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Imaging Mass Spectrometry: A New Technology

for the Analysis of Protein Expression in
Mammalian Tissues

Article in Nature Medicine May 2001

DOI: 10.1038/86573 Source: PubMed

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 2001 Nature Publishing Group http://medicine.nature.com
NEW TECHNOLOGY

Imaging mass spectrometry: A new technology for the

analysis of protein expression in mammalian tissues
MARKUS STOECKLI, PIERRE CHAURAND, DENNIS E. HALLAHAN & RICHARD M. CAPRIOLI
Mass Spectrometry Research Center, Vanderbilt University School of Medicine, Nashville, Tennessee, USA
Correspondence should be addressed to R.M.C.; email: r.caprioli@vanderbilt.edu
2001 Nature Publishing Group http://medicine.nature.com

The molecular specificity and sensitivity of mass spectrometry Geneva, Switzerland). With a laser frequency of 20 Hz, the time
(MS) has been employed in a new technology for direct map- cycle was about 2.5 seconds per data point, including acquisi-
ping and imaging of biomolecules present in tissue sections. tion, data download to the computer, data processing and repo-
This technology has been developed using matrix-assisted laser sitioning of the sample stage. A typical data array was
desorption/ionization MS (MALDI MS)1 and has been initially 1,00030,000 spots depending on the desired image resolution,
targeted for the analysis of peptides and proteins present on or which contains the intensity of ions desorbed at each spot in a
near the surface of tissue sections2. Imaging MS brings a new molecular weight range of 500 D to over 80 kD. For most tissue
tool to bear on the problem of unraveling and understanding sections, we recorded over 200 protein and peptide peaks in the
the molecular complexities of cells. It joins techniques such as mass spectrum from each spot ablated by the laser. We could
immunochemistry and fluorescence microscopy for the study of produce an MS image or molecular weight-specific map of the
the spatial arrangement of molecules within biological tissues. sample at any desired molecular weight value. It is commonly
Many previous experiments using MS to image samples have fo- possible to generate individual maps to verify the presence, mol-
cused on the measurement of the distribution of elements and ecular weight and location of proteins. In the fullest extent,
small molecules in biological specimens, including tissue slices from a single raster of a piece of tissue, imaging MS could pro-
and individual cells35. An extensive review on imaging by MS duce hundreds of image maps each at a discrete molecular
can be found in the article by Pacholski and Winograd6. weight value.

Technological aspects Application to mammalian tissue

For the molecular image analysis, tissue samples can be pre- We used imaging MS to study normal tissue sections from
pared using several protocols: direct analysis of fresh frozen sec- mouse brain and human brain tumor xenograph sections. These
tions79, individual cells or clusters of cells isolated by samples contained well-defined regions, many of which had
laser-capture microdissection or contact blotting of a tissue on a subsets of proteins and peptides in a unique distribution or
membrane target10. In a typical preparation procedure (Fig. 1), array. The bilateral symmetry of the brain provides an internal
we mounted a frozen section of tissue on a stainless steel target confirmation of the localized distribution of proteins and the
plate, coated it with a solution of matrix (for example, sinapinic homogeneity of the prepared tissue sections. An optical image
acid), then dried and introduced into the vacuum inlet of the of the normal mouse brain section fixed on a metal plate and
mass spectrometer (Voyager Elite DE, Applied Biosystems, coated with matrix is shown in Fig. 2a. We scanned the section
Framingham, Massachusetts). The instrument was controlled by by acquiring 170 90 spots with a spot-to-spot center distance
MS imaging software written in our laboratory11. We created of 100 m in each direction. We recorded ions occurring in 82
molecular images from a raster over the surface of the sample different mass ranges and created images by integrating the
with consecutive laser spots (25 m in diameter). The laser po- peak areas and plotting the relative values using a color scale.
sition was fixed and the sample plate was repositioned for con- For specific molecular images, we acquired data in a window de-
secutive spots. Each spot produced a mass spectrum obtained limited by two mass-to-charge (m/z) units on either side of the
from molecules present within the irradiated area. Typically, molecular peak. Although many of the protein signals were
each mass spectrum was the average of 50 laser shots acquired common to all areas of the brain, some were found to be highly
using a fast transient recorder PC board (DP211, Acqiris, specific for a given brain region. For example, the protein de-
tected at m/z 8258 1 (Fig. 2b) was present in the regions of the
cerebral cortex and the hippocampus; the protein at m/z 6716
1 (Fig. 2c) was localized in the regions of the substantia nigra
and medial geniculate nucleus; and the peptide at m/z 2564 1
was in the midbrain (Fig. 2d). These ions are [M+H]+ species, and
the molecular weights of the compounds were obtained by sub-
tracting the weight of a proton, nominally 1 m/z unit from the

Fig. 1 Methodology developed for the spatial analysis of tissue by

MALDI mass spectrometry. Frozen sections are mounted on a metal plate,
coated with an UV-absorbing matrix and placed in the mass spectrome-
ter. A pulsed UV laser desorbs and ionizes analytes from the tissue and
their m/z values are determined using a time-of-flight analyzer. From a
raster over the tissue and measurement of the peak intensities over thou-
sands of spots, mass spectrometric images are generated at specific
molecular weight values.

NATURE MEDICINE VOLUME 7 NUMBER 4 APRIL 2001 493

 2001 Nature Publishing Group http://medicine.nature.com
NEW TECHNOLOGY

Fig. 2 Mass spectrometric images of a

a b mouse brain section. a, Optical image
of a frozen section mounted on a gold-
coated plate. b, m/z 8,258 in the re-
gions of the cerebral cortex and the
hippocampus. c, m/z 6,716 in the re-
gions of the substantia nigra and medial
geniculate nucleus d, m/z 2,564 in the
midbrain.

c d
2001 Nature Publishing Group http://medicine.nature.com

measured m/z value. Identification of the proteins can be done in Fig. 3a. The orientation in the figure is such that the actively
through extraction, HPLC fractionation, proteolysis, mass spec- growing area of the tumor is at the top of the figure, and the
trometric sequencing of one or more of the fragments and pro- point where the tumor was attached to the healthy tissue at the
tein database searching. This procedure is illustrated below for bottom. The fine line (cross-hatched) pattern on the optical
proteins in tumor sections. image was produced by laser ablation of the surface during the
scan. Mass spectrometric images were produced from a raster
Molecular imaging of tumor sections over an area of 8.5 mm 8 mm (image spots 100 m apart on
One our aims is the molecular analysis and imaging of peptides center). During the scan, we recorded images of ions in 45 mass
and proteins in brain tumors, specifically in human glioblas- ranges and the mass spectra were saved for further analysis.
toma. Such an analysis would be an important if not essential Three mass spectrometric images of molecules present in dis-
part of strategies designed to locate specific proteins that are tinct areas of the tumor are shown in Fig. 3bd. In this figure,
more highly expressed in tumors and those greatly diminished color is used to represent different ions, with color saturation a
in expression, relative to normal tissue. Currently, brain tumors function of the relative intensity (see color reference bar).
account for 2% of all cancer deaths, or about 11,000 deaths an- Overall, we detected over 150 different proteins, with many
nually in the United States. Gliomas account for 50% of all pri- being present in all parts of the tissue. Individual selected pro-
mary brain tumors, with glioblastomas compromising half of teins were identified as described below. We took three different
those12.
Here, tumor-bearing tissues were generated a b
by subcutaneous implantation of human
glioblastoma cells (D54) into the hind limb of
a nude mouse. After tumors grew to about 1
cm in diameter, we surgically removed them
from the mouse and immediately froze them
using liquid nitrogen. For image analysis, we
cut the tumor tissue using a microtome in 12-
m thick sections orthogonal to the point of
attachment to normal tissue. Frozen sections
were processed following the protocol de-
scribed above before image analysis by MS.
The optical image of a frozen human c d
glioblastoma section taken immediately fol-
lowing mass spectrometric imaging is shown

Fig. 3 Selected protein images from a glioblastoma

section. a, Human glioblastoma slice mounted on a
metal plate, coated with matrix (the lines are from
ablation of matrix with the laser). bd, Mass spectro-
metric images of proteins showing high concentra-
tion in the proliferating area of the tumor (d) and
other proteins present specifically in the ischemic and
necrotic areas (b and c).

494 NATURE MEDICINE VOLUME 7 NUMBER 4 APRIL 2001

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NEW TECHNOLOGY

Fig. 4 MALDI mass spectra taken at different locations within a glioblas-

toma slice (Fig. 3). Over 150 different peaks could be detected, with some
of them having a distinct spatial distribution in the tissue. Top, distal and
most active area of tumor proliferation; middle, an ischemic area; bottom,
a necrotic area of the tumor. The inset shows an expanded portion of the
spectrum in the region of thymosin .4.

ion spectrum (MS/MS analysis) using an electrospray quadru-

pole TOF mass spectrometer (Q-Star, Applied Biosystems/SCIEX,
2001 Nature Publishing Group http://medicine.nature.com

Foster City, California) from one of the tryptic fragments. The

MS/MS spectrum of the N-terminal tryptic peptide obtained
from a similar digest of T.4 purified from a mouse with prostate
cancer is shown in Fig. 6. Fragment ions were matched by iden-
mass spectra from different regions of the glioblastoma during tifying portions of the y and b ion series17, covering the com-
the scan (Fig. 4). These spectra clearly show differences in pro- plete sequence of the peptide. This spectrum confirmed the
tein expression in different parts of the tumor. presence of T.4 in mouse models of prostate cancer.
The proliferating area of the tumor was of particular interest Furthermore, from the MS/MS spectrum, the presence of an
with many proteins being expressed at higher levels relative to acetyl group at the N-terminal end of the T.4 peptide was con-
normal tissue. For example, the protein of molecular weight firmed. The protein of molecular weight 11,639 2 (Fig. 3c) was
4,964 (Fig. 3d) is localized only in the outer area of the tumor. similarly identified as S100 calcium-binding protein A4
Other proteins, such as that of molecular weight 41,662 (Fig. (S100A4), and the protein of measured molecular weight 41,659
3b), were localized in the necrotic area. In addition, other pro- 4 to be cytoplasmic actin.
teins were localized in the ischemic area between the necrotic
center and proliferating periphery, as shown for the protein Discussion
with a molecular weight of 11,639. To identify the mapped pro- The identification of specific tumor markers, for example T.4,
teins, we made an extract of the appropriate portion of the in the proliferating area of the tumors demonstrates the poten-
glioblastoma tissue, and then fractionated the proteins by tial of this technique to be used in intra-operative assessment of
HPLC. The UV chromatogram of such an extract is shown in the surgical margins of tumors. Currently, frozen sections and
Fig. 5. The on-line mass spectrometric analysis (Ion Trap, light microscopy are required for rapid decisions, but are, at
Finnigan Company, San Jose, California) performed using elec- times, inaccurate18,19. There is presently a need to develop tech-
trospray ionization MS easily permitted localization of the frac- nology to improve the accuracy of such decisions20,21. For exam-
tion containing the proteins of interest. For example, one of the ple, cancer invasion into muscle indicates that more extensive
proteins of molecular weight 4,964 eluted at 28.35 min in the surgery or adjuvant therapy is needed22,23 and intra-operative di-
chromatogram. We spotted a sample of this fraction onto a agnosis of central nerve system neoplasia is required for surgical
MALDI target plate and performed an on-target digestion by management2426. Clinical validation will determine the useful-
trypsin. We analyzed the digest by MALDI MS followed by a ness of imaging MS to demonstrate these pathologic criteria
database search in SwissProt using the software MoverZ accurately for more aggressive management of cancer.
(ProteoMetrics, New York, New York). Thymosin .4 (T.4) was Beyond the application of imaging MS to brain cancer research,
found to match the digest data precisely. The sequence analysis we are currently using this technology to study prostate and colon
of the amino-terminal peptide confirmed the identification of cancer development and progression. In both cases, numerous
the protein as T.4 in this human glioblastoma xenograft. tumor-specific markers have been identified and specifically local-
Increased expression of T.4 has been reported in a variety of ized within the tumors. Protein profiling and imaging MS are also
different tumors13. The localization of T.4 in the proliferating proving to be of prime importance in our current research aiming
area of the tumor correlates with previous findings of higher at a better understanding of prostate development. Overall, imag-
levels of T.4 in embryonic/neoplastic tissue compared with ing MS can be a valuable molecular tool in a wide variety of studies
normal/benign tissue14. One of the known activities of this im- and applications involving animal tissues.
munoregulatory peptide is its ability to sequester cytoplasmic
monomeric actin15. Moreover, actin filaments have been shown
to change into clump formation in apoptosis induced by anti-
tumor drugs, a process thought to be the result of decreased T.4
concentrations16.
We also observed the increased expression of T.4 in other tu-
mors as well. For example, in some mouse models of prostate
cancer, high levels of this protein have been found using imag-
ing MS. To confirm the identification, we generated a fragment

Fig. 5 UV chromatogram of a LC separation on a glioblastoma

xenograft extract. The analyte of molecular weight 4,964 was detected by
online electrospray mass spectrometry (inset shows mass spectrum) at a
retention time of 28.3 min.

NATURE MEDICINE VOLUME 7 NUMBER 4 APRIL 2001 495

 2001 Nature Publishing Group http://medicine.nature.com
NEW TECHNOLOGY

Methods
Tissue preparation. 12-m sections were cut from a frozen
mouse brain on a Leica CM 3000 cryostat at 15 C and di-
rectly picked up onto a gold-coated stainless steel plate. The
sections were immediately transferred to a cold room (4 C),
where 10 l of matrix (sinapinic acid, 10 mg/ml in acetoni-
trile/0.05% trifluoroacetic acid 50:50) were deposited with a
pipette in a line adjacent to the tissue and mechanically
spread over the tissue using a small plastic spatula. After air-
2001 Nature Publishing Group http://medicine.nature.com

drying for 45 min, the sections were dried for 2 h in a desic-

cator before mass spectrometric analysis. This application
technique results in formation of crystals of the organic ma-
trix on the surface of the tissue while minimizing the spread-
ing of sample molecules.

Glioblastoma extraction and protein fractionation by

Fig. 6 The mass spectrometric analysis by electrospray MS/MS of the N-
terminal tryptic fragment of T.4. The complete sequence of the frag- HPLC. A portion of the glioblastoma (82 mg) was immersed
ment was confirmed from the mass spectrometric data. in 500 l extraction buffer (0.25 M sucrose, 0.01 M Tris-HCl
and inhibitor mix; (Roche Molecular Biochemicals,
Switzerland), homogenized using a Duall homogenizer and
centrifuged 3 times (10 min at 680g, 10 min at 10,000g and
Acknowledgments 1 h at 55,000g), each time transferring the soluble fraction
We thank S. Schroeter, E. Sierra-Rivera, B. DaGue and Darell Bigner to a new tube. The final fraction (50 l) was separated over a
for help with this study. This work was supported by NIH grants GM C4 microbore column (Vydac, Hesperia, California), samples
58008 (to R.M.C.), CA 58506 (to D.H.) and C.A. 70937 (to D.H.). were collected and the separation run was recorded with a
UV detector set at 214 nm. Solvent A was 0.1 trifluoro acetic
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