovic et al., 2001; Puolitaival et al., 2008;Schiller et al., 1999), DHA (Jackson et al., 2005; Shimma et al., 2007),p-nitroaniline (PNA) (Estrada and Yappert, 2004; Rujoi et al., 2004), and9-aminoacridine (Eibisch and Schiller, 2011; Teuber et al., 2010). However,PNA and dihydroxyacetone phosphate (DHAP) were unstable under highvacuum conditions and started to evaporate after their introduction intothe MALDI-MS instrument (Jackson et al., 2005; Rujoi et al., 2004; Shrivaset al., 2010). DHB matrix exhibited a lower sensitivity for the detectionof PA, PS, PE, PI, and PG in negative-ion mode, possibly due to its acid-ity (Estrada and Yappert, 2004; Petkovic et al., 2001). DHA can be usedin both positive and negative ionization modes for analysis of phospho-lipids (Woods et al., 2006). PNA is another good matrix for the analysis ofphospholipids in either positive-ion or negative-ion modes (Estrada andYappert, 2004). Recently, 2-mercaptobenzothiazole (MCT) was added asan alternative to the use of DHB for MALDI-MS analysis of phospho-lipids in brain and liver tissue samples (Astigarraga et al., 2008). The mainadvantages of MCT over the commonly used matrices DHB, DHA. andPNA are low vapor pressure, low acidity, and homogenous crystal forma-tion, which allowed for detection of more lipid species in negative mode,with high sensitivity and high detection reproducibility. Ionic matriceshave also been used in MALDI-IMS owing to the good vacuum stability,homogenous crystal formation, and good solubility of analytes for effi-cient ionization and desorption of molecules (Chana et al., 2009; Lemaireet al., 2006a; Shrivas et al., 2010). Shrivas et al. (2010) used an ionic matrixof CHCAB to image and identify lipids in mouse cerebellum and foundthat this ionic matrix yields a higher number of ion images comparedwith the use of DHB matrix in MALDI-IMS (Figure 11). Use of NPs isanother good approach for selective and sensitive analysis of lipids andsmall metabolites in tissue samples without matrix-oriented peaks in thelow-molecular-mass range (Cha and Yeung, 2007; Goto-Inoue et al., 2010a;Hayasaka et al., 2010; Shrivas et al., 2011; Taira et al., 2008).

Sugiura et al. (2009) showed the imaging of polyunsaturated fatty acid–containing PC in mouse brain using MALDI-IMS. The results demon-strated that arachidonic acid (AA) and DHA-containing PC were foundin the hippocampal neurons and cerebellar Purkinje cells, respectively.Figure 12 shows the localization of PC species in different layers of themouse brain (Sugiura et al., 2009). The distribution of PC species also hasbeen reported in the mouse retinal section via MALDI-IMS analysis. Thelocalization of PC (16:0/18:1) was found in the inner nuclear layer and


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GL

ML

WM

(a)

(b)

m/z 734.5 m/z 772.5 m/z 826.5 m/z 846.5

m/z 756.5 m/z 798.5 m/z 832.5 m/z 850.6

m/z 760.5 m/z 810.5 m/z 834.5 m/z 864.6

m/z 769.5 m/z 822.6 m/z 835.6

m/z 770.5 m/z 824.5 m/z 844.5

m/z 870.5

(c)

m/z 734.5 m/z 772.5 m/z 826.5 m/z 846.5

ND

ND

ND

ND

ND

ND

ND

m/z 756.5 m/z 798.5 m/z 832.5 m/z 850.6

m/z 760.5 m/z 810.5 m/z 834.5 m/z 864.6

m/z 769.5 m/z 822.6 m/z 835.6

m/z 770.5 m/z 824.6 m/z 844.5

m/z 870.5

FIGURE 11 (a) H&E-stained mouse cerebellum showing three layers with 1.5-mm scalebar (white color). The ion images of lipids in mouse cerebellum tissue section obtainedby using (b) CHCAB and (c) DHB as a matrix at m/z 734.5 [(PC(16:0/16:0)+H)]+, 770.5[PC(16:0/16:1)+K]+, 772.5 [PC(16:0/16:0)+K]+, 798.5 [PC(16:0/18:1)+K]+, 834.5[PC(18:0/22:6)+H]+, and 870.5 [PC(18:1/22:6)+K]+ were localized in the molecular layerof cerebellum; at m/z 760.5 [PC(16:0/18:1)+H]+, 832.5 [PC(18:0/20:4)+Na]+, 844.5[PC(16:0/22:6)+K]+, and 846.5 [PC(18:1/20:4)+K]+ were specific to the granular layer;and at m/z 756.50 [PC(16:0/16:0)+Na]+, 810.5 [PC(18:0/18:1)+Na]+, 824.5[PC(18:0/18:2)+K]+, and 826.5 [PC(18:0/18:1)+K]+ and were found to be concentrated inthe white matter of cerebellum. The ion images at m/z 769.5 [SM(d18:1/18:0)+K]+ and835.6 [SM(d18:1/24:1)+Na]+ illustrated that the molecules were distributed in the regionof molecular layer of tissue. The ion images at m/z822.6 [GalCer(d18:1/22:0)+K]+ and850.6 GalCer(d18:1/24:0)+K]+ were localized in the white matter of mouse cerebellum.ND indicates the molecules were not detected. GL, granular layer; ML, molecular layer;WM, white matter. Reprinted from Shrivas et al. (2010) with permission from AmericanChemical Society.

the outer plexiform layer; PC (16:0/16:0) in the outer nuclear layer andinner segment; and PC (16:0/22:6) and PC (18:0/22:6) in the outer segmentand pigment epithelium (Hayasaka et al., 2008). Differential localizationof PC (16:0/20:4) species was observed between terminal and stem villi of


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16:0

18:0

16:0

18:1

(O

A)

20:4

(A

A)

22:6

(D

HA

)

PC (diacyl-16:0/16:0)

m/z 772.4

m/z 798.4

m/z 820.5

m/z 844.5

0% 100%

m/z 826.5

m/z 848.5

m/z 872.6

PC (diacyl-16:0/18:1)

PC (diacyl-16:0/20:4)

PC (diacyl-16:0/22:6)

PC (diacyl-18:0/18:1)

PC (diacyl-18:0/20:4)

PC (diacyl-168:0/22:6)

m/z 846.5

m/z 870.6

PC (diacyl-18:1/20:4)18:1 (OA)

CBX HPF

TH

CTX

Cp

ON+O−

POH2C

H2C

HC

CH3

CH3

CH3

O

O

OR2 (sn–2)

R1 (sn–1)

Structure of PCs

PC (diacyl-18:1/22:6)

FIGURE 12 Identification of molecular species of PC in sagittal mouse brain sectionsby MALDI-IMS. Among the PC, AA-PC showed characteristic localization in thehippocampal cell layers (arrowheads). Among DHA-containing species, two abundantspecies, PC (diacyl-16:0/22:6) and PC (diacyl-18:1/22:6), were commonly enriched in thegranule layer of the cerebellum, while PC (diacyl-18:0/22:6) showed a characteristicdotted distribution pattern near the cell layer (arrows). CBX, cerebellar cortex;CP, corpus striatum; CTX, cerebral cortex; HPF, hippocampal formation; TH, thalamus.Reprinted from Sugiura et al. (2009) with permission from the American Societyfor Biochemistry and Molecular Biology.

human placenta, which could be helpful in understanding the patholog-ical involvement of fetal growth restriction and fetal hypoxia (Kobayashiet al., 2010). The accumulation of lipid molecules, such as LPC (1-acyl 16:0),PC (1-acyl 36:4), and shingomyelin (SM) (d18:1/16:0) around the damagedvalvular region was investigated and suggested an association of thesemolecules with tissue inflammation and resultant valvular incompetence(Tanaka et al., 2010). PC (diacyl-16:0/20:4) containing an AA was foundat higher concentration in prefrontal cortex tissue compared with occipi-tal cortex tissue in the brains of patients with schizophrenia (Matsumotoet al., 2011). The specific localization of five PC species in the cochleawas also examined using the mass microscope. A differential distribu-tion of PC species was observed; (16:0/18:1) in the organ of Corti and thestria vascularis, (16:0/18:2) in the spiral ligament, and (16:0/16:1) in theorgan of Corti (Takizawa et al., 2010). Recently Goto-Inoue et al. (2009a)


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investigated use of a TLC-Blot-MALDI-IMS for analysis and characteri-zation of acidic, neutral glycosphingolipids and PC in sample mixtures.In TLC-Blot, the lipids are separated and transferred to a polyvinylidenefluoride (PVDF) membrane without any change in the stability of themolecules. PVDF membranes with the sample may then be placed on thetarget plate for MALDI-IMS analysis. This method might be useful forthe detection of minor components that could not be detected by the con-ventional TLC method. SIMS is also used for imaging of lipids at highspatial resolution and sensitive detection. The combined approaches ofMALDI-IMS and SIMS-IMS have been used for imaging of PC in culturedmammalian neuron. The data obtained from MALDI and SIMS supportedthat the signals of small molecules in the low molecular region, such asPC head groups and fatty acids (detected in SIMS) were obtained fromthe intact lipids (Yang et al., 2010). DESI-IMS has been used for imaging ofmost commonly encountered brain lipid species such as PE and PI in ratspinal cord cross sections in negative-ion mode. The ion image of PI (38:4)was shown in grey matter regions such as the cortex and hippocampus.The ion image of PE (at m/z 888) showed the white specific region in thebrain (Dill et al., 2009).

7.1.2. Sphingolipids

The sphingolipid is a type of lipid obtained from the aliphatic amino alco-hol sphingosine. The main functions of sphingolipids are transmissionand cell recognition. The investigation of sphingolipids is very impor-tant because they are indicative of aging and may function as a diseasemarker. Sphingolipids contain a sphingoid base backbone and includesphingomyelins (SM), sulfatides (ST), ceramides, cerebrosides, and gan-gliosides (Merrill et al., 2009) (Figure 13). Changes in the levels of lipids,

HO

HO

HO

O

Ceramide Cerebroside

Sphingomyelin Sulfatide

P

O

O

OO

O OO

O OR

R

R

C13H27

C13H27

C13H27

OH

OH

OH

SOH

OH

HO HO

HO

HO

HH

H

H HN

HO

O

O

O

O

R

C13H27

H

H HN

H HN

H HN

−ON+

FIGURE 13 Structure of sphingolipids. Reprinted from Jackson and Woods (2009) withpermission from Elsevier Science.


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725 727

(a) (b) (c)

729

NormalCancer−7

25.5

Rel

ativ

e in

tens

ity

(m/z)

m/z 725.5

616 618 620

NormalCancer

616.

1

Rel

ativ

e in

tens

ity(m/z)

m/z 616.1 Merged image

Green: m/z 616.1 Red: m/z 725.5

FIGURE 14 Imaging of normal and cancerous cells in human liver sample. (a) The mostprominent signal at m/z 725 showed the higher expression for cancerous cells thannormal cells. (b) The signal at m/z 616 showed the higher distribution of this moleculein normal cells. (c) Merged images at m/z 725 and 616. Reprinted from Shimma et al.(2007) with permission from Elsevier Science.

in particular ceramide, also have been observed in apoptosis or cell death(Fuchs et al., 2007; Thomas et al., 1999). MALDI-MS/MS analyses wereused to image liver tissue samples at a thickness 3 µm from a patient withcolon cancer. A higher expression of sphingomyelin (SM, 16:0) at m/z 725was observed in cancerous tissue than in normal tissue by MS/MS analy-ses (Figure 14). In contrast, a strong distribution of an ion at m/z 616 wasobserved in the normal but not cancerous tissue sample (Shimma et al.,2007). IMS has also been used to detect seminolipid, a glycolipid syn-thesized in sperm. Here, seminolipid localization was performed in micetestis during testicular maturation Goto-Inoue et al. (2009b). In anotherstudy, the distribution pattern of ganglioside molecular species (C-18and C-20) in mouse hippocampus was demonstrated using MALDI-IMS.The localization of C-18 species was found in the frontal brain and C-20species contained in the entorhinal-hippocampus projections of the molec-ular layer (ML) of the dentate gyrus. Figure 15 shows the distributionof C-20-sphingosine–containing gangliosides in the hippocampal forma-tion (Sugiura et al., 2008). In a study using gold NPs in Nano-PALDI-IMSand comparing it with the use of DHB matrix, the PI, ST, and ganglio-side species (GM3, GM2, GM1, GD1, and GD3) were all detected withhigher sensitivity. This is the first report of the visualization of minorsphingolipids by IMS analyses using gold NPs in tissue sections (Goto-Inoue et al., 2010). Higher expression of sulphatides in ovarian cancer cellswas reported compared with a normal sample with MALDI-IMS anal-ysis and similar results were obtained by a transcriptomic approach oflipid analysis (Liu et al., 2010). The high spatial resolution localizationof glucosylceramide in spleen of a mouse model of Gaucher disease was


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(a) (b) m/z 878.6

ST (22:0 OH) ST (24:0 OH)

[GD1(18:0/d20:1) + K - 2H] −[GD1(18:0/d18:1) + K - 2H] −

[GD1(18:0/d20:1) + Na - 2H] −[GD1(18:0/d18:1) + Na - 2H] −

GM

1

(B)

(A)

GD

1

[GM1(18:0/d20:1) - H] −[GM1(18:0/d18:1) - H] −

(c) m/z 906.6

(d) m/z 1874

C18

Mad/PMad/P

MBMB

SOSOSRSR

SLMSLM

MLML

MLML

SOSR

SLM

ML

ML

THTH

CTXCTX

CpCp

C20

(e) m/z 1902 (f) Merged

(g) m/z 1858 (h) m/z 1886 (i) Merged

(j) m/z 1544 (k) m/z 1572 (l) Merged

HPF

(a) (b) m/z 878.6

ST (22:0 OH) ST (24:0 OH)

[GD1(18:0/d20:1) + K - 2H] −[GD1(18:0/d18:1) + K - 2H] −

[GD1(18:0/d20:1) + Na - 2H] −[GD1(18:0/d18:1) + Na - 2H] −

GM

1G

D1

[GM1(18:0/d20:1) - H] −[GM1(18:0/d18:1) - H] −

(c) m/z 906.6

(d) m/z 1874

C18

DGCA3

CA1

DGCA3

CA1

C20

(e) m/z 1902 (f) Merged

(g) m/z 1858 (h) m/z 1886 (i) Merged

(j) m/z 1544 (k) m/z 1572 (l) Merged

Mad/P

MB TH

CTX

Cp

FIGURE 15 (Continued)


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FIGURE 15 Localization of C20-sphingosine–containing gangliosides in thehippocampal formation. IMS at 50-mm raster step size was used to gain an overview ofganglioside distribution in different brain regions (A), and IMS at 15-mm raster size wasused to study in detail the distribution pattern of gangliosides in the hippocampus (B). Inboth panels, schematic diagram of the brain section (a) and ion images of STs (b)–(c) arepresented. For ions corresponding to the GD1 molecular species, we observed the iondistributions of both sodium and potassium complexes; that is, the ions at m/z 1858 (f)and m/z 1886 (g), which correspond to the [M+Na-H]− form of C18- and C20-GD1, andthose at m/z 1874 (h) and m/z 1902 (i), which correspond to the [M+K-H]− form of C18-and C20-GD1, respectively. The ion distribution patterns corresponding to the GD1-Nasalts and GD1-K salts are fairly uniform for both C18- and C20-species. For GM1, m/z 1544(d) and m/z 1572 (e), which correspond to C18- and C20-sphingosines–containing GM1,respectively, are shown. HPF, hippocampus formation; MadP, ———————-; CTX,cerebral cortex; MB, ————————-; TH, Thalamus; SO,————————-; SR, stratumradiatum; SLM, stratum lacunosum molecular; ML, molecular layer. Reprinted fromSugiura et al. (2008) with permission from Public Library of Science.

also demonstrated using MALDI-IMS (Snel and Fuller, 2010). GangliosideGM2, asialo-GM2 (GA2), and sulfatides in brain from a mouse model ofTay-Sachs/Sandhoff disease (Chen et al., 2008) and sulfatides in mousekidney (Marsching et al., 2011) have been reported.

7.1.3. Nonpolar Lipids

Imaging and identification of nonpolar lipid in tissue sections is noteasy, perhaps because of the difficulty in the ionization of molecules inMALDI-MS. Thus only a few species of nonpolar lipids have been suc-cessfully reported. One example is cholesterol, a highly abundant lipidin many tissues. It is usually detected at m/z 369 after the loss of awater molecule using an organic matrix in MALDI-MS. Cholesterol isa vital constituent of the cell membrane, required for lipid organiza-tion and cell signaling. Changes in the quantity of cholesterol in tissuecan cause myocardial infarctions and stroke, as well as other disorders(Fernandez et al., 2011). SIMS has been used for imaging of cholesterolwith the capability to analyze single cells. In this setup one drawback wasthat cholesterol was fragmenting (Piehowski et al., 2008). However, theuse of NIMS could directly analyze the brain cholesterol metabolites inSmith-Lemli-Opitz syndrome without the fragmentation of molecules inMS (Patti et al., 2010). The distribution of triglycerides (TAG) in mouseembryo was also investigated using MALDI-IMS (Hayasaka et al., 2009).TAG is an ester derived from glycerol and three fatty acids and is the mainconstituent of vegetable oil and animal fats. Figure 16 is illustrates the dis-tribution of different molecular species of TAG [(16:0/18:2/18:1)+Na]+,[(16:0/18:1/18:1)+Na]+, [(16:0/20:3/18:1)+K]+ in mouse embryo. Theion images of TAG were concentrated mainly around the brown adiposeand liver tissue (Hayasaka et al., 2009).


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Merged

(e)(d)

(c)(b)

(a)

(TAG (16:0/20:3/18:1) + K)+

(TAG (16:0/18:1/18:1) + Na)+(TAG (16:0/18:2/18:1) + Na)+

Lung

Liver

Intestine

Heart

Thymus

Brown adiposetissue

Brian

Hypoglottis

100%

0%

100%

0%

100%

0%

100%

0%

FIGURE 16 MALDI-IMS of neutral lipids. Distribution of triglycerides (TAG) inmouse embryo. (a) H&E staining, (b) ion images of [TAG (16:0/18:2/18:1)+Na]+,[TAG 16:0/18:1/18:1)+Na]+, and [TAG (16:0/20:3/18:1)+K]+ are shown. (b)–(d) The ionimages were merged with the optical image of an H&E-stained section. (e) Three mergedTAG images demonstrate the same distribution. Reprinted from Hayasaka et al. (2009)with permission from Springer.

7.2. IMS for Proteomics

The study of proteomics is useful for biomarker discovery of a large num-ber of diseases, using tissue samples such as vascular tissue, heart, brain,lung or bone, with a current major focus on cancer and malignant tissues(MacAleese et al., 2009). Today MALDI-IMS is increasingly being usedfor direct analysis of peptides and proteins from tissue sections; the mainadvantage is that it requires no labeling reagents (McDonnell and Heeren,2007; Stoeckli et al., 2001). Immunohistochemistry (IHC) has long been thestandard technology for imaging of peptide and protein distribution intissue. The sensitivity of IHC is usually excellent. However, this methodrequires a specific binder, usually an antibody, to detect a previouslydefined protein from the sample. Only a very small number of moleculesmay be detected in parallel, and these all need to be known beforehand.


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Antibody availability and specificity are other constraints with IHC thatlimit its capacity (Luongo de Matos et al., 2010). Thus the introduction ofIMS has provided complementary resources.

What IMS lacks in terms of sensitivity, it compensates for by enablinguntargeted studies with the possibility to detect hundreds of moleculesin a single run. In the so-called bottom-up approach, the proteins presentin the tissue sample must first be subjected to in situ digestion by a pro-teolytic enzyme. Usually trypsin is used since it renders peptides thatcontain at least one lysine or arginine amino acid and hence are easilyionized. Setou et al. (2008) investigated whether the addition of deter-gent into the trypsin solution could improve the digestion efficiency ofproteins for direct analysis of tissue section in MALDI-.IMS. Trypsin solu-tion can be spotted directly onto the tissue sections, rendering spot sizes∼150–200 µm. Considering the possibility for peptide migration withinthis spot area, the tryptic spot size can also be said to determine theresolution of the IMS experiment, although the spatial resolution fromthe actual data acquisition is determined by the instrument used and isusually lower. Organic matrices such as DHB and CHCA are used forionization.

For biomarker studies, the tissues available through biobanks aroundthe world have generally been treated with formalin for increased tissuestability over time. Formalin fixation and subsequent paraffin embeddingallows for stable histomorphology, but it also causes difficulty in IMS sinceit cross-links proteins and hampers protein mining. This problem has beenovercome by deparaffinization methods followed by the same antigen-retrieval methods used in IHC experiments (enzymatic or heat-mediated)(Aoki et al., 2007). Recently formalin-fixed, paraffin-embedded tissuemicroarrays were analyzed in MALDI-IMS and MS/MS experiments tostudy the gastric carcinoma tissue, thereby identifying the histone (H4)-specific signal in poorly differentiated cancer tissue samples (Morita et al.,2010). Other groups have demonstrated the direct analysis and identifi-cation of tryptically digested proteins from tissue samples of lung cancer(Groseclose et al., 2008), breast cancer (Ronci et al., 2008), prostate cancer(Cazares et al., 2009), and pancreatic adenocarcinoma (Djidja et al., 2009).Chaurand et al. (2004) showed the level of the binding protein (S100B)in tissue samples using MALDI imaging to distinguish a high-grade andlow-grade glioma. In addition, the combined approach of MALDI-IMSand MS/MS analyses of digested myelin basic protein (MBP) in a coro-nal section of rat brain has been demonstrated (Figure 17a–c) (Grosecloseet al., 2007). After digestion, a total of eight tryptic peptides from MBPwere detected (Figure 17d). This protein is essential for the formation ofmyelin in the central nervous system. MALDI-IMS also has been used toclassify a pancreatic cancer tissue microarray where a number of proteinsthat appear to discriminate between different tumor classes were detected(Djidja et al., 2009). Direct proteomic-based imaging was also performed


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High

Low

High

(d)

(a) (b) (c)

m/z 699.47(NIVTPR)

m/z 726.46(HGFLPR)

m/z 1067.60(FSWGGRDSR)

m/z 1909.92(FSWGGRDSRSGSPMARR)

m/z 1131.84(TTHYGSLPQK)

m/z 1336.69(YLATASTMDHAR)

m/z 1460.90(TQDENPVVHFFK)

m/z 1502.98(TTHYGSLPQKSQR)

Low

FIGURE 17 (a) H&E stain of rat brain tissue section serial to the sections used fordigestion and imaging. (b) Tissue section spotted with a sinapinic acid matrix solution.(c) Image of the 14.2-kDa isoform of myelin basic protein. (d) Images of 8-trypticpeptides generated from the digestion of the 14.2-kDa isoform of myelin basic protein.Reprinted from Groseclose et al. (2007) with permission from John Wiley and Sons.

on a gene knockout mice tissue section of rat that could be useful for thediagnosis of human diseases (Yao et al., 2008). Figure 18 shows the PCAof mass spectra from Scrapper-knockout (SCR-KO) and WT mouse brainsanalyzed by MALDI-IMS.

7.3. IMS for Pharmacokinetic Studies

Imaging of pharmaceuticals samples is performed to examine pharmaco-kinetics—that is, the absorption, distribution, metabolism, and excre-tion of drugs in laboratory animals and humans. HPLC combined withMS/MS is used to analyze and characterizze most drugs. However,HPLC-MS/MS analyses cannot provide the distribution of drugs in differ-ent organs or tissues of laboratory animal experiments (Hsieh et al., 2003).Whole-body autoradiography (WBA) is normally used for the visualiza-tion of drug candidates in all tissues; however, it requires the compoundof interest to be radioactively labeled (Kertesz et al., 2008). This disad-vantage of WBA can be overcome by using MALDI-IMS to analyze thedrugs in tissue samples. The drug distribution profile obtained by IMStells whether the oral administration of an exogenous compound affectsthe endogenous metabolites (Rubakhin et al., 2005). Reyzer et al. (2003)reported images of two antitumor drugs in mouse tissue samples using


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WT

SCR-KO

(a)

(c)

(b)

Cerebral cortex hypothalamus

Cerebral corteWT

HypothalamusWT

Cerebral corteSCR-KO

HypothalamusSCR-KOm/z

m/z 7420 m/z 7109

m/z 7420

m/z 5004

m/z 5004

m/z 4285

m/z 7109

m/z 4285

100% 0%

100% 0% 100% 0%

100% 0%

2500 5000 7500 10000Corpus striatum pons/medullary

Corpus striatum8 15

−15

0.3

−15

−12 −0.8

−1

−20

−1 0.6

0.4

−0.6

10

0.8

10

10

−20

0.8

−0.8

10

−8−0.6

0.8

−15 10−0.8 0.4

25−15

−0.8 0.8

PC 1

PC 1

PC 1

PC 1

PC 2

Load 1

Load 1

Load 1

Load 1

Load 2

Load 2 Load 2

Load 2

PC 2

PC 2 PC 2

WT

WT

SCR-KO

SCR-KO

Pons/medullary

Cerebral cortex Hypothalamus

Rel

ativ

e in

tens

ity

FIGURE 18 In situ proteomics of the SCR-KO mouse brain using IMS and PCA.(a) H&E-stained images of the WT and SCR-KO mouse brain. The regions focused inIMS analyses are indicated by colors. (b) Mass spectra obtained from each region of theWT or SCR-KO mouse brain sections. Specific signals of the regions are indicated byarrowheads. (c) Distributions of principal component scores of mass spectra fromvarious brain regions (left spray graphs; WT, blue; KO, red) and the loading factorsplot (right graphs). The signal intensities of mass spectra of the substances with indicatedm/z are shown in the reconstructed images of the mouse brain analyzed by IMS.Reprinted from Yao et al. (2008) with permission from John Wiley and Sons.


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MALDI-IMS. The results showed the spatial distributions of drugs inbrain tissue section were elucidated using a Q-TOF instrument operated inselective reaction monitoring (SRM) mode to provide good sensitivity fortissue analysis. This work demonstrated the proof of MALDI-IMS in mon-itoring a drug distribution in different parts of body organs. MALDI-IMScan provide the spatial information for both drugs and their metabo-lites. Figure 19a–d shows the distributions of the drug olanzapine and itsmetabolites (N-desmethyl metabolite and 2-hydroxymethyl) in tissue afterpost dosing of 2 hours and 6 hours (Khatib-Shahidi et al., 2006). Further,

Fur(a)

(b)

(c)

(d)

Kidney Liver Lung Spinal cord Brain

Testis Bladder Spleen Thoraciccavitv

Thymus

0 100%

0 100%

0 100%

FIGURE 19 Detection of drug and metabolite distribution at 2 hours after dosing in awhole rat sagittal tissue section using IMS analysis. (a) Optical image of a 2-hr postolanzapine-dosed rat tissue section across four gold MALDI target plates Organs areoutlined in red. A pink dot used as a time point label. (b) MS/MS ion image ofolanzapine (m/z 256). (c) MS/MS ion image of N-desmethyl metabolite (m/z 256).(d) MS/MS ion image of 2-hydroxymethyl metabolite (m/z 272). Scale bar, 1 cm.Reprinted from Khatib-Shahidi et al. (2006) with permission from American ChemicalSociety.


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SIMS and NIMS were used for imaging of drugs in tissue samples and themass spectrum obtained was free of the matrix-oriented peaks. The directanalysis of clozapine and its metabolites in dosed rat brains has been illus-trated using TOF/TOF mass analyzers (Yanes et al., 2009). NIMS-IMS iscompatible with both ion beam and laser sources available on commercialSIMS and MALDI instruments. In addition, fewer laser shots are requiredper spot compared with the MALDI technique.

7.4. IMS for Metabolomics

Metabolomics is the study of metabolites, including metabolic interme-diates such as lipids, amino acids, organic acids, and small signalingmolecules. Concentration changes of metabolites in tissue samples mightreflect a specific physiological or pathological condition of the organism(Dunn, 2008; Nicholson and Lindon, 2008). Liquid chromatography–mass spectrometry and gas chromatography–mass spectrometry are well-known techniques for metabolite analysis (Griffiths and Wang, 2009;Novotny et al., 2008). Here, the tissue samples are homogenized beforeanalysis and thus it is impossible to assess their actual tissue distri-bution. However, IMS can be directly used to profile a broad rangeof small molecules, including nucleotides, amino acids, proteins, lipids,and carboxylic acids, in tissue samples with their unique distributions.MALDI-IMS has been used for imaging and identification of 13 pri-mary metabolites, such as adenosine monophosphate (AMP), adenosinediphosphate (ADP), adenosine triphosphate (ATP), uridine diphosphate(UDP), or N-acetyl-D-glucosamine (GlcNAc) in rat brain sections (Benab-dellah et al., 2009). The distribution pattern of lipids such as cholesterol,cholesterol sulfate, vitamin E, and glycosphingolipids in skin and kidneysections of patients with Fabry disease using the combined approaches ofMALDI-TOF and cluster-TOF-SIMS was demonstrated by Touboul et al.(2007). The MALDI-based imaging technique was also used to visualizeenergy metabolism in the mouse hippocampus via imaging of energy-related metabolites. Cellular metabolic processes use ATP as an energysource and converting it into ADP or AMP. Thus the imaging of thesemolecules in tissue samples can provide useful information about energyproduction and how it can be used in the function of tissue (Sugiura et al.,2011). The phenomenon of energy metabolism is shown in Figure 20.

Metabolomics studies of plants have also been performed to elucidatethe structure, function, and biosynthetic pathways (Lisec et al., 2006). Car-bohydrates, amino acids, vitamins, hormones, flavonoids, phenolics, andglucosinolates are the main metabolites found in plants and are neededfor growth, stress adaptation, and defense (Hounsome et al., 2008). Incombination with soft ionization methods such as ESI and MALDI, MSproved useful for direct analysis of plant tissue sections. The spatialdistribution of sugars, metabolites, and lipids in plant tissue samples wasinvestigated using MALDI-IMS. Cha et al. (2009) exploited the use of


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ATP

Con

trol

Sei

zure

(30

min

)

ADP AMP

ATP

ADP

AMP

Succinyl AMP

IMP

Inosine

Adenosine

Hypoxanthine

Adenine

ATP

nmol

/g ti

ssue 120

0

40Sham

Seizure (30 min)

80

0

200

400

0

1000

2000**

**

ADP AMP

Energy chargeHigh

LowControl Seizure

CA1

CA3

(a)

(b)

(e)

(d)

(c)

Log

((in

t.(sh

am/K

A))

Incr

ease

Dec

reas

e

−2

−1

0

Incr

ease

Dec

reas

e

−2

−1

0

Incr

ease

Dec

reas

e

−2

−1

0

**** **

* *

ATP

Entire region

CA3 cell layer

CA1 cell layerDG cell layer

ADP AMP

FIGURE 20 (Continued)


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FIGURE 20 CA3 cell-selective consumption of adenosine triphosphate (ATP) andadenosine diphosphate (ADP) during a kainate-induced seizure. (a) MALDI imaging ofadenosine nucleotides in a mouse hippocampus. (b) Absolute quantification of ATP, ADP,and adenosine monophosphate (AMP) in a mouse cerebrum using CE-MS. Massivereductions in the levels of ATP and ADP, but not AMP, were observed duringkainate-induced seizures. (c) Results of the relative quantification of ion intensity forATP, ADP, and AMP calculated from the averaged mass spectra of each hippocampalsubregion obtained using MALDI imaging. The values shown are logarithmic ratios of ionintensities between sham-operated (sham) and kainate-treated mice (KA). (d) Mapping ofenergy-charge index values on tissue sections. The region-specific reduction of thesevalues in the CA3 region (arrows) suggests massive energy metabolism in CA3 neurons.(e) Relative quantitative comparison of adenosine nucleotides and related metabolitesusing CE-MS. Each result is mapped on the metabolic pathway and clearly shows thedepletion of ATP and ADP due tp their conversion into downstream metabolites. Thecolored graphs indicate significant increases (orange) and decreases (blue). IMP, inosine5′-monophosphate. Reprinted from Sugiura et al. (2011) with permission from PublicLibrary of Science.

colloidal silver NPs for direct profiling of an epicuticular wax on leavesand flowers from Arabidopsis thaliana in LDI-IMS. Recently, Goto-Inoueet al. (2010b) illustrated the spatial distribution of gamma-aminobutyricacid (GABA) in the seed of eggplant and the presence of GABA wasconfirmed by MS/MS analysis. The localization of GABA in eggplant isshown in Figure 21. Zhang et al. (2007) showed imaging and identificationof fatty acids, sugars, and other small metabolites using colloidal graphiteNPs in GALDI-IMS, which was free from matrix background noise in thelow molecular region. The distribution of lysophosphatidylcholine andPC in rice endosperm and bran and alpha-tocopherol in the germ has alsobeen reported (Zaima et al., 2010).

8. SUMMARY

Several advances in sample preparation, ionization, and MS instrumenta-tion have been achieved, steadily improving sensitivity, spatial resolution,and identification capabilities for MALDI-IMS. These improvements arebroadening the MS imaging applications for lipid, peptide, and proteinbiomarker identification, as well as drug and metabolite imaging. Nano-PALDI, the use of ionic matrices, and the mass sicroscope techniquesare new developments that could be powerful tools in obtaining high-resolution images for biomolecular distribution in biological samples. Inthe future, MALDI-IMS has the potential to become a routine tool forimaging of tissues, helping us to understand the link between the localiza-tion of certain molecules and their function during pathogenesis, diseaseprogression, or treatment.


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(a)

(d)

GABA (standard)

104.0

×10058.1

100

030 40 50 60 70 80 90

(m/z)100 110 120130 140

Rel

ativ

e in

tens

ity (

%)

(e)

58.0

×100

30 40 50 60 70 80 90(m/z)

100 110 120 130 140

100 104.0

0Rel

ativ

e in

tens

ity (

%)

m/z 104.0 on tissue

5 cm

3 mm

(b)

High-power field ofred rectangle area

100%

0%2.5 mm

(c)

100%

0%

Optical image ofeggplant section m/z 104.07

0.5 mm

m/z 104.07Optical image ofeggplant section

FIGURE 21 Optical images of eggplant, the results of IMS and tandem mass analyses.(a) Optical images of eggplant, vertically cut eggplant, and round-cut eggplant. A greyrectangle in a round-cut image shows the region of analyses by IMS. (b) Optical image ofeggplant section and ion image of the m/z values at 104.07. The red arrows in the opticalimage show seed locations. Scale bar: 2.5 mm. Reproducibility was confirmed (n = 3).(c) Optical image of eggplant section and ion image of the m/z values at 104.07 withhigher spatial resolution at 25 µm on a seed. Scale bar: 0.5 mm. (d) The tandem massspectrum of standard gamma-aminobutyric acid (GABA) and (e) m/z 104.0 on eggplanttissue. Reprinted from Goto-Inoue et al. (2010b) with permission from The Japan Societyfor Analytical Chemistry.

ACKNOWLEDGMENTS

We thank the Japanese Society for the Promotion of Science, Japan,for a postdoctoral fellowship (to K.S.). This work was also supportedby a grant-in-aid for SENTAN from the Japan Science and Technology


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Agency (to M.S.). Cecilia Eriksson (Medical Mass Spectrometry, UppsalaUniversity) is acknowledged for assistance in developing this chapter.

REFERENCES

Adachi, J., Kumar, C., Zhang, Y., Olsen, J. V., & Mann, M. (2006). The human urinaryproteome contains more than 1500 proteins, including a large proportion of membraneproteins. Genome Biology, 7, R80.

Aerni, H. R., Cornett, D. S., & Caprioli, R. M. (2006). Automated acoustic matrix depositionfor MALDI sample preparation. Analytical Chemistry, 78, 827–834.

Altelaar, A. F. M., Luxembourg, S. L., McDonnell, L. A., Piersma, S. R., & Heeren, R. M. (2007).Imaging mass spectrometry at cellular length scales. Nature Protocol, 2, 1185–1196.

Ametamey, S. M., Honer, M., & Schubiger, P. A. (2008). Molecular imaging with PET. ChemicalReview, 108, 1501–1516.

Aoki, Y., Toyama, A., Shimada, T., Sugita, T., Aoki, C., Umino, Y., . . . Sato, T. A.(2007). A novel method for analyzing formalin-fixed paraffin-embedded (FFPE) tissuesections by mass spectrometry imaging. Proceedings of the Japan Academy, Series B, 83,205–214.

Armstrong, D. W., Li-Kang, Z., He, L., & Gross, M. L. (2001). Ionic liquids as matrixes formatrix-assisted laser desorption/ionization mass spectrometry. Analytical Chemistry, 73,3679–3686.

Astigarraga, E., Barreda-Gomez, G., Lombardero, L., Fresnedo, O., Castano, F., Giralt,M. T., . . . Fernandez, J. A. (2008). Profiling and imaging of lipids on brain andliver tissue by matrix-assisted laser desorption/ionization mass spectrometry using2-mercaptobenzothiazole as a matrix. Analytical Chemistry, 80, 9105–9114.

Baluya, D. L., Garrett, T. J., & Yost, R. A. (2007). Automated MALDI matrix depositionmethod with inkjet printing for imaging mass spectrometry. Analytical Chemistry, 79,6862–6867.

Benabdellah, F., Touboul, D., Brunelle, A., & Laprevote, O. (2009). In situ primary metabo-lites localization on a rat brain section by chemical mass spectrometry imaging. AnalyticalChemistry, 81, 5557–5560.

Benninghoven, A. (1973). Surface investigation of solids by the statical method of secondaryion mass spectroscopy (SIMS). Surface Science, 35, 427–457.

Brown, H. A. (2007). Lipidomics and Bioactive Lipids: Mass-Spectrometry–Based Lipid Analysis(Methods in Enzymology, Vol. 432). Academic Press, Boston.

Bruker Daltonics GmbH. Bremen, Germany. Retrived from http://www.bdal.com GmbH.Bunch, J., Clench, M. R., & Richards, D. S. (2004). Determination of pharmaceutical com-

pounds in skin by imaging matrix-assisted laser desorption/ionization mass spectrome-try. Rapid Communications for Mass Spectrometry, 18, 3051–3060.

Caprioli, R. M., Farmer, T. B., & Gile, J. (1997). Molecular imaging of biological samples:Localization of peptides and proteins using MALDI-TOF-MS. Analytical Chemistry, 69,4751–4760.

Cazares, L. H., Troyer, D., Mendrinos, S., Lance, R. A., Nyalwidhe, J. O., Beydoun, H. A., . . .Semmes, O. J. (2009). Imaging mass spectrometry of a specific fragment of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 2 discrimi-nates cancer from uninvolved prostate tissue. Clinical Cancer Research, 15, 5541–5551.

Cha, S., Song, Z., Nikolau, B. J., & Yeung, E. S. (2009). Direct profiling and imaging of epicu-ticular waxes on Arabidopsis thaliana by laser desorption/ionization mass spectrometryusing silver colloid as a matrix. Analytical Chemistry, 81, 2991–3000.

http://www.bdal.com


Hawkes 171 09-ch04-145-194-9780123942975 2012/4/17 15:35 Page 185 #41


Cha, S., & Yeung, E. S. (2007). Colloidal graphite-assisted laser desorption/ionization massspectrometry and MSn of small molecules. 1. Imaging of cerebrosides directly from ratbrain tissue. Analytical Chemistry, 79, 2373–2385.

Chana, K., Lanthiera, P., Liua, X., Sandhua, J. K., Stanimirovica, D., & Li, J. (2009). MALDImass spectrometry imaging of gangliosides in mouse brain using ionic liquid matrix.Analytica Chimica Acta, 639, 57–61.

Chaurand, P., Latham, J. C., Lane, K. B., Mobley, J. A., Polosukhin, V. V., Wirth, P. S., . . .Caprioli, R. M. (2008). Imaging mass spectrometry of intact proteins from alcohol-preserved tissue specimens: Bypassing formalin fixation. Journal of Proteome Research, 7,3543–3555.

Chaurand, P., Norris, J. L., Cornett, D. S., Mobley, J. A., & Caprioli, R. M. (2006). Newdevelopments in profiling and imaging of proteins from tissue sections by MALDI massspectrometry. Journal of Proteome Research, 5, 2889–2900.

Chaurand, P., Sanders, M. E., Jensen, R. A., & Caprioli, R. M. (2004). Proteomics in diagnosticpathology profiling and imaging proteins directly in tissue sections. American Journal ofPathology, 165, 1057–1068.

Chaurand, P., Schriver, K. E., & Caprioli, R. M. (2007). Instrument design and charac-terization for high resolution MALDI-MS imaging of tissue sections. Journal of MassSpectrometry, 42, 476–489.

Chen, Y., Allegood, J., Liu, Y., Wang, E., Cachon-Gonzalez, B., Cox T. M., . . . Sullards, M. C.(2008). Imaging MALDI mass spectrometry using an oscillating capillary nebulizer matrixcoating system and its application to analysis of lipids in brain from a mouse model ofTay-Sachs/Sandhoff disease. Analytical Chemistry, 80, 2780–2788.

Chen, R., Hui, L., Sturm, R. M., & Li, L. (2009). Three dimensional mapping of neuropeptidesand lipids in crustacean brain by mass spectral imaging. Journal of the American Society forMass Spectrometry, 20, 1068–1077.

Chou, Y. L. (1975). Statistical Analysis, with Business and Economic Applications (p. 17.9). Holt,Rinehart and Winston, New York.

Colliver, T. L., Brummel, C. L., Pacholski, M. L., Swanek, F. D., Ewing, A. G., & Winograd, N.(1997). Atomic and molecular imaging at the single-cell level with TOF-SIMS. AnalyticalChemistry, 69, 2225–2231.

Cornett, D. S., Frappier, S. L., & Caprioli, R. M. (2008). MALDI-FTICR imaging massspectrometry of drugs and metabolites in tissue. Analytical Chemistry, 80, 5648–5653.

Cottrell, J. S., & Greathead, R. J. (1986). Extending the mass range of a sector massspectrometer. Mass Spectrometry Reviews, 5, 215–247.

Deininger, S. O., Ebert, M. P., Futterer, A., Gerhard, M., & Rocken, C. (2008). MALDI imagingcombined with hierarchical clustering as a new tool for the interpretation of complexhuman cancers. Journal of Proteomic Research, 7, 5230–5236.

Dill, A. L., Ifa, D. R., Manicke, N. E., Ouyang, Z., & Cooks, R. G. (2009). Mass spectrometricimaging of lipids using desorption electrospray ionization. Journal of Chromatography B,Analytical Technologies for the Biomedical and Life Sciences, 877, 2883–2889.

Djidja, M. C., Claude, E., Snel, M. F., Francese, S., Scriven, P., Carolan, V., & Clench, M. R.(2010). Novel molecular tumour classification using MALDI–mass spectrometry imagingof tissue micro-array. Analytical and Bioanalytical Chemistry, 397, 587–601.

Djidja, M. C., Claude, E., Snel, M. F., Scriven, P., Francese, S., Carolan, V., & Clench, M. R.(2009). MALDI-ion mobility separation-mass spectrometry imaging of glucose-regulatedprotein 78 kDa (Grp78) in human formalin-fixed, paraffin-embedded pancreaticadenocarcinoma tissue sections. Journal of Proteome Research, 8, 4876–4884.

Douglas, D. J., Frank, A. J., & Mao, D. (2005). Linear ion traps in mass spectrometry. MassSpectrometry Reviews, 24, 1–29.

Dreisewerd, K. (2003). The desorption process in MALDI. Chemical Reviews, 103, 395–426.


Hawkes 171 09-ch04-145-194-9780123942975 2012/4/17 15:35 Page 186 #42


Dunn, W. B. (2008). Current trends and future requirements for the mass spectrometricinvestigation of microbial, mammalian and plant metabolomes. Physical Biology, 5,11001.

Eibisch, M., & Schiller, J. (2011). Sphingomyelin is more sensitively detectable as a neg-ative ion than phosphatidylcholine: A matrix-assisted laser desorption/ionizationtime-of-flight mass spectrometric study using 9-aminoacridine (9-AA) as matrix. RapidCommunications in Mass Spectrometry, 25, 1100–1106.

Enomoto, H., Sugiura, Y., Setou, M., & Zaima, N. (2011). Visualization of phosphatidyl-choline, lysophosphatidylcholine and sphingomyelin in mouse tongue body bymatrix-assisted laser desorption/ionization imaging mass spectrometry. Analyticaland Bioanalytical Chemistry, 400, 1913–1921.

Estrada, R., & Yappert, M. C. (2004). Alternative approaches for the detection of variousphospholipid classes by matrix-assisted laser desorption/ionization time-of-flight massspectrometry. Journal of Mass Spectrometry, 39, 412–422.

Fahy, E., Subramaniam, S., Murphy, R., Nishijima, M., Raetz, C., Shimizu, T., . . .Dennis, E. A.(2009). Update of the LIPID MAPS comprehensive classification system for lipids. Journalof Lipid Research, 50, S9–S14.

Fales, H. M., Milne, G. W., Pisano, J. J., Brewer, H. B., Blum, M. S., MacConnell, J. G., . . .Law, N. (1972). Biological applications of electron ionization and chemical ionizationmass spectrometry. Recent Progress in Hormone Research, 28, 591–626.

Fenn, J. B., Mann, M., Meng, C. K., Wong, S. F., & Whitehouse, C. M. (1989). Electrosprayionization for mass spectrometry of large biomolecules. Science, 246, 64–71.

Fernandez, J. A., Ochoa, B., Fresnedo, O., Giralt, M. T., & Rodriguez-Puertas, R. (2011).Matrix-assisted laser desorption ionization imaging mass spectrometry in lipidomics.Analytical and Bioanalytical Chemistry, 401, 29–51.

Fournier, I., Marinach, C., Tabet, J. C., & Bolbach, G. (2003). Irradiation effects inMALDI, ablation, ion production, and surface modifications. PART II: 2,5-dihydroxy-benzoic acid monocrystals. Journal of the American Society for Mass Spectrometry, 14,893–899.

Fuchs, B., Schiller, J., & Cross, M. A. (2007). Apoptosis-associated changes in the glyc-erophospholipid composition of hematopoietic progenitor cells monitored by 31P NMRspectroscopy and MALDI-TOF mass spectrometry. Chemistry and Physics of Lipids, 150,229–238.

Fuchs, B., Schiller, J., Wagner, U., Hantzschel, H., & Arnold, K. (2005). The phosphatidyl-choline/lysophosphatidylcholine ratio in human plasma is an indicator of the severityof rheumatoid arthritis: Investigations by 31P NMR and MALDI-TOF MS. ClinicalBiochemistry, 38, 925–933.

Fuchs, B., Sus, R., & Schiller, J. (2010). An update of MALDI-TOF mass spectrometry in lipidresearch. Progress in Lipid Research, 49, 450–475.

Garrett, T. J., Prieto-Conaway, M. C., Kovtoun, V., Bui, H., Izgarian N., Stafford, G., &Yost, R. A. (2007). Imaging of small molecules in tissue sections with a new intermediate-pressure MALDI linear ion trap mass spectrometer. International Journal of MassSpectrometry, 260, 166–176.

Goodwin, R. J. A., Pennington, S. R., & Pitt, A. R. (2008). Protein and peptides in pictures:Imaging with MALDI mass spectrometry. Proteomics, 8, 3785–3800.

Goto-Inoue, N., Hayasaka, T., Takib, T., Gonzalez, T. V., & Setou, M. (2009a). Newlipidomics approaches by thin-layer chromatography-blot-matrix-assisted laser des-orption/ionization imaging mass spectrometry for analyzing detailed patterns ofphospholipid molecular species. Journal of Chromatography A, 1216, 7096–7101.

Goto-Inoue, N., Hayasaka, T., Zaima, N., Kashiwagi, Y., Yamamoto, M., Nakamoto, M., &Setou, M. (2010a). The detection of glycosphingolipids in brain tissue sections by imagingmass spectrometry using gold nanoparticles. Journal of the American Society for MassSpectrometry, 21, 1940–1943.


Hawkes 171 09-ch04-145-194-9780123942975 2012/4/17 15:35 Page 187 #43


Goto-Inoue, N., Hayasaka, T., Zaima, N., & Setou, M. (2009b). The specific localization ofseminolipid molecular species on mouse testis during testicular maturation revealed byimaging mass spectrometry. Glycobiology, 19, 950–957.

Goto-Inoue, N., Hayasaka, T., Zaima, N., & Setou, M. (2011). Imaging mass spectrometry forlipidomics. Biochimica et Biophysica Acta, 1811, 961–969.

Goto-Inoue, N., Setou, M., & Zaima, N. (2010b). Visualization of spatial distributionof γ -aminobutyric acid in eggplant (Solanum melongena) by matrix-assisted laserdesorption/ionization imaging mass spectrometry. Analytical Sciences, 26, 821–825.

Griffiths, W. J., & Wang, Y. (2009). Mass spectrometry: From proteomics to metabolomicsand lipidomics. Chemical Society Reviews, 38, 1882–1896.

Groseclose, M. R., Andersson, M., Hardesty, W. M., & Caprioli, R. M. (2007). Identificationof proteins directly from tissue: In situ tryptic digestions coupled with imaging massspectrometry. Journal Mass Spectrometry, 42, 254–262.

Groseclose, M. R., Massion, P. P., Chaurand, P., & Caprioli, R. M. (2008). High-throughputproteomic analysis of formalin-fixed paraffin-embedded tissue microarrays using MALDIimaging mass spectrometry. Proteomics, 8, 3715–3724.

Gross, J. H. (2004). Mass Spectrometry. Springer-Verlag, Berlin.Han, X., Holtzman, D. M., & McKeel, D. W., Jr. (2001). Plasmalogen deficiency in early

Alzheimer’s disease subjects and in animal models: Molecular characterization usingelectrospray ionization mass spectrometry. Journal of Neurochemistry, 77, 1168–1180.

Han, X., Holtzman, D. M., McKeel, D. W., Jr., Kelley, J., & Morris, J. C. (2002). Substantialsulfatide deficiency and ceramide elevation in very early Alzheimer’s disease: Potentialrole in disease pathogenesis. Journal of Neurochemistry, 82, 809–818.

Han, X., Yang, J., Yang, K., Zhao, Z., Abendschein, D. R., & Gross, R. W. (2007). Alterationsin myocardial cardiolipin content and composition occur at the very earliest stages ofdiabetes: A shotgun lipidomics study. Biochemistry, 46, 6417–6428.

Hankin, J. A., Barkley, R. M., & Murphy, R. C. (2007). Sublimation as a method of matrixapplication for mass spectrometric imaging. Journal of the American Society for MassSpectrometry, 18, 1646–1652.

Harada, T., Yuba-Kubo, A., Sugiura, Y., Zaima, N., Hayasaka, T., Goto-Inoue, N., . . .Setou, M. (2009). Visualization of volatile substances in different organelles with anatmospheric-pressure mass microscope. Analytical Chemistry, 81, 9153–9157.

Hayasaka, T., Goto-Inoue, N., Sugiura, Y., Zaima, N., Nakanishi, H., Ohishi K., . . .Setou, M. (2008). Matrix-assisted laser desorption/ionization quadrupole ion traptime-of-flight (MALDI-QIT-TOF)-based imaging mass spectrometry reveals a layereddistribution of phospholipid molecular species in the mouse retina. Rapid Communicationsin Mass Spectrometry, 22, 3415–3426.

Hayasaka, T., Goto-Inoue, N., Zaima, N., Kimura, Y., & Setou, M. (2009). Organ-specificdistributions of lysophosphatidylcholine and triacylglycerol in mouse embryo. Lipids, 44,837–848.

Hayasaka, T., Goto-Inoue, N., Zaima, N., Shrivas, K., Kashiwagi, Y., Yamamoto, M., . . .Setou, M. (2010). Imaging mass spectrometry with silver nanoparticles reveals thedistribution of fatty acids in mouse retinal sections. Journal of the American Society for MassSpectrometry, 21, 1446–1454.

He, X., Chen, F., McGovern, M. M., & Schuchman, E. H. (2002). A fluorescence-based,high-throughput sphingomyelin assay for the analysis of Niemann-Pick disease andother disorders of sphingomyelin metabolism. Analytical Biochemistry, 306, 115–123.

Heeren, R. M. A., McDonnell, L. A., Amstalden, E., Luxembourg, S. L., Altelaar, A. F. M.,& Piersma, S. R. (2006). Why don’t biologists use SIMS? A critical evaluation of imagingMS. Applied Surface Science, 252, 6827–6835.

Herring, K. D., Oppenheimer, S. R., & Caprioli, R. M. (2007). Direct tissue analysis bymatrixassisted laser desorption ionization mass spectrometry: application to kidneybiology. Seminars Nephrology, 27, 597–608.


Hawkes 171 09-ch04-145-194-9780123942975 2012/4/17 15:35 Page 188 #44


Hiltunen, Y., Kaartinen, J., Pulkkinen, J., Hakkinen, A. M., Lundbom, N., & Kauppinen,R. A. (2002). Quantification of Human Brain Metabolites from in Vivo 1H NMR Magni-tude Spectra Using Automated Artificial Neural Network Analysis. Journal of MagneticResonance, 154, 1–5.

Hopfgartner, G., Varesio, E., & Stoeckli, M. (2009). Matrix-assisted laser desorption/ionization mass spectrometric imaging of complete rat sections using a triple quadrupolelinear ion trap. Rapid Communications in Mass Spectrometry, 23, 733–736.

Hopfgartner, G., Varesio, E., Tschappat, V., Grivet, C., Bourgogne, E., & Leuthold, L. A.(2004). Triple quadrupole linear ion trap mass spectrometer for the analysis of smallmolecules and macromolecules. Journal of Mass Spectrometry, 39, 845–855.

Hounsome, N., Hounsome, B., Tomos, D., & Edwards-Jones, G. (2008). Plant metabolitesand nutritional quality of vegetables. Journal of Food Science, 73, R48–R65.

Hsieh, Y., Wang, G., Wang, Y., Chackalamannil, S., & Korfmacher, W. A. (2003). Directplasma analysis of drug compounds using monolithic column liquid chromatographyand tandem mass spectrometry. Analytical Chemistry, 75, 1812–1818.

Huang, R., Zhang, B., Zou, D., Hang, W., He, J., & Huang, B. (2011). Elemental imagingvia laser ionization orthogonal time-of-flight mass spectrometry. Analytical Chemistry, 83,1102–1107.

Hurd, E., & Freeman, D. M. (1989). Metabolite specific proton magnetic resonance imaging.Proceedings of the National Academy of Sciences USA, 86, 4402–4406.

Jackson, S. N., Wang, H. Y., & Woods, A. S. (2005). In situ structural characterization ofphosphatidylcholines in brain tissue using MALDI-MS/MS. Journal of the American Societyfor Mass Spectrometry, 16, 2052–2056.

Jackson, S. N., Woods, A. S. (2009). Direct profiling of tissue lipids by MALDI-TOFMS,Journal of Chromatography B, 877, 2822–2829.

Jones, E. A., Lockyera, N. P., & Vickerman, J. C. (2007). Mass spectral analysis and imagingof tissue by ToF-SIMS–the role of buckminsterfullerene, C+60, primary ions. InternationalJournal of Mass Spectrometry, 260, 146–157.

Karas, M., Bachmann, D., & Hillenkamp, F. (1985). Influence of the wavelength in highirradiance ultraviolet laser desorption mass spectrometry of organic molecules. AnalyticalChemistry, 57, 2935–2939.

Kertesz, V., Van Berkel, G. J., Vavrek, M., Koeplinger, K. A., Schneider, B. B., & Covey, T. R.(2008). Comparison of drug distribution images from whole-body thin tissue sectionsobtained using desorption electrospray ionization tandem mass spectrometry andautoradiography. Analytical Chemistry, 80, 5168–5177.

Khatib-Shahidi, S., Andersson, M., Herman, J., Gillespie, T., & Caprioli, R. (2006). Directmolecular analysis of whole-body animal tissue sections by imaging MALDI massspectrometry. Analytical Chemistry, 78, 6448–6456.

Kobayashi, Y., Hayasaka, T., Setou, M., Itoh, H., & Kanayama, N. (2010). Comparison ofphospholipid molecular species between terminal and stem villi of Human term placentaby imaging mass spectrometry. Placenta, 31, 245–248.

Landgraf, R. R., Conaway, M. C. P., Garrett, T. J., Stacpoole, P. W., & Yost, R. A. (2009).Imaging of lipids in spinal cord using intermediate pressure MALDI-LIT/Orbitrap MS.Analytical Chemistry, 81, 8488–8495.

Lane, A. L., Nyadong, L., Galhena, A. S., Shearer, T. L., Stout, E. P., Parry, R. M., . . .Kubanek, J.(2009). Desorption electrospray ionization mass spectrometry reveals surface-mediatedantifungal chemical defense of tropical seaweed. Proceedings of the National Academy ofSciences USA, 106, 7314–7319.

Laremore, T. N., Zhang, F., & Linhardt, R. J. (2007). Ionic liquid matrix for direct UV-MALDI-TOF-MS analysis of dermatan sulfate and chondroitin sulfate oligosaccharides. AnalyticalChemistry, 79, 1604–1610.

Lee, S. H., Williams, M. V., DuBois, R. N., & Blair, I. A. (2003). Targeted lipidomics usingelectron capture atmospheric pressure chemical ionization mass spectrometry. RapidCommunication in Mass Spectrometry, 17, 2168–2176.


Hawkes 171 09-ch04-145-194-9780123942975 2012/4/17 15:35 Page 189 #45


Lemaire, R., Tabet, J. C., Ducoroy, P., Hendra, J. B., Salzet, M., & Fournier, I. (2006a). Solidionic matrixes for direct tissue analysis and MALDI imaging. Analytical Chemistry, 78,809–819.

Lemaire, R., Wisztorski, M., Desmons, A., Tabet, J. C., Day, R., Salzet, M., & Fournier, I.(2006b). MALDI-MS direct tissue analysis of proteins: Improving signal sensitivity usingorganic treatments. Analytical Chemistry, 78, 7145–7153.

Lisec, J., Schauer, N., Kopka, J., Willmitzer, L., & Fernie, A. R. (2006). Gas chromatog-raphy mass spectrometry-based metabolite profiling in plants. Nature Protocols, 1,387–396.

Liu, Y., Chen, Y., Momin, A., Shaner, R., Wang, E., Bowen, N. J., . . .Merrill, A. H., Jr. (2010).Elevation of sulfatides in ovarian cancer: An integrated transcriptomic and lipidomicanalysis including tissue-imaging mass spectrometry. Molecular Cancer, 9, 186.

Luongo de Matos, L., Trufelli D. C., Luongo de Matos, M. G., & da Silva Pinhal, M. A. (2010).Immunohistochemistry as an important tool in biomarkers detection and clinical practice.Biomarker Insights, 5, 9–20.

MacAleese, L., Stauber, J., & Heeren, R. M. A. (2009). Perspectives for imaging massspectrometry in the proteomics landscape. Proteomics, 9, 819–834.

Makarov, A. A., Denisov, E., Lange, O., & Horning, S. (2006). Dynamic range of massaccuracy in LTQ orbitrap hybrid mass spectrometer. Journal of the American Society forMass Spectrometry, 17, 977–982.

Manicke, N. E., Dill, A. L., Ifa, D. R., & Cooks, R. G. (2010). High resolution tissue imaging onan orbitrap mass spectrometer by desorption electro-spray ionization mass spectrometry(DESI-MS). Journal of Mass Spectrometry, 45, 223–226.

Marsching, C., Eckhardt, M., Grone, H. J., Sandhoff, R., & Hopf, C. (2011). Imaging ofcomplex sulfatides SM3 and SB1a in mouse kidney using MALDI-TOF/TOF massspectrometry. Analytical and Bioanalytical Chemistry, 401, 53–64.

Matsumoto, J., Sugiura, Y., Yuki, D., Hayasaka, T., Goto-Inoue, N., Zaima, N., . . .Niwa, S.(2011). Abnormal phospholipids distribution in the prefrontal cortex from apatient with schizophrenia revealed by matrix-assisted laser desorption/ionizationimaging mass spectrometry. Analytical and Bioanalytical Chemistry, 400, 1933–1943.

McCluer, R. H., Ullman, M. D., & Jungalwala, F. B. (1986). HPLC of glycosphingolipids andphospholipids. Advances in Chromatography, 25, 309–353.

McDonnell, L. A., & Heeren, R. M. A. (2007). Imaging mass spectrometry. Mass SpectrometryReviews, 26, 606–643.

Merrill, A. H., Jr., Stokes, T. H., Momin, A., Park, H., Portz, B. J., Kelly, S., . . .Wang, M. D.(2009). Sphingolipidomics: A valuable tool for understanding the roles of sphingolipidsin biology and disease. Journal of Lipid Research, 50, S97–S102.

Morita, Y., Ikegami, K., Goto-Inoue, N., Hayasaka, T., Zaima, N., Tanaka, H., . . .Konno, H. (2010). Imaging mass spectrometry of gastric carcinoma in formalin-fixedparaffin-embedded tissue microarray. Cancer Science, 101, 267–273.

Morris, H. R., Panico, M., Barber, M., Bordoli, R. S., Sedgwick, R. D., & Tyler, A. (1981). Fastatom bombardment: A new mass spectrometric method for peptide sequence analysis.Biochemical and Biophysical Research Communications, 101, 623–631.

Murphy, E. J., Schapiro, M. B., Rapoport, S. I., & Shetty, H. U. (2000). Phospholipidcomposition and levels are altered in Down syndrome brain. Brain Research, 867, 9–18.

Nemes, P., Barton, A. A., Li, Y., & Vertes, A. (2008). Ambient molecular imaging anddepth profiling of liver tissue by infrared laser ablation electrospray ionization massspectrometry. Analytical Chemistry, 80, 4575–4582.

Nemes, P., Barton, A. A., & Vertes, A. (2009). Three-dimensional imaging of metabolitesin tissues under ambient conditions by laser ablation electrospray ionization massspectrometry. Analytical Chemistry, 81, 6668–6675.

Nemes, P., & Vertes, A. (2007). Laser ablation electrospray ionization for atmosphericpressure, in vivo, and imaging mass spectrometry. Analytical Chemistry, 79, 8098–8106.


Hawkes 171 09-ch04-145-194-9780123942975 2012/4/17 15:35 Page 190 #46


Nemes, P., Woods, A. S., & Vertes, A. (2010). Simultaneous imaging of small metabolites andlipids in rat brain tissues at atmospheric pressure by laser ablation electrospray ionizationmass spectrometry. Analytical Chemistry, 82, 982–988.

Nicholson, J. K., & Lindon, J. C. (2008). Systems biology: Metabonomics. Nature, 455,1054–1056.

Northen, T. R., Yanes, O., Northen, M. T., Marrinucci, D., Uritboonthai, W., & Apon, J. (2007).Clathrate nanostructures for mass spectrometry. Nature, 449, 1033–1036.

Novartis, Basel, Switzerland. Retrived from http://www.maldi-msi.org.Novotny, M. V., Soini, H. A., & Mechref, Y. (2008). Biochemical individuality reflected in chro-

matographic, electrophoretic and mass-spectrometric profiles. Journal of Chromatography B,Analytical Technologies for the Biomedical and Life Sciences, 866, 26–47.

Oresic, M., Hanninen V. A., & Vidal-Puig, A. (2008). Lipidomics: A new window tobiomedical frontiers. Trends in Biotechnology, 26, 647–652.

Patti, G. J., Shriver, L. P., Wassif, C. A., Woo, H. K., Uritboonthai, W., Apon, J., . . . Siuzdak, G.(2010). Nanostructure-initiator mass spectrometry (NIMS) imaging of brain cholesterolmetabolites in Smith-Lemli-Opitz syndrome. Neuroscience, 170, 858–864.

Petkovic, M., Schiller, J., Muller, M., Benard, S., Reichl, S., Arnold, K., & Arnhold, J. (2001).Detection of individual phospholipids in lipid mixtures by matrix-assisted laser des-orption/ionization time-of-flight mass spectrometry: Phosphatidylcholine prevents thedetection of further species. Analytical Biochemistry, 289, 202–216.

Piehowski, P. D., Carado, A. J., Kurczy, M. E., Ostrowski, S. G., Heien, M. L., Winograd, N., &Ewing, A. G. (2008). MS/MS methodology to improve subcellular mapping of cholesterolusing TOF-SIMS. Analytical Chemistry, 80, 8662–8667.

Pol, J., Strohalm, M., Havlicek, V., & Volny, M. (2010). Molecular mass spectrometry imagingin biomedical and life science research. Histochemistry and Cell Biology, 134, 423–443.

Pulfer, M., & Murphy, R. C. (2003). Electrospray mass spectrometry of phospholipids. MassSpectrometry Reviews, 22, 332–364.

Puolitaival, S. M., Burnum, K. E., Cornett, D. S., & Caprioli, R. M. (2008). Solvent-free matrixdry-coating for MALDI imaging of phospholipids. Journal of the American Society for MassSpectrometry, 19, 882–886.

Reyzer, M. L., Hsieh, Y., Ng, K., Korfmacher, W. A., & Caprioli, R. M. (2003). Direct anal-ysis of drug candidates in tissue by matrix-assisted laser desorption/ionization massspectrometry. Journal of Mass Spectrometry, 38, 1081–1092.

Ronci, M., Bonanno, E., Colantoni, A., Pieroni, L., Di Ilio, C., Spagnoli, L. G., . . .Urbani, A.(2008). Protein unlocking procedures of formalin-fixed paraffin-embedded tissues:Application to MALDI-TOF imaging MS investigations. Proteomics, 8, 3702–3714.

Rubakhin, S. S., Jurchen, J. C., Monroe, E. B., & Sweedler, J. V. (2005). Imaging mass spectrom-etry: Fundamentals and applications to drug discovery. Drug Discovery Today, 10, 823−837.

Rujoi, M., Estrada, R., & Yappert, M. C. (2004). In situ MALDI-TOF MS regional analysis ofneutral phospholipids in lens tissue. Analytical Chemistry, 76, 1657–1663.

Schiller, J., Arnhold, J., Benard, S., Muller, M., Reichl, S., & Arnold, K. (1999). Lipid analysisby matrix-assisted laser desorption and ionization mass spectrometry: A methodologicalapproach. Analytical Biochemistry, 267, 46–56.

Schmitz, G., & Ruebsaamen, K. (2010). Metabolism and atherogenic disease association oflysophosphatidylcholine. Atherosclerosis, 208, 10–18.

Schwamborn, K., Krieg, R. C., Reska, M., Jakse, G., Knuechel, R., & Wellmann, A. (2007).Identifying prostate carcinoma by MALDI-Imaging. International Journal of MolecularMedicine, 20, 155–159.

Schwartz, S. A., Reyzer, M. L., & Caprioli, R. M. (2003). Direct tissue analysis using matrix-assisted laser desorption/ionization mass spectrometry: Practical aspects of samplepreparation. Journal of Mass Spectrometry, 38, 699–708.

http://www.maldi-msi.org


Hawkes 171 09-ch04-145-194-9780123942975 2012/4/17 15:35 Page 191 #47


Schwartz, J. C., Senko, M. W., & Syka, J. E. P. (2002). A two-dimensional quadrupoleion trap mass spectrometer. Journal of the American Society for Mass Spectrometry, 13,659–669.

Seeley, E. H., Oppenheimer, S. R., Mi, D., Chaurand, P., & Caprioli, R. M. (2008). Enhancementof protein sensitivity for MALDI imaging mass spectrometry after chemical treatment oftissue sections. Journal of the American Society for Mass Spectrometry, 19, 1069–1077.

Setou, M., Hayasaka, T., Shimma, S., Sugiura, Y., & Matsumoto, M. (2008). Protein denat-uration improves enzymatic digestion efficiency for direct tissue analysis using massspectrometry. Applied Surface Science, 255, 1555–1559.

Setou, M., Shrivas, K., Sroyraya, M., Yang, H., Sugiura, Y., Moribe, J., . . .Konishi, Y. (2010).Developments and applications of mass microscopy. Medical Molecular Morphology, 43,1–5.

Shimma, S., Sugiura, Y., Hayasaka, T., Hoshikawa, Y., Noda, T., & Setou, M. (2007). MALDI-based imaging mass spectrometry revealed abnormal distribution of phospholipids incolon cancer liver metastasis. Journal of Chromatography B, Analytical Technologies in theBiomedical and Life Sciences, 855, 98–103.

Shimma, S., Sugiura, Y., Hayasaka, T., Zaima, N., Matsumoto, M., & Setou, M. (2008).Mass imaging and identification of biomolecules with MALDI-QIT-TOF-based system.Analytical Chemistry, 80, 878–885.

Shrestha, B., Nemes, P., Nazarian, J., Hathoutn, Y., Hoffman, E. P., & Vertes, A. (2010). Directanalysis of lipids and small metabolites in mouse brain tissue by AP-IR-MALDI andreactive LAESI mass spectrometry. Analyst, 135, 751–758.

Shrivas, K., Hayasaka, T., Goto-Inoue, N., Sugiura, Y., Zaima, N., & Setou, M. (2010).Ionic matrix for enhanced MALDI imaging mass spectrometry for identification ofphospholipids in mouse liver and cerebellum tissue sections. Analytical Chemistry, 82,8800–8806.

Shrivas, K., Hayasaka, T., Sugiura, Y., & Setou, M. (2011). Method for simultaneouslyimaging of low molecular metabolites in mouse brain using TiO2 nanoparticles in nano-particle assisted laser desorption/ionization mass spectrometry. Analytical Chemistry, 83,7283–7289.

Slaveykova, V. I., Guignard, C., Eybe, T., Migeon, H. N., & Hoffmann, L. (2009). DynamicNanoSIMS ion imaging of unicellular freshwater algae exposed to copper. Analytical andBioanalytical Chemistry, 393, 583–589.

Slodzian, G., Daigne, B., Girard, F., Boust, F., & Hillion, F. (1992). Scanning secondary ionanalytical microscopy with parallel detection. Biology of the Cell, 74, 43–50.

Snel, M. F., & Fuller, M. (2010). High-spatial resolution matrix-assisted laser desorptionionization imaging analysis of glucosylceramide in spleen sections from a mouse modelof Gaucher disease. Analytical Chemistry, 82, 3664–3670.

Sripadi, P., Shrestha, B., Easley, R. L., Carpio, L., Kehn-Hall, K., Chevalier, S., . . .Vertes, A.(2010). Direct detection of diverse metabolic changes in virally transformed andtax-expressing cells by mass spectrometry. PLoS ONE, 5, e12590.

Stauber, J., MacAleese, L., Franck, J., Claude, E., Snel, M., Kaletas, B. K., . . .Heeren, R. M.(2010). On-tissue protein identification and imaging by MALDI-ion mobility massspectrometry. Journal of the American Society for Mass Spectrometry, 21, 338–347.

Stoeckli, M., Chaurand, P., Hallahan, D. E., & Caprioli, R. M. (2001). Imaging mass spec-trometry: A new technology for the analysis of protein expression in mammalian tissues.Nature Medicine, 7, 493–496.

Stuebiger, G., & Belgacem, O. (2007). Analysis of lipids using 2,4,6-trihydroxyacetophenoneas a matrix for MALDI mass spectrometry. Analytical Chemistry, 79, 3206–3213.

Sugiura, Y., Konishi, Y., Zaima, N., Kajihara, S., Nakanishi, H., Taguchi, R., & Setou, M. (2009).Visualization of the cell-selective distribution of PUFA-containing phosphatidylcholinesin mouse brain by imaging mass spectrometry. Journal of Lipid Research, 50, 1776–1788.


Hawkes 171 09-ch04-145-194-9780123942975 2012/4/17 15:35 Page 192 #48


Sugiura, Y., Shimma, S., Konishi, Y., Yamada, M. K., & Setou, M. (2008). Imaging massspectrometry technology and application on ganglioside study; visualization ofage-dependent accumulation of C20-ganglioside molecular species in the mousehippocampus. PLoS One, 3, e3232.

Sugiura, Y., Taguchi, R., & Setou, M. (2011). Visualization of spatiotemporal energy dynamicsof hippocampal neurons by mass spectrometry during a kainate-induced seizure. PLoSOne, 6, e17952.

Sunner, J., Dratz, E., & Chen, Y. C. (1995). Graphite surface-assisted laser desorption/ionization time-of-flight mass spectrometry of peptides and proteins from liquidsolutions. Analytical Chemistry, 67, 4335–4342.

Taban, I. M., Altelaar, A. F., van der Burgt, Y. E., McDonnell, L. A., Heeren, R. M.,Fuchser, J., & Baykut, G. (2007). Imaging of peptides in the rat brain using MALDI-FTICRmass spectrometry. Journal of the American Society for Mass Spectrometry, 18, 152–161.

Taira, S., Sugiura, Y., Moritake, S., Shimma, S., Ichiyanagi, Y., & Setou, M. (2008).Nanoparticle-assisted laser desorption/ionization based mass imaging with cellularresolution. Analytical Chemistry, 80, 4761–4766.

Takats, Z., Wiseman, J. M., Gologan, B., & Cooks, R. G. (2004). Mass spectrometry samplingunder ambient conditions with desorption electrospray ionization. Science, 306, 471–473.

Takizawa, Y., Mizuta, K., Hayasaka, T., Nakanishi, H., Okamura, J., Mineta, H., & Setou, M.(2010). Specific localization of five phosphatidylcholine species in the cochlea by massmicroscopy. Audiology and Neurotology, 16, 315–322.

Tanaka, K., Waki, H., Ido, Y., Akita, S., Yoshida, Y., & Yoshida, T. (1988). Protein and polymeranalyses up to m/z 100000 by laser ionization time-of-flight mass spectrometry. RapidCommunications in Mass Spectrometry, 2, 151–153.

Tanaka, H., Zaima, N., Yamamoto, N., Sagara, D., Suzuki, M., Nishiyama, M., . . . Setou, M.(2010). Imaging mass spectrometry reveals unique lipid distribution in primary varicoseveins. European Journal of Vascular Endovascular surgery, 40, 657–663.

Teuber, K., Schiller, J., Fuchs, B., Karas, M., & Jaskolla, T. W. (2010). Significant sensitivityimprovements by matrix optimization: A MALDI-TOF mass spectrometric study of lipidsfrom hen egg yolk. Chemistry and Physics of Lipids, 163, 552–560.

Tholey, A., & Heinzle, E. (2006). Ionic (liquid) matrices for matrix assisted laser des-orption/ionization mass spectrometry applications and perspectives. Analytical andBioanalytical Chemistry, 386, 24–37.

Thomas R. L., Jr., Matsko, C. M., Lotze, M. T., & Amoscato, A. A. (1999). Mass spectrometricidentification of increased C16 ceramide levels during apoptosis. Journal of BiologicalChemistry, 274, 30580–30588.

Touboul, D., Roy, S., Germain, D. P., Chaminade, P., Brunelle, A., & Laprevote, O. (2007).MALDI-TOF and cluster-TOF-SIMS imaging of Fabry disease biomarkers. InternationalJournal of Mass Spectrometry, 260, 158–165.

Touchstone, J. C. (1995). Thin-layer chromatographic procedures for lipid separation. Journalof Chromatography B, Analytical Technologies in the Biomedical and Life Sciences, 671, 169–195.

Verbeck, G., Ruotolo, B., Sawyer, H., Gillig, K., & Russell, D. (2002). A fundamental introduc-tion to ion mobility mass spectrometry applied to the analysis of biomolecules. Journal ofBiomolecular Techniques, 13, 56–61.

Verhaert, P. D., Pinkse, M. W., Strupat, K., & Conaway, M. C. (2010). Imaging of similarmass neuropeptides in neuronal tissue by enhanced resolution MALDI MS with an iontrap-Orbitrap hybrid instrument. Methods in Molecular Biology, 656, 433–449.

Vidova, V., Novak, P., Strohalm, M., Pol, J., Havlicek, V., & Volny, M. (2010). Laser desorption-ionization of lipid transfers: Tissue mass spectrometry imaging without MALDI matrix.Analytical Chemistry, 82, 4994–4997.

Walch, A., Rauser, S., Deininger, S. O., & Hofler, H. (2008). MALDI imaging mass spectrom-etry for direct tissue analysis: A new frontier for molecular histology. Histochemistry andCell Biology, 130, 421–434.


Hawkes 171 09-ch04-145-194-9780123942975 2012/4/17 15:35 Page 193 #49


Wang, H. Y., Chu, X., Zhao, Z. X., He, X. S., & Guo, Y. L. (2011). Analysis of low molec-ular weight compounds by MALDI-FTICR-MS. Journal of Chromatography B, AnalyticalTechnologies in the Biomedical and Life Sciences, 879, 1166–1179.

Wei, J., Buriak, J. M., & Siuzdak, G. (1993). Desorption-ionization mass spectrometry onporous silicon. Nature, 399, 243–246.

Wiseman, J. M., Ifa, D. R., Zhu, Y., Kissinger, C. B., Manicke, N. E., & Kissinger P. T. (2008).Desorption electrospray ionization mass spectrometry: Imaging drugs and metabolites intissues. Proceedings of the National Academy of Sciences USA, 105, 18120–18125.

Wisztorski, M., Franck, J., Salzet, M., & Fournier I. (2010). MALDI direct analysis andimaging of frozen versus FFPE tissues: What strategy for which sample? Methods inMolecular Biology, 656, 303–322.

Woods, A. S., Ugarov, M., Jackson, S. N., Egan, T., Wang, H. Y., Murray, K. K., & Schultz, J. A.(2006). IR-MALDI-LDI combined with ion mobility orthogonal time-of-flight massspectrometry. Journal of Proteome Research, 5, 1484–1487.

Wu, L., Lu, X., Kulp, K. S., Knize, M. G., Berman, E. S., Nelson, E. J., . . .Wu, K. J. (2007).Imaging and differentiation of mouse embryo tissues by ToF-SIMS. International Journal ofMass Spectrometry, 260, 137–145.

Yanes, O., Woo, H. K., Northen, T. R., Oppenheimer, S. R., Shriver, L., Apon, A., . . .Siuzdak, G. (2009). Nanostructure initiator mass spectrometry: Tissue imaging and directbiofluid analysis. Analytical Chemistry, 81, 2969–2975.

Yang, H. J., Sugiura, Y., Ishizaki, I., Sanada, N., Ikegami, K., Zaima, N., . . . Setou, M. (2010).Imaging of lipids in cultured mammalian neurons by matrix assisted laser/desorptionionization and secondary ion mass spectrometry. Surface and Interface Analysis, 42,1606–1611.

Yao, I., Sugiura, Y., Matsumoto, M., & Setou, M. (2008). In situ proteomics with imagingmass spectrometry and principal component analysis in the Scrapper-knockout mousebrain. Proteomics, 8, 3692–3701.

Zaima, N., Goto-Inoue, N., Hayasaka, T., & Setou, M. (2010). Application of imagingmass spectrometry for the analysis of Oryza sativa rice. Rapid Communication in MassSpectrometry, 24, 2723–2729.

Zaima, N., Matsuyama, Y., & Setou, M. (2009). Principal component analyses of directmatrix-assisted laser desorption/ionization mass spectrometric data related metabolitesof fatty liver. Journal of Oleo Science, 58, 267–273.

Zhang, H., Cha, S., & Yeung, E. S. (2007). Colloidal graphite-assisted laser desorption/ionization MS and MSn of small molecules. 1. Direct profiling and MS imaging of smallmetabolites from fruits. Analytical Chemistry, 79, 6575–6584.

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