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Imaging Mass Spectrometry:
Sample Preparation,
Instrumentation, and
Applications
Kamlesh Shrivas , and Mitsutoshi Setou
Contents
1. Introduction
2. Ionization Methods for Imaging Mass Spectrometry
2.1. Desorption Electrospray Ionization
2.2. Secondary Imaging Mass Spectrometry
2.3. Laser Ablation Electrospray Ionization
2.4. Matrix-Assisted Laser Desorption/Ionization
3. MALDI Imaging
3.1. Sample Handling
3.2. Choice of Matrix
3.3. Application of Matrix Solution
4. Instrumentation
4.1. Quadrupole Mass Analyzer
4.2. Time-of-Flight Mass Analyzer
4.3. Sector-Type Mass Analyzer
4.4. Ion Trap Mass Analyzer
4.5. Orbitrap Mass Analyzer
4.6. Ion Cyclotron Resonance Mass Analyzer
5. IMS Measurements
6. Data Analysis
7. Applications of IMS for Direct Analysis of Tissue
7.1. IMS for Lipidomics
7.2. IMS for Proteomics
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Department of Cell Biology and Anatomy, Hamamatsu University School of Medicine, 1-20-1 Handayama,
Advances in Imaging and Electron Physics, Volume 171, ISSN 1076-5670, DOI: 10.1016/B978-0-12-394297-5.00004-0.
c 2012 Elsevier Inc. All rights reserved.
Copyright
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1. INTRODUCTION
The ability to visualize the molecular distribution in biological material
such as tissue samples has helped scientists to provide a better understanding of the principles of life. The study of biomolecule distribution in
organs and its alterations with disease remains one of the most challenging and intriguing scientific issues of recent times. Various techniques are
used in laboratories around the world to visualize molecular systems
techniques such as magnetic resonance imaging (MRI) technology (Hurd
and Freeman, 1989) and positron electron tomography (PET) (Ametamey
et al., 2008). The Nobel Prizewinning MRI and PET technologies are
known as noninvasive techniques for medical diagnosis. Nuclear magnetic resonance spectroscopy (NMRS) is also helpful for imaging and
identification of biomolcules in tissue sample (Hiltunen et al., 2002). The
limitations of these techniques are the relatively poor resolution, sensitivity, and requirement of labeling of molecules for detection (in the case of
the PET method).
Imaging mass spectrometry (IMS) was introduced for spatial distribution analysis of biomolecules without the need for extraction, purification,
separation, or labeling of biological samples. Recent developments in
molecular imaging have created new opportunities to perform molecular
diagnostic and therapeutic procedures. The technique can be exploited to
visualize cellular and molecular processes that occur in two-dimensional
(2D) or three-dimensional (3D) fashion without perturbing the structure
of the system (Caprioli et al., 1997; Setou et al., 2010).
Mass spectrometry (MS) is a technique based on the measurement
of the charged ions in an electric or magnetic field. Generally, a mass
spectrometer contains three distinct parts: (1) an ion source producing
ions from sample molecules; (2) a mass analyzer separating the different 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 the
ions were detected. Data are collected and visualized in a mass spectrum where the different m/z ratios are displayed as a function of their
signal intensity (Gross, 2004). MS is a great scientific tool because of the
wide range of molecules that can be accurately detected and identified:
large organic compounds and biomolecules of low molecular weight.
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Inlet of mass
spectrometer
Solvent
N2
Secondary
droplets
Spray droplets
Sample
(a)
Inlet of mass
spectrometer
Secondary
ions
Primary ions
Sample
(b)
Laser
attenuator
UV or IR laser
Inlet of mass
spectrometer
Analyte/matrix
mixture
(c)
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150
to date. In traditional MALDI, an organic matrix is mixed with the sample on the target plate and irradiated by a ultraviolet or IR light generated
by a pulsed and focused laser. The matrix absorbs the light at the wavelength of the laser, leading to a soft desorption/ionization of the intact
compounds 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 and
registering individual mass spectra for each pixel, a 2D ion density map
can be reconstructed using appropriate software. Direct MALDI-IMS analysis of clinical samples offers a unique approach to reveal the spatial
expression of biomolecules linked with pathological disease and other
clinical information. MALDI imaging is also suitable as a biomarker
discovery tool by comparing the relative quantities and/or spatial distribution patterns of molecules in pathological and normal samples. The
localization and abundance of biomarkers identified in tissue sections
are used to understand disease progression at a molecular level. The
main advantages of a direct biomarker analysis using MALDI imaging
are that it provides spatial distribution patterns and is free from extraction, purification, or separation steps, hence avoiding procedures that are
both 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 packages, we can construct multiplexed imaging maps of selected biomolecules within tissue sections. The laser energy is used in a raster scan
pattern to ionize the molecules, which are present as discrete spots or
pixel. For each pixel the full mass spectrum is represented. The data acquisition time for IMS was shortened by the introduction of N2 (337-nm) or
neodymium-doped yttrium aluminum garnet (Nd:YAG) (355-nm) lasers
with repetition rates of 2001000 Hz with pulse lengths of 3 ns. The laser
spot size of MALDI-MS is decreased from 100150 to 20 m, rendering
higher spatial resolution of biomolecules on the tissue surface. Further,
a higher spatial resolution can be attained with a MALDI instrument
equipped with a highly focused laser. Chaurand et al. (2007) used a laser
beam at 7 m, which is in the order of the diameter of a single cell to
detect protein ions. However, a decrease in sensitivity is observed while
increasing the resolution in this manner. Figure 2 shows an example of
MALDI-IMS analysis of protein from tissue section.
In addition to increased sample integrity, the great advantage of IMS
is that it allows the construction of numerous ion images of molecules
detected in a single run. This technique does not require previous
Profiling
151
Imaging
High
density
droplet
array
Apply
matrix
Acquire
mass spectra
r
se
La
Molecular
profiles
r
se
La
Molecular
images
FIGURE 2 Scheme presenting the protein profiling and imaging analytical strategies
from thin tissue sections. Reprinted from Chaurand (2006) with permission from
American Chemical Society.
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153
Intensity
(a)
Tissue slice
(b)
OCT
Intensity
OCT
4500
6000
7500
m/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 but
does not come into contact with the sliced tissue. The resulting spectrum shows many
intense signals between m/z 4500 and 10500. (b) The tissue was embedded in OCT and
attached to the sample stage. The resulting tissue slice is surrounded by OCT on the
MALDI plate, and the resulting spectrum contains of only about half of the signals as
in (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. The
tissue is thereby thaw-mounted on the target plate. In the second method,
the plate is kept at room temperature and placed over the sliced frozen
section, and the tissue is thereby simply thawed on the target plate.
Great care should be taken with both methods to retain the shape of the
tissue. Obviously, folding or stretching caused during the sectioning of
tissue section may affect the molecule distribution analysis and prevents
detection of some of the molecules from the tissue surface.
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Relative intensities
Acetone
Chloroform
Ethanol
Water
Hexane
Isopropanol
Acetic acid
Methanol
t-MBE
Toluene
Xylene
No wash
0
5000
10000
15000
Mass-to-charge (m/z)
(a)
6226
Intensity
2000
7928
900
1000
600
500
300
9912
13777
7936
1200
1500
6000
20000
9980
4000
7885
500
2000
0
6200 6220 6240 6260
m/z
0
7860 7900 7940 7980
m/z
(b)
(c)
1000
0
9850 9900 9950 10000
m/z
(d)
0
13700
13800
m/z
13900
(e)
FIGURE 4 Average MALDI-IMS protein profiles directly acquired from serial mouse
liver tissue sections not washed or washed with different solvent systems. (a) Full mass
range; (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 samples (Lemaire et al., 2006b). Seeley et al. (2008) reported a new washing
procedure to enhance protein detection in terms of both the number of
observed peaks and the signal intensity. They demonstrated that the use
of 12 different washing solvents established the most effective condition for direct protein analysis from the surface of tissue section. They
also obtained a high detection sensitivity of protein signals, matrix crystal formations, and histological integrity of the tissues by washing with
70% isopropanol for 30 seconds followed by a 90% isopropanol wash for
30 seconds. Figure 4 shows the MALDI-IMS results for protein detection in
mouse liver tissue sections after washing with different organic solvents.
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m/z 6651
1 mm
(a)
m/z 2897
(b)
m/z 9685
(c)
1 mm
(d)
m/z 2897
Pars distalis
(e)
Pars intermedia
Pars neuralis
m/z 9685
a.u.
12
10
8
6
4
2
m/z 6651
3000
4000
5000
6000
7000
(f)
8000
9000
10000
m/z
FIGURE 5 MALDI-IMS of a tissue section of rat pituitary gland. (a) Optical microscopic
image of an H&E-stained tissues section. The staining was done after the MALDI
measurement of the tissue section. (b)(d) Visualized selected m/z species representing
features to pars distalis (m/z 6,651; green), pars intermedia (m/z 2,897; red), and pars
neuralis (m/z 9,685; yellow). (e) Merge of (ad). (f) MALDI-TOF-MS spectra obtained
from this case from pars distalis (green), pars intermedia (red), and pars neuralis (yellow)
showing the molecular differences between the histological regions. Reprinted from
Walch et al. (2008) with permission from Springer.
and another section is cut for histological staining. They can then be superimposed on each other and provide an absolute value of the molecular
distribution (Figure 5). Recently a new approach for tissue section staining after the MALDI measurement has been reported. The results obtained
from IMS analysis were correlated with the H&E staining of the tissue
section (Schwamborn et al., 2007).
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Applications
References
2,5-Dihydroxybenzoic
acid (DHB)
Lipids, Sugars,
peptides, nucleotides,
glycopeptides,
glycoproteins, and
small proteins
-Cyano-4hydroxycinnamic acid
(CHCA)
Peptides, small
proteins and
glycopeptides
2,6Dihydroxyacetophenone
(DHA)
Phospholipids
2,4,6Trihydroxyacetophenone
(THAP)
Lipids
p-nitroaniline (PNA)
Phospholipids
2-mercaptobenzothiazole
(MBT)
Sinapinic acid (SA)
Phospholipids
CHCA/aniline, ionic
matrix
Peptides
CHCA/n-butylamine,
ionic matrix
Phospholipids
157
786
786
45
4
760
760
30
810
810
(a)
Signal intensity 10
15
(b)
731
703
500
1000
800
731
703
500
800
(i)
1000
(k)
786
786
350
120
760
760
90
810
210
810
60
731
703
30
(c)
731
703
70
(d)
500
1000
800
500
(l)
60
703
100%
3
760
0%
8
703
731
760
786
40
Signal intensity 10
731
20
0
1
2
0
703
731
760
786
0
0
(n)
50
(f) CHCA
703
731
760
786
(m)
100
(e) DHB
0
0
150
786
1000
800
(j)
400
300
200
100
0
703
731
760
786
0
(o)
(p)
Number of sample analyses
FIGURE 6 The crystal formation of (a) DHB, (b) CHCA, (c) DHBB, and (d) CHCAB
matrixes with phospholipids on to a MALDI target plate. The pictures were taken with an
Ultraflex II TOF/TOF. The scale bar (white color line) is 100 m; images (e) to (h) show
the 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) show
the 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-fold
improvement 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 relative
standard deviation, % by using (m) DHB: 20.540.8%, (n) CHCA: 29.545.8%,
(o) DHBB: 14.521.8%, and (p) CHCAB: 7.510.0%. Reprinted from Shrivas et al. (2010)
with permission from American Chemical Society.
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159
40
35
30
25
20
15
10
5
200
220
240
260
280
300
320
340 m/z
40
35
30
25
20
15
10
5
200
220
240
40
35
30
25
20
15
10
5
200
Only AgNPs
220
240
260
280
300
320
340 m/z
280
300
320
340 m/z
Only DHB
40
35
30
25
20
15
10
5
200
220
m/z 255.4
(16:0)
240
260
280
300
320
340
m/z
(d)
(b)
Optical
image
260
(c)
Signal intensity (a.u.)
(a)
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)
100%
AgNPs
DHB
0%
(e)
160
MALDI
plate with
tissue slice
(a)
(b)
(c)
(d)
FIGURE 8 Apparatus used to deposit matrix on the tissue section. (a) Thin-layer
chromatography sprayer, (b) sublimation apparatus, (c) air brush sprayer, and (d) chemical
inkjet 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 maintained
at fixed direct current (DC) with an alternating radiofrequency (RF). With
this setup molecular ions formed at the source pass through the middle
of the quadrupoles in the electric field region and the ions of a specific
m/z have a stable trajectory path and may pass all the way to the detector, while the remaining ions collide with the electrodes and never reach
the detector (Gross, 2004). Using a continuous and controlled manner to
change the frequency and potential, the quadropole transmits molecules
at certain m/z values. Figure 9a shows a diagram of quadrupole mass analyzer. The sensitivity of the instrument can be enhanced by increasing the
number of qaudupoles from two to three (triple quadrupole) in series. In
triple-quadrupole analyzers, the first (Q1 ) and third (Q3 ) quadrupoles act
as filters, and the second (Q2 ) quadrupole functions as a collision cell. The
third (Q3 ) quadrupole is worked at normal RF/DC or in the linear ion
trap (LIT) mode (Douglas et al., 2005). Hopfgartner et al. (2009) demonstrated the fast imaging of complete rat sections using MALDI coupled
with a triple-quadrupole LIT where the precursor ion mode can be used
to monitor the presence of the parent drug in the tissue section.
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Ring
electrode
Entrance
slit
Quadrupole
rods
Exit
slit
(a)
Flight
tube
Entrance
endcap
electrode
Linear
detector
Exit
endcap
electrode
(d)
Detector
electrode
Detector
electrode
Central
electrode
Reflector Reflector
detector (lon mirror)
(b)
Superconductive
magnet
Excitation
plates
Magnet
Flight
tube
(e)
Exit
slit
Trapping
plates
(c)
Detector
plates
(f)
ions and ions of the same charge state is thus possible through their different mobility in the IM spectrometer. The combined techniques of IM and
TOF-MS were used for imaging and identification of digested proteins.
IM separates isobaric ions that cannot be distinguished by MALDI-TOF
alone, providing mass- and time-selected ion images of biomolecules in
tissue samples (Stauber et al., 2010).
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specific tissue section prior to IMS and has spatial resolution (10 m)
higher than the naked eye.
In a linear quadrupole ion trap (LIT) or 2D traps (2D-IT), the ions are
trapped in a 2D quadrupole field and then pass axially. The 2D-IT ion
trap produces reasonable mass accuracy, mass resolution, and sensitivity
(Schwartz et al., 2002). LIT has a better ion storage capacity and a higher
trapping efficiency compared with 3D-IT. However, the disadvantage of
LIT is the relatively narrow mass range of small molecule analysis. Garrett
et al. (2007) described a new MALDI-LIT-MS for imaging of tissue samples and also used for MSn analyses to confirm the molecules. Enomoto
et 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).
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5. IMS MEASUREMENTS
MALDI-IMS experiments can be performed after the deposition of matrix
on the tissue section and using different types of MS instruments as discussed above. The setting of the laser energy, detector gain, and random
walk function are optimized in order to obtain better signal intensity of the
target molecules during the IMS analysis. Either a particular region of
the tissue or the entire tissue section is selected for analysis, depending on
the particular interest. At present, the commercially available instruments
can perform IMS analyses with the highest spatial resolution of 25 m
(Goto-Inoue et al., 2009a). Recently we developed a mass microscope that
can move a sample stage by 1 m, and the finest size of the laser diameter is approximately 10 m (Harada et al., 2009). The measurement time of
IMS experiments depends on the number data spots, the frequency of the
laser, and the number of shots per spot.
6. DATA ANALYSIS
Due to the large (gigabytes) size of the dataset, high-capacity visualization
software is required to visualize the ion image and distribution pattern of
biomolecules in tissue samples. New computing methods are required for
both rapid, accurate data acquisition and the interpretation of the IMS
analysis results. Therefore, in addition to instrumental improvements,
data acquisition and software development have been important for the
production of reliable data. The databases used are based on algorithms
that perform analysis through statistical evaluation of observed and
theoretical 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 used
to identify biomolecules in various sample types. The intensity of the
different color images obtained by both software packages can relate
the distribution of biomolecules in the tissue section. These software
packages also help in understanding the localization of biomolecules at
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1995), and MS have been used to analyze lipids in tissue samples. However, the sample preparation procedures in chromatographic techniques
are lengthy and the localization of biomolecules in tissue sample surface
cannot be established. Therefore, different IMS systems are successfully
used for imaging of lipids. The analysis of glycerophospholipids, sphingolipids, and neutral lipids is discussed in detail in the following
sections.
7.1.1. Glycerophospholipids
GPLs are glycerol-based phospholipids and essential components of cell
membranes. They act as second messengers involved in cell proliferation, apoptosis, and metabolism. The determination of GPL content in
tissue samples is useful for finding potential biomarkers for diseases such
as atherosclerosis or rheumatism (Fuchs et al., 2005; Schmitz and Ruebsaamen, 2010). Altered levels of lipids are found in many pathological
conditions such as Alzheimers disease (Han et al., 2001, 2002), Down syndrome (Murphy et al., 2000), diabetes (Han et al., 2007), and NiemannPick
disease (He et al., 2002). Figure 10 illustrates the basic structures of common classes of GPLs such as PC, phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylserine (PS) (Jackson and Woods,
2009). PC is easily ionized due to its charged quaternary ammonium head
group and has thus become a popular lipid species to investigate (Pulfer
and Murphy, 2003). The ionized molecules observed in the mass spectrum
are usually either protonated [M+H]+ , sodiated [M+Na]+ , or pottasiated
[M+K]+ in positive-ion mode. Phospholipids such as PE, PS, PA, PG, and
PI may generate negative ions due to the presence of the phosphodiester
moiety and display molecular anions [M-H] (Fuchs et al., 2010). The
addition of potassium acetate (Sugiura et al., 2009) or LiCl (Jackson et al.,
O
P
O O
O
+
N
O
O
H O
R1
H 2N
R2
HO
OH
O
OH P
O
O
OH
O
O
H O
R1
R2
O
H2N H
P
HO
O
O
OH
C
O
R2
O
O
H O
O
Phosphatidylinositol
R1
O
H O
O
Phosphatidylethanolamine
O
Phosphatidylcholine
HO
HO
O
P
O
O
OH
Phosphatidylserine
R1
R2
168
2005) to the matrix solution has also been reported for effective ionization
of molecules in tissue samples.
The selection of MALDI matrices is an important issue. For MALDIIMS, the matrix should have good vacuum stability and homogenous
crystal formation containing analyte molecules. Various matrices have
been reported for the identification and characterization of lipids in
MALDI-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), and
9-aminoacridine (Eibisch and Schiller, 2011; Teuber et al., 2010). However,
PNA and dihydroxyacetone phosphate (DHAP) were unstable under high
vacuum conditions and started to evaporate after their introduction into
the MALDI-MS instrument (Jackson et al., 2005; Rujoi et al., 2004; Shrivas
et al., 2010). DHB matrix exhibited a lower sensitivity for the detection
of PA, PS, PE, PI, and PG in negative-ion mode, possibly due to its acidity (Estrada and Yappert, 2004; Petkovic et al., 2001). DHA can be used
in both positive and negative ionization modes for analysis of phospholipids (Woods et al., 2006). PNA is another good matrix for the analysis of
phospholipids in either positive-ion or negative-ion modes (Estrada and
Yappert, 2004). Recently, 2-mercaptobenzothiazole (MCT) was added as
an alternative to the use of DHB for MALDI-MS analysis of phospholipids in brain and liver tissue samples (Astigarraga et al., 2008). The main
advantages of MCT over the commonly used matrices DHB, DHA. and
PNA are low vapor pressure, low acidity, and homogenous crystal formation, which allowed for detection of more lipid species in negative mode,
with high sensitivity and high detection reproducibility. Ionic matrices
have also been used in MALDI-IMS owing to the good vacuum stability,
homogenous crystal formation, and good solubility of analytes for efficient ionization and desorption of molecules (Chana et al., 2009; Lemaire
et al., 2006a; Shrivas et al., 2010). Shrivas et al. (2010) used an ionic matrix
of CHCAB to image and identify lipids in mouse cerebellum and found
that this ionic matrix yields a higher number of ion images compared
with the use of DHB matrix in MALDI-IMS (Figure 11). Use of NPs is
another good approach for selective and sensitive analysis of lipids and
small metabolites in tissue samples without matrix-oriented peaks in the
low-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 demonstrated that arachidonic acid (AA) and DHA-containing PC were found
in the hippocampal neurons and cerebellar Purkinje cells, respectively.
Figure 12 shows the localization of PC species in different layers of the
mouse brain (Sugiura et al., 2009). The distribution of PC species also has
been reported in the mouse retinal section via MALDI-IMS analysis. The
localization of PC (16:0/18:1) was found in the inner nuclear layer and
169
GL
ML
WM
(a)
ND
m/z 734.5
m/z 772.5
m/z 826.5
m/z 846.5
m/z 734.5
m/z 772.5
m/z 826.5
ND
m/z 756.5
m/z 798.5
m/z 832.5
m/z 850.6
m/z 756.5
m/z 798.5
m/z 832.5
ND
m/z 760.5
m/z 810.5
m/z 834.5
m/z 864.6
m/z 760.5
m/z 810.5
m/z 834.5
ND
m/z 769.5
m/z 822.6
m/z 835.6
m/z 770.5
m/z 824.5
m/z 844.5
(b)
m/z 870.5
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 846.5
ND
m/z 850.6
ND
m/z 864.6
ND
m/z 870.5
(c)
FIGURE 11 (a) H&E-stained mouse cerebellum showing three layers with 1.5-mm scale
bar (white color). The ion images of lipids in mouse cerebellum tissue section obtained
by 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 layer
of 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 in
the white matter of cerebellum. The ion images at m/z 769.5 [SM(d18:1/18:0)+K]+ and
835.6 [SM(d18:1/24:1)+Na]+ illustrated that the molecules were distributed in the region
of molecular layer of tissue. The ion images at m/z822.6 [GalCer(d18:1/22:0)+K]+ and
850.6 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 American
Chemical Society.
the outer plexiform layer; PC (16:0/16:0) in the outer nuclear layer and
inner segment; and PC (16:0/22:6) and PC (18:0/22:6) in the outer segment
and pigment epithelium (Hayasaka et al., 2008). Differential localization
of PC (16:0/20:4) species was observed between terminal and stem villi of
170
16:0
PC (diacyl-16:0/16:0)
16:0
CBX
CTX
HPF
TH
m/z 772.4
18:0
PC (diacyl-18:0/18:1)
Structure of PCs
CH3
O
N+ CH3
H2C O P O
CH3
O
HC O R2 (sn2)
18:1 (OA)
PC (diacyl-16:0/18:1)
H2C O
m/z 826.5
m/z 798.4
PC (diacyl-16:0/20:4)
Cp
20:4 (AA)
PC (diacyl-18:0/20:4)
R1 (sn1)
18:1 (OA)
PC (diacyl-18:1/20:4)
m/z 848.5
m/z 820.5
PC (diacyl-16:0/22:6)
PC (diacyl-168:0/22:6)
m/z 846.5
22:6 (DHA)
PC (diacyl-18:1/22:6)
m/z 844.5
0%
m/z 872.6
m/z 870.6
100%
human placenta, which could be helpful in understanding the pathological involvement of fetal growth restriction and fetal hypoxia (Kobayashi
et 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 damaged
valvular region was investigated and suggested an association of these
molecules with tissue inflammation and resultant valvular incompetence
(Tanaka et al., 2010). PC (diacyl-16:0/20:4) containing an AA was found
at higher concentration in prefrontal cortex tissue compared with occipital cortex tissue in the brains of patients with schizophrenia (Matsumoto
et al., 2011). The specific localization of five PC species in the cochlea
was also examined using the mass microscope. A differential distribution of PC species was observed; (16:0/18:1) in the organ of Corti and the
stria vascularis, (16:0/18:2) in the spiral ligament, and (16:0/16:1) in the
organ of Corti (Takizawa et al., 2010). Recently Goto-Inoue et al. (2009a)
171
investigated use of a TLC-Blot-MALDI-IMS for analysis and characterization of acidic, neutral glycosphingolipids and PC in sample mixtures.
In TLC-Blot, the lipids are separated and transferred to a polyvinylidene
fluoride (PVDF) membrane without any change in the stability of the
molecules. PVDF membranes with the sample may then be placed on the
target plate for MALDI-IMS analysis. This method might be useful for
the detection of minor components that could not be detected by the conventional TLC method. SIMS is also used for imaging of lipids at high
spatial resolution and sensitive detection. The combined approaches of
MALDI-IMS and SIMS-IMS have been used for imaging of PC in cultured
mammalian neuron. The data obtained from MALDI and SIMS supported
that the signals of small molecules in the low molecular region, such as
PC head groups and fatty acids (detected in SIMS) were obtained from
the intact lipids (Yang et al., 2010). DESI-IMS has been used for imaging of
most commonly encountered brain lipid species such as PE and PI in rat
spinal 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 the
brain (Dill et al., 2009).
7.1.2. Sphingolipids
The sphingolipid is a type of lipid obtained from the aliphatic amino alcohol sphingosine. The main functions of sphingolipids are transmission
and cell recognition. The investigation of sphingolipids is very important because they are indicative of aging and may function as a disease
marker. Sphingolipids contain a sphingoid base backbone and include
sphingomyelins (SM), sulfatides (ST), ceramides, cerebrosides, and gangliosides (Merrill et al., 2009) (Figure 13). Changes in the levels of lipids,
HO
OH
HO
HO
C13H27
H HN
HO
OH
O
+
N
O
O P
O O
C13H27
H HN
O
Ceramide
HO H
HO
H
C13H27
H HN
OH
OH O S
O O
HO
OH
O
HO H
C13H27
H HN
O
Sphingomyelin
R
O
Cerebroside
Sulfatide
R
O
FIGURE 13 Structure of sphingolipids. Reprinted from Jackson and Woods (2009) with
permission from Elsevier Science.
m/z 725.5
Relative intensity
Normal
Cancer
616.1
725.5
Relative intensity
172
Normal
Cancer
m/z 616.1
Merged image
(b)
(c)
FIGURE 14 Imaging of normal and cancerous cells in human liver sample. (a) The most
prominent signal at m/z 725 showed the higher expression for cancerous cells than
normal cells. (b) The signal at m/z 616 showed the higher distribution of this molecule
in normal cells. (c) Merged images at m/z 725 and 616. Reprinted from Shimma et al.
(2007) with permission from Elsevier Science.
HPF
(a)
CTX
MB
TH
Mad/P
Cp
ST (22:0 OH)
(e) m/z 1902
[GD1(18:0/d18:1) + K - 2H]
(f) Merged
[GD1(18:0/d20:1) + K - 2H]
(i) Merged
[GD1(18:0/d18:1) + Na - 2H]
[GD1(18:0/d20:1) + Na - 2H]
(l) Merged
GM1
GD1
ST (24:0 OH)
C20
C18
[GM1(18:0/d18:1) - H]
[GM1(18:0/d20:1) - H]
(A)
(a)
SO
SR
SLM
ML
CA1
ML
DG
ST (22:0 OH)
CA3
C18
[GD1(18:0/d18:1) + K - 2H]
[GD1(18:0/d18:1) + Na - 2H]
[GD1(18:0/d20:1) + Na - 2H]
[GM1(18:0/d18:1) - H]
[GM1(18:0/d20:1) - H]
(B)
FIGURE 15
(Continued)
(f) Merged
[GD1(18:0/d20:1) + K - 2H]
GM1
GD1
ST (24:0 OH)
C20
(i) Merged
(l) Merged
173
174
175
Lung
Brown adipose
tissue
Liver
Brian
Intestine
Hypoglottis
Heart
Thymus
(a)
(TAG (16:0/18:2/18:1) + Na)+
100%
0%
0%
(b)
(c)
Merged
100%
100%
0%
0%
(d)
(e)
176
Antibody availability and specificity are other constraints with IHC that
limit its capacity (Luongo de Matos et al., 2010). Thus the introduction of
IMS has provided complementary resources.
What IMS lacks in terms of sensitivity, it compensates for by enabling
untargeted studies with the possibility to detect hundreds of molecules
in a single run. In the so-called bottom-up approach, the proteins present
in the tissue sample must first be subjected to in situ digestion by a proteolytic enzyme. Usually trypsin is used since it renders peptides that
contain at least one lysine or arginine amino acid and hence are easily
ionized. Setou et al. (2008) investigated whether the addition of detergent into the trypsin solution could improve the digestion efficiency of
proteins for direct analysis of tissue section in MALDI-.IMS. Trypsin solution can be spotted directly onto the tissue sections, rendering spot sizes
150200 m. Considering the possibility for peptide migration within
this spot area, the tryptic spot size can also be said to determine the
resolution of the IMS experiment, although the spatial resolution from
the actual data acquisition is determined by the instrument used and is
usually lower. Organic matrices such as DHB and CHCA are used for
ionization.
For biomarker studies, the tissues available through biobanks around
the world have generally been treated with formalin for increased tissue
stability over time. Formalin fixation and subsequent paraffin embedding
allows for stable histomorphology, but it also causes difficulty in IMS since
it cross-links proteins and hampers protein mining. This problem has been
overcome by deparaffinization methods followed by the same antigenretrieval methods used in IHC experiments (enzymatic or heat-mediated)
(Aoki et al., 2007). Recently formalin-fixed, paraffin-embedded tissue
microarrays were analyzed in MALDI-IMS and MS/MS experiments to
study 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 identification 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 and
low-grade glioma. In addition, the combined approach of MALDI-IMS
and MS/MS analyses of digested myelin basic protein (MBP) in a coronal section of rat brain has been demonstrated (Figure 17ac) (Groseclose
et al., 2007). After digestion, a total of eight tryptic peptides from MBP
were detected (Figure 17d). This protein is essential for the formation of
myelin in the central nervous system. MALDI-IMS also has been used to
classify a pancreatic cancer tissue microarray where a number of proteins
that appear to discriminate between different tumor classes were detected
(Djidja et al., 2009). Direct proteomic-based imaging was also performed
177
High
Low
(a)
(b)
(c)
High
m/z 699.47
(NIVTPR)
Low
m/z 1131.84
(TTHYGSLPQK)
m/z 726.46
(HGFLPR)
m/z 1067.60
m/z 1909.92
(FSWGGRDSR) (FSWGGRDSRSGSPMARR)
m/z 1336.69
m/z 1460.90
(YLATASTMDHAR) (TQDENPVVHFFK)
m/z 1502.98
(TTHYGSLPQKSQR)
(d)
FIGURE 17 (a) H&E stain of rat brain tissue section serial to the sections used for
digestion 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-tryptic
peptides 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 the
diagnosis of human diseases (Yao et al., 2008). Figure 18 shows the PCA
of mass spectra from Scrapper-knockout (SCR-KO) and WT mouse brains
analyzed by MALDI-IMS.
178
Relative intensity
WT
SCR-KO
Cerebral corte
WT
Cerebral corte
SCR-KO
Hypothalamus
WT
m/z 2500
5000
(a)
7500
(b)
Pons/medullary
Corpus striatum
0.3
0.4
15
Load 1
1
PC 1
15
m/z 7109
0.8
10
WT
SCR-KO
m/z 7420
Hypothalamus
SCR-KO
10000
1Load 1
PC 1
20
0.6
10
m/z 7420
Load 2
0.8
12 PC 2
100%
Load 2
0.6
15 PC 2
m/z 7109
0%
100%
0%
SCR-KO
WT
Cerebral cortex
Hypothalamus
0.8
10
0.8
10
m/z 5004
PC 1
15
25
Load 1
0.8
PC 2
20
m/z 5004
0.8
15
PC 1
Load 2
0.8
100%
100.8
m/z 4285
m/z 4285
0.4
Load 2
0.6
PC 2
0%
Load 1
100%
0%
(c)
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 in
IMS analyses are indicated by colors. (b) Mass spectra obtained from each region of the
WT or SCR-KO mouse brain sections. Specific signals of the regions are indicated by
arrowheads. (c) Distributions of principal component scores of mass spectra from
various brain regions (left spray graphs; WT, blue; KO, red) and the loading factors
plot (right graphs). The signal intensities of mass spectra of the substances with indicated
m/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.
179
(b)
(c)
(d)
Kidney
Fur
(a)
Testis
Bladder
Spleen
Liver Lung
Thoracic
cavitv
Thymus
100%
100%
100%
180
SIMS and NIMS were used for imaging of drugs in tissue samples and the
mass spectrum obtained was free of the matrix-oriented peaks. The direct
analysis of clozapine and its metabolites in dosed rat brains has been illustrated using TOF/TOF mass analyzers (Yanes et al., 2009). NIMS-IMS is
compatible with both ion beam and laser sources available on commercial
SIMS and MALDI instruments. In addition, fewer laser shots are required
per spot compared with the MALDI technique.
ADP
AMP
Seizure
(30 min)
Control
ATP
nmol/g tissue
(a)
ATP
120
ADP
**
400
80
**
40
200
AMP
2000
Sham
Seizure (30 min)
1000
0
**
**
**
Increase
ADP
AMP
0
1
*
*
2
Decrease
Decrease
Increase
Increase
ATP
Decrease
Log ((int.(sham/KA))
(b)
Entire region
CA1 cell layer
DG cell layer
CA3 cell layer
(c)
Energy charge
High
CA1
CA3
Control
Seizure
Low
(d)
ATP
Succinyl AMP
IMP
AMP
ADP
Inosine
Adenine
Adenosine
(e)
FIGURE 20 (Continued)
Hypoxanthine
182
8. SUMMARY
Several advances in sample preparation, ionization, and MS instrumentation have been achieved, steadily improving sensitivity, spatial resolution,
and identification capabilities for MALDI-IMS. These improvements are
broadening the MS imaging applications for lipid, peptide, and protein
biomarker identification, as well as drug and metabolite imaging. NanoPALDI, the use of ionic matrices, and the mass sicroscope techniques
are new developments that could be powerful tools in obtaining highresolution images for biomolecular distribution in biological samples. In
the future, MALDI-IMS has the potential to become a routine tool for
imaging of tissues, helping us to understand the link between the localization of certain molecules and their function during pathogenesis, disease
progression, or treatment.
183
5 cm
3 mm
(a)
Optical image of
eggplant section
m/z 104.07
2.5 mm
100%
Optical image of
eggplant section
0%
High-power field of
red rectangle area
m/z 104.07
0.5 mm
0%
(b)
(c)
m/z 104.0 on tissue
104.0
58.1
100
(d)
GABA (standard)
100
100%
104.0
100
100
58.0
0
(e)
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 grey
rectangle in a round-cut image shows the region of analyses by IMS. (b) Optical image of
eggplant section and ion image of the m/z values at 104.07. The red arrows in the optical
image 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 with
higher spatial resolution at 25 m on a seed. Scale bar: 0.5 mm. (d) The tandem mass
spectrum of standard gamma-aminobutyric acid (GABA) and (e) m/z 104.0 on eggplant
tissue. Reprinted from Goto-Inoue et al. (2010b) with permission from The Japan Society
for 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 supported
by a grant-in-aid for SENTAN from the Japan Science and Technology
184
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