Deuterated matrix-assisted laser desorption ionization matrix uncovers masked mass spectrometry imaging signals of small molecules

Article abstract of DOI:10.1021/ac301498m

D⁴-α-Cyano-4-hydroxycinnamic acid (D⁴-CHCA) has been synthesized for use as a matrix for matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) and MALDI-MS imaging (MSI) of small molecule drugs and endogenous compounds. MALDI-MS analysis of small molecules has historically been hindered by interference from matrix ion clusters and fragment peaks that mask signals of low molecular weight compounds of interest. By using D⁴-CHCA, the cluster and fragment peaks of CHCA, the most common matrix for analysis of small molecules, are shifted by + 4, + 8 and + 12 Da, which expose signals across areas of the previously concealed low mass range. Here, obscured MALDI-MS signals of a synthetic small molecule pharmaceutical, a naturally occurring isoquinoline alkaloid, and endogenous compounds including the neurotransmitter acetylcholine have been unmasked and imaged directly from biological tissue sections.

Full text of DOI:10.1021/ac301498m

Article
pubs.acs.org/ac
Deuterated Matrix-Assisted Laser Desorption Ionization Matrix
Uncovers Masked Mass Spectrometry Imaging Signals of Small
Molecules
Mohammadreza Shariatgorji,† Anna Nilsson,† Richard J. A. Goodwin,†,‡ Per Svenningsson,§
Nicoletta Schintu,§ Zoltan Banka,∥ Laszlo Kladni,∥ Tibor Hasko,∥ Andras Szabo,∥ and Per E. Andren*,†
†Department of Pharmaceutical Biosciences, Medical Mass Spectrometry, Uppsala University, Box 591 BMC, SE-751 24 Uppsala,
Sweden
‡Global Distribution Imaging, DMPK IM, AstraZeneca R&D, Sodertalje, Sweden
̈
̈
§Translational Neuropharmacology, Department of Clinical Neuroscience, Center for Molecular Medicine, Karolinska Institute,
SE-171 76 Stockholm, Sweden
∥Ubichem Research Ltd., Illatos ut 33, 1097 Budapest, Hungary
S
* Supporting Information
ABSTRACT: D4-α-Cyano-4-hydroxycinnamic acid (D4-
CHCA) has been synthesized for use as a matrix for matrix-
assisted laser desorption ionization-mass spectrometry
(MALDI-MS) and MALDI-MS imaging (MSI) of small
molecule drugs and endogenous compounds. MALDI-MS
analysis of small molecules has historically been hindered by
interference from matrix ion clusters and fragment peaks that
mask signals of low molecular weight compounds of interest.
By using D4-CHCA, the cluster and fragment peaks of CHCA,
the most common matrix for analysis of small molecules, are shifted by + 4, + 8 and + 12 Da, which expose signals across areas of
the previously concealed low mass range. Here, obscured MALDI-MS signals of a synthetic small molecule pharmaceutical, a
naturally occurring isoquinoline alkaloid, and endogenous compounds including the neurotransmitter acetylcholine have been
unmasked and imaged directly from biological tissue sections.
atrix-assisted laser desorption ionization (MALDI) mass
spectrometry (MS) is a soft ionization technique which,
weight (typically <600 Da). These ions can significantly mask
the signal of any analytes with a similar mass-to-charge (m/z)
ratio, hence limiting the ability to analyze some endogenous
small molecules, pharmaceutical compounds, and metabolites.10
To overcome the problem, several strategies can be
attempted. The simplest is to switch to an alternative MALDI
matrix which will generate a different set of matrix cluster ions.
However, different MALDI matrices have different ionization
properties and will often not enable sufficient ionization of the
target analyte. More complex approaches have been developed
which includes desorption ionization from porous silicon
(DIOS),11 nanostructure initiator mass spectrometry
(NIMS),12 high molecular weight matrices,13 and surface-
assisted laser desorption ionization.14 Despite some success,
these methods have never been as widely used as MALDI
because of the technical and handling difficulties for conven-
tional MS and especially MS imaging.
M
when coupled to one of a broad range of mass analyzers,
enables high sensitivity analyte detection and is used across a
diverse variety of research areas.1 Since the development of the
technique by Hillenkamp and Karas,2 it has been utilized for the
analysis of biological macromolecules (peptides,3 proteins, and
protein complexes4), synthetic polymers,5 and even nano-
particles.6 MALDI-MS imaging (MALDI-MSI) is an application
of the technology that enables label-free compound distribution
mapping of molecular species directly from tissue sections.7 By
performing MALDI-MSI analysis directly on the surface of a
tissue section, the technique has quickly been established as a
powerful in situ visualization tool for measuring relative
abundance and spatial distribution of endogenous and
pharmaceutical small molecule compounds, lipids, and
proteins.8,9
MALDI works through the cocrystallization of the MALDI
matrix, an organic compound, with analytes in the sample. The
matrix is able to absorb the laser energy pulse (typically at a
wavelength of 355 or 337 nm) which then causes the
subsequent desorption and ionization of the analyte and
matrix. However, during the ionization process the matrix
generates fragment and cluster ions which are low in molecular
Following the development of α-cyano-4-hydroxycinnamic
acid (CHCA),15 it has become the most commonly used
Received: June 1, 2012
Accepted: July 18, 2012
Published: July 18, 2012
© 2012 American Chemical Society
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a
Scheme 1. Synthesis of D4-CHCA
a
(a) Br2/dioxane, 0 °C; (b) TBDMSCl/DMF, imidazole/DMF, RT; (c) n-BuLi, THF, N2, −65°C, DMF then aq HCl, 0 °C; (d) methanol/HCl, N2,
RT; (e) cyanoacetic acid ethyl ester, piperidine, toluene, 5h; (f) LiOH, RT, 2 h, water then aq HCl.
MALDI matrix for both spotted sample and tissue imaging
MALDI analysis and it is very effective for the ionization of
small molecules. However, there are a large number of cluster
peaks detected <600 Da, causing complications in analyte
identification or quantitation. By synthesizing D4-CHCA that
retains the same physicochemical properties of the standard
CHCA (the acidity and light absorption properties) means that
the m/z ratios of matrix cluster and fragments peaks are shifted.
Therefore, by alternating between D4-CHCA and standard
CHCA the entire lower mass range can be analyzed without
interference of the matrix cluster, while retaining the effective
ionization properties of the CHCA matrix.
The C57Bl/6 male mouse, 3 months old, was housed in air-
conditioned rooms (12 h dark−light cycles) at 20 °C with a
humidity of 53%. The mouse was euthanized, and the brain was
rapidly removed, frozen in dry ice-cooled isopentane, and
stored at −80 °C.
The frozen lung and brain tissues were cut by a cryostat-
microtome (Leica CM3050S, Leica Microsystems, Germany).
Lung sections were taken from the long flat frontal plane of the
left lobe, providing sections of the central airways at the
thickness of 12 μm. Control lung tissue sections and a lung
tissue section from a dosed animal were placed on the same
MALDI target glass slide. Sagittal brain sections were cut at a
thickness of 12 μm. Tissue sections were transferred by thaw
mounting onto conductive indium tin oxide (ITO) glass slides
(Bruker Daltonics, Bremen, Germany), prior to storage at −80
°C. Sections were desiccated at room temperature for 15 min
prior to MALDI matrix application. Tissue sections were not
washed prior to matrix application.
MALDI-MS and MALDI-MSI Analysis. For MALDI-MS
analyses, 0.5 μL of matrices, (CHCA and D4-CHCA, 7 mg/mL,
50% acetonitrile, 0.2% TFA) were spotted and dried on a
stainless steel MALDI plate (Bruker Daltonics). Berberine (0.5
μL of 2 pmol/μL in 50% acetonitrile, 0.2% TFA) was spotted
on dried spots of CHCA and D4-CHCA. Collected spectra
were summed from 500 laser shots (355 nm Nd:YAG
Smartbeam laser, Ultraflex II MALDI time-of-flight (TOF)/
TOF mass spectrometer, Bruker Daltonics, Bremen, Germany).
For MALDI-MS imaging analyses the MALDI matrices
(CHCA and D4-CHCA, 7 mg/mL in 50% acetonitrile, 0.2%
TFA) were applied to the MALDI glass targets using an
automatic matrix sprayer (ImagePrep, Bruker Daltonics).
MALDI-MS and MALDI-MSI analyses were performed using
an Ultraflex II TOF/TOF mass spectrometer (Bruker
Daltonics) in positive ion reflectron mode using a Smartbeam
II 200 Hz laser. The mass spectrometer parameters were as
manufacturer’s recommended settings adjusted for optimal
acquisition performance. The laser spot size was set at medium
focus (∼50 μm laser spot diameter), and laser power was
optimized at the start of each run and then fixed for the
MALDI-MSI experiment. Tissue sections were analyzed in a
random order to prevent any possible bias due to such factors
as matrix degradation or variation in mass spectrometer
sensitivity. MSI data was analyzed and normalized using
FlexImaging, version 2.0 (Bruker Daltonics). Regions of
interest were manually defined in the analysis software using
both the optical image and MSI data image. Masses were
selected with a mass precision of 0.1 Da.
EXPERIMENTAL SECTION
■
Chemicals and Reagents. All chemicals and reagents were
purchased from Sigma Aldrich (St. Louis, MO) unless
otherwise noted and were used without further purification.
Phenol-2,3,4,5,6-d5 (D5-phenol) was purchased from CDN
Isotopes Inc. (Essex, U.K.). Water, methanol, trifluoroacetic
acid (TFA), and TLC plates were obtained from Merck
(Darmstadt, Germany).
Synthesis of D4-CHCA. D4-CHCA was synthesized using
phenol-2,3,4,5,6-d5 (D5-phenol) as the starting material as
shown in Scheme 1. In short, D5-phenol was brominated, and
the product was then protected by tert-butyldimethylsilyl
(TBDMS) in dimethylformamide (DMF) at ambient temper-
ature in the presence of imidazole. The product was purified by
distillation under reduced pressure. 4-Hydroxybenzaldehyde
was synthesized in tetrahydrofuran (THF) at −65 °C using n-
butyllithium (n-BuLi) and DMF. 4-Hydroxycinnamic ester was
prepared using methyl cyanoacetate in the presence of
piperidine in toluene. The final product was obtained by
hydrolysis with lithium hydroxide (LiOH) followed by
crystallization from 2-propanol. The detailed synthesis
procedure is described in the Supporting Information.
Animal Experiments and Tissue Preparation. Animal
experiments were conducted in accordance with the European
Communities Council Directive of November 24, 1986 (86/
609/EEC) approved by the Ethical Committees on Animal
Experiments in Lund/Malmo, Sweden (no. M84-05) and at
̈
Karolinska Institute, Stockholm, Sweden (no. N351/08). Adult
male wistar rats weighing approximately 350 g were used. The
animals were kept in a climate controlled facility (22 3 °C, 55
10% relative humidity) with 12 h light−dark cycles. Food
and water were given ad libitum. Amiloride was administered
(800 μg/kg) through intratracheal injection. Control and dosed
animals were anesthetized and sacrificed 2 h post admin-
istration. The lung and brain were rapidly dissected out and
immediately placed in a fulminating bath of isopentane/dry ice
on a hard plastic dish. The samples were stored at −80 °C until
required for sectioning.
RESULTS AND DISCUSSION
■
The study reported here describes the synthesis and use of D4-
CHCA for the detection of pharmaceutical and endogenous
compounds that could not previously be detected due to
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Figure 1. MALDI mass spectra of CHCA (black) and D4-CHCA (red). The fragment/cluster peaks in the low mass range are shifted by + 4, + 8,
and + 12 Da when D4-CHCA is used as an alternative matrix to uncover the hidden m/z ratios.
Figure 2. (a) MALDI mass spectra of CHCA, (b) D4-CHCA, and (c) berberine acquired by D4-CHCA as matrix. The overlapping peak of CHCA at
336.1 Da masks the signal of berberine while the clean background of D4-CHCA in this region facilitates uncovering the berberine signal.
interference by CHCA matrix cluster peaks. D4-CHCA is used
for the analysis of both spotted sample and tissue sample
analysis.
More than 50 fragment/cluster peaks of CHCA in the low
molecular weight region (100−600 Da) of the mass spectra
were found to be shifted when using the deuterated matrix. A
ChemSpider chemical database search (Royal Society of
Chemistry, Cambridge, U.K.) resulted in approximately 19
million known compounds in the target mass region (100−600
Da), and of that 19 million, approximately 1.5 million would
potentially be masked by the interfering fragments/clusters of
CHCA given the centroid masses of fragment/cluster peaks of
0.1 Da (typical of mass analyzers such as quadrupole, ion trap,
and time-of-flight). Therefore, by shifting fragment/cluster
peaks it can be anticipated that a significant number of
endogenous and synthetic compounds may well be detected
Following the synthesis of D4-CHCA matrix, the phys-
icochemical properties of the product were analyzed to confirm
that no detrimental effect had occurred. The D4-CHCA
produced was found to have the same light absorption pattern
as standard CHCA as shown in the Supporting Information
(Figure S1). Initial comparison between CHCA and D4-CHCA
was made by spotting of each matrix onto a stainless steel
MALDI target and collecting spectra as described in the
Experimental Section. The CHCA clusters and fragments were
shifted by + 4, + 8, and + 12 Da (Figure 1).
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Figure 3. MALDI-MSI relative abundance and distribution of m/z 230.0 0.1 in rat lung tissue sections. (a) The cluster product of CHCA at m/z
230.0 is observed on the control tissue covered by CHCA as the matrix. (b) No signal at m/z 230.0 is observed on the control tissue when D4-
CHCA is used as the matrix, which facilitates an appropriate condition for imaging of amiloride. (c) The spatial distribution of amiloride is imaged in
a rat lung tissue section of an in vivo amiloride administered rat using D4-CHCA as the matrix. Difficulty in sectioning lung tissue produced samples
with different morphology. (Data displayed in the rainbow scale over the same range, scale bar 2 mm, and the dashed line marks the outline of the
tissue section).
when using D4-CHCA as the MALDI matrix. Absolute intensity
values are not always reliable during MALDI analysis. Different
factors such as matrix crystal structures and the amount of salts
in the sampling spots can affect the detected abundance of
target compounds. However, from Figure 1 it is clear that D4-
CHCA gives a higher detected abundance for molecular ions
and fragments while the signal abundance for matrix clusters
are almost the same. This could be attributed to C−D, C−H
bond energy values and gas phase reactivity differences.
Berberine (m/z 336.1), a naturally occurring potent
therapeutic isoquinoline alkaloid,16 is an example of a small
molecule pharmaceutical compound which is masked from
detection by MALDI by a matrix cluster of CHCA at m/z
336.1, as demonstrated in Figure 2. The use of D4-CHCA shifts
the matrix cluster by +8 Da and allows the unmasked detection
of berberine. Therefore, by spotting a sample twice, once with
standard and once with D4-CHCA, it is possible to mitigate the
risk of matrix cluster interference and unfettered access to the
whole mass spectrum is obtained.
acceptable MS/MS spectra and identification was based on the
corresponding m/z ratio.
The suitability of D4-CHCA for use in MALDI-MSI
detection and the mapping of the relative abundance of small
molecule endogenous compounds across tissue sections was
examined and shown to be an effective strategy in enabling the
detection of compounds that were previously obscured by
standard CHCA matrix cluster and fragment peaks. An example
of how the use of D4-CHCA can allow a previously masked
endogenous compound in a mouse brain section to be detected
is given in Figure 4. When the distribution of m/z 250.1 is
mapped using the standard CHCA matrix, the strong signal
obtained from the matrix cluster masks any possible detection
of endogenous compounds with closely related masses (Figure
4a,c). However, when using D4-CHCA the matrix cluster peak
is shifted by +4 Da enabling detection of endogenous
compounds at m/z 250.1 (Figure 4b,c). Subsequent MS/MS
fragmentation of m/z 250.1 generated product ions of m/z 184
and m/z 136 that may indicate a phosphocholine functional
group.
D4-CHCA can also be demonstrated to provide a means to
analyze small molecule synthetic pharmaceutical compounds
that have m/z ratios that are masked by the standard CHCA
matrix. Amiloride is an antikaliuretic-diuretic pharmaceutical
used for management of congestive heart failure and hyper-
tension. The third isotopic peak of a CHCA cluster overlaps
with protonated amiloride (m/z 230.0) and hence prohibits its
detection and distribution to be determined by MALDI
analyses. Following the administration of amiloride to rats,
tissue sections were coated in either standard or D4-CHCA
prior to MALDI-MSI analysis. Figure 3a shows the distribution
of m/z 230.0 across a drug treated lung tissue section coated by
standard CHCA. Because of the overlapping between the
matrix cluster and the amiloride peak, no discernible
distribution can be observed. However, when D4-CHCA is
applied to the control lung tissue section, no matrix cluster peak
is detected at m/z 230.0 (Figure 3b) but in drug treated lung
tissue the distribution of protonated amiloride (m/z 230.0) is
clearly detected without the interference of any matrix cluster
peaks (Figure 3c). We submitted amiloride ions to MS/MS
analysis directly from tissue sections in an attempt to acquire a
specific identification of the drug, but we were unable to obtain
Acetylcholine (ACh) is an endogenous neurotransmitter that
plays important roles in the peripheral and central nervous
system17 but has a mass (m/z 146.1) preventing MALDI-MSI
analysis with CHCA. When the abundance and distribution of
m/z 146.1 is mapped across a tissue section coated by standard
CHCA matrix, the strong signal obtained from the matrix
fragment masks any possible mapping of ACh. However, when
using D4-CHCA as the matrix the fragment peak is shifted by +
4 Da meaning that any detected signal is as a result of
endogenous compounds. The resulting distribution map shows
the molecular distribution of ACh in brain tissue sections
(Figure 5). The confirmation that m/z 146.1 is ACh was
achieved by on-tissue tandem MS (MS/MS) fragmentation of
m/z 146.1 (Figure S2 in the Supporting Information), where
product ions of m/z 87.0 and m/z 104.1 are generated. The
MALDI-MSI data shows that ACh is most abundant in brain
structures such as the cerebral cortex, corpus callosum, ventral
hippocampal commissure, thalamus, and cerebellum (Figure 5).
An alternative approach to overcome matrix cluster and
fragment peak overlap would be the use of higher spectral
resolution mass analyzers able to resolve the cluster and
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Figure 5. MALDI-MSI relative abundance and spatial distribution of
the neurotransmitter acetylcholine (m/z 146.1 0.1) in a rat brain
sagittal tissue sections. The image corresponds to the tissue section
covered by D4-CHCA as the matrix. Unlike in Figures 2 and 4, a mass
spectrum with no interference at m/z 146.1 is not presented for this
example.
matrix cluster peaks when analyzed using standard CHCA.
Through a combination of standard and D4-CHCA matrices,
researchers will be able to eliminate matrix interferences but
still keeping the advantages of CHCA ionization efficiency for
both MALDI-MS and MALDI-MSI analyses. It is believed that
the matrix we have developed can be readily implemented in
targeted and nontargeted MALDI-MS and MALDI-MSI
workflows and will be of substantial benefit for researchers
working within the pharmaceutical, metabolomics, and environ-
mental fields. While an alternative method of overcoming
analyte masking by overlapping matrix peaks could be the use
of higher spectral resolution mass analyzers, there is often a lack
of sensitivity in the low mass range where such masking occurs
that makes detection of low abundance masked compounds
impossible. There is also often limited availability of high-
resolution instruments for MSI, compared to the more
prolifically employed lower resolution instruments. Hence,
the use of D4-CHCA is clearly demonstrated as a powerful
addition to matrices used for the analysis of small molecule
compounds.
Figure 4. MALDI-MSI relative abundance and distribution of an
endogenous compound with m/z 250.1 0.1 in mouse brain tissue
sections. (a) The cluster product of CHCA at m/z 250.1 is observed
on the tissue covered by CHCA as the matrix. (b) The spatial
distribution of the endogenous compound is imaged in a mouse brain
tissue section using D4-CHCA as the matrix. (c) MALDI mass spectra
of CHCA (red-dashed line), D4-CHCA (black-solid line), and the
endogenous compound unmasked by D4-CHCA (green-dashed line).
(Data displayed in the rainbow scale over the same range, scale bar 2
mm, and the dashed line marks the outline of the tissue section). The
overlapping peak of CHCA at m/z 250.1 masks the signal of the
endogenous compound appearing in the same mass while the clean
background of D4-CHCA in this region facilitates uncovering and
imaging of the endogenous compound.
ASSOCIATED CONTENT
* Supporting Information
Additional information as noted in text. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*E-mail: per.andren@farmbio.uu.se. Phone: +46 − 18 471
7206.
■
compound masses. Attempts were made to detect ACh using a
12 T Fourier transform ion cyclotron resonance-mass
spectrometer (FTICR-MS, Bruker Daltonics) equipped with
the same type of MALDI laser; however, because of the lack of
sensitivity toward the very low mass range region it was not
possible to detect ACh. Hence, the use of D4-CHCA is clearly
demonstrated as a powerful addition to matrices used for the
analysis of endogenous small molecule compounds.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work is financially supported by Swedish Research Council
under Grant Numbers 2008-5597, 2009-6050, 2010-5421, and
2010-0881 and the Knut and Alice Wallenberg Foundation.
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