Direct ionization of biological tissue for mass spectrometric analysis

Abstract

Analysis of biological tissue at a molecular level is of great importance in biological, medical and clinical studies. In this manuscript, we report that both plant and animal tissues can be directly ionized and analyzed by mass spectrometry under ambient conditions. By adding some solvents and applying a high voltage, spray ionization can be induced at the tip of biological tissue and a mass spectrum can be observed. Various plant and animal tissues have been tested and compounds such as lipids, alkaloids, glucosides, lignans, pharmaceuticals and proteins could be detected in the spectra. This new technique provides a simple and rapid method for tissue analysis and allows observation of compounds that cannot be detected by other ionization techniques.

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Huanwen Chen, Zhongping Yao et al.
Direct ionization of biological tissue
for mass spectrometric analysis

Direct ionization of biological tissue for mass spectrometric analysis†
Bin Hu,
abc
Ying-Han Lai,
ab
Pui-Kin So,
ab
Huanwen Chen*
c
and Zhong-Ping Yao*
ab
Received 7th December 2011, Accepted 16th April 2012
DOI: 10.1039/c2an16223g
Analysis of biological tissue at a molecular level is of great impor-
tance in biological, medical and clinical studies. In this manuscript,
we report that both plant and animal tissues can be directly ionized
and analyzed by mass spectrometry under ambient conditions. By
adding some solvents and applying a high voltage, spray ionization
can be induced at thetip of biological tissue and a mass spectrum can
be observed. Various plant and animal tissues have been tested and
compounds such as lipids, alkaloids, glucosides, lignans, pharma-
ceuticals and proteins could be detected in the spectra. This new
technique provides a simple and rapid method for tissue analysis and
allows observation of compounds that cannot be detected by other
ionization techniques.
Introduction
Analysis of biological tissue at a molecular level is an important task
in biological, medical and clinical studies.
1–3
Understanding molec-
ular compositions of tissue allows us to monitor growth, develop-
ment and variation of biological individuals, discover markers for
disease diagnosis and gain insight into the mechanism of diseases.
Conventional approaches for tissue analysis typically involve
homogenization, extraction and analysis of extracts, and are usually
time-consuming and labor intensive.
Mass spectrometry (MS) is a rapid and sensitive tool for qualitative
and quantitative analyses of various samples. The ambient ionization
techniques introduced in recent years have greatly facilitated sample
preparation for MS analysis.
4–7
Direct analysis of tissues by MS has
been achieved mainly with techniques including secondary ion mass
spectrometry (SIMS), desorption electrospray ionization (DESI) and
matrix-assisted laser desorption/ionization (MALDI).
8,9
These
techniques employ high energy ions,
10
charged microdroplets
11
and
laser (with the assistance of a matrix)
12
respectively to desorb and
ionize analytes on a tissue surface, and can be used for tissue imaging.
Among these three techniques, DESI imaging can be performed at
atmospheric pressure.
13
Other techniques for ambient imaging
include electrospray-assisted laser desorption/ionization (ELDI)
14
and probe electrospray ionization (PESI).
15
In ELDI, analytes on
a tissue surface are desorbed by laser then postionized by an ESI
fume;
14
while in PESI, a tissue surface is probed by a solid needle, and
the trace biological fluid adhering to the needle is then analyzed in
awaysimilartoESI.
15
Recently, paper spray was also used for tissue
analysis.
16
In this method, a tissue sample is loaded on a paper. When
solvent is added and a high voltage is applied to the paper, spray
ionization occurs at the paper tip, and compounds such as hormones,
lipids and therapeutic drugs could be detected from animal tissue.
We recently reported the electrospray ionization using wooden
tips.
17
Upon applying a high voltage, sample solution adhering to
a wooden tip (toothpick) can be sprayed out to generate a charac-
teristic mass spectrum. Wood is a plant material in nature. The
successful utilization of wooden tips for ionization led us to further
investigate direct ionization (DI) of plant tissue and other similar
materials. In this study, we report that both plant and animal tissues
can be directly ionized and analyzed by mass spectrometry.
Experimental methods
Materials
Herbal medicines, including Coptis chinensis Franch, Schisandra
sphenanthera,Schisandra chinensis, and crude and processed Polyg-
onum multiflorum, were purchased from pharmacy stores in Hong
Kong. Spinach leaves and animal tissues used in this study were
purchased from supermarkets in Hong Kong. Methanol, acetone and
formic acid were purchased from Fisher Scientific (New Hampshire,
U.S.), a-cyano-4-hydroxycinnamic acid (CHCA) from Fluka, los-
artan from Gracure Pharmaceuticals Ltd. (New Delhi, India), and
filter paper from Macherey Nagel (D€
uren, Germany). Extraction of
spinach leaf and herbal medicines was performed by vortexing 10 mg
of the homogenized sample and 500 ml of methanol–water (1/1, v/v)
for 1 min, and the supernatants were used for analysis.
Setup for direct ionization analysis of tissue samples
The experimental setup for DI analysis of tissue samples is shown in
Fig. 1. A tissue sample was held typically with a clip connected to the
a
State Key Laboratory of Chirosciences, Food Safety and Technology
Research Centre and Department of Applied Biology and Chemical
Technology, The Hong Kong Polytechnic University, Hung Hom,
Kowloon, Hong Kong Special Administration Region, China. E-mail:
bczpyao@polyu.edu.hk; Fax: +852 2364-9932; Tel: +852 34008792
b
State Key Laboratory of Chinese Medicine and Molecular Pharmacology,
Shenzhen Research Institute of The Hong Kong Polytechnic University,
Shenzhen 518057, China
c
Jiangxi Key Laboratory for Mass Spectrometry and Instrumentation, East
China Institute of Technology, Nanchang, Jiangxi Province 330013, China.
E-mail: chw8868@gmail.com; Fax: +86-791-3896-370; Tel: +86-791-
3896-370
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c2an16223g
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high voltage supply of a mass spectrometer. The tissue sample had
been cut to produce a sharp end, which was then placed pointing to
the MS inlet (see Fig. S1 in the ESI† for photos of the experimental
setup for analysis of tissue samples). The shape and size of the
analyzed tissue sample and the holding and connecting device are
variable, as long as the tissue sample can be steadily held with a sharp
end pointing to the MS inlet and is effectively connected to the high
voltage. After adding some solvents (usually 2 mL, may skip this for
very wet samples) and applying a high voltage, a plume of spray was
induced at the sharp end of the analyzed tissue, and a mass spectrum
could be observed. The high voltage applied was typically 3 kV,
under which desirable mass spectra could be generally obtained.
Further increasing the high voltage was found to increase the
tendency of undesirable electric discharge.
Mass spectrometry
Mass spectra were acquired on a QToF II mass spectrometer
(Waters, Milford, MA) using positive ion mode unless specified
elsewhere. DESI experiments were performed using a home-made
DESIionsourcewithspraysolvent methanol–water–formic acid
(1/1/0.1%) at a flow rate of 5 mLmin
1
. Paper spray experiments were
performed in a way similar to the literature
16,18
and with methanol–
water–formic acid (1/1/0.1%) as the solvent. Capillary voltages were
typically set at 3.0 kV for direction ionization, 3.5 kV for DESI,
3.5 kV for paper spray, and 1.5 kV for nano-ESI. These voltages were
chosen after optimization of each technique. LC-MS experiments
were performed using the CapLC liquid chromatography (Waters,
Milford, MA) coupled to the Q-ToF II mass spectrometer. Other
settings were similar to those for normal ESI analysis.
17
MALDI spectra were obtained using a MALDI Micro-MX time-
of-flight mass spectrometer equipped with a 337 nm UV laser source
(Waters, Milford, MA). For MALDI analysis of spinach leaf, a small
piece of spinach leaf was attached to a target plate with double-faced
adhesive tape, and a matrix solution of CHCA was loaded on the
leaf. The plate was introduced into the mass spectrometer for
MALDIanalysisafterthematrixsolutionbecamedry.Other
experimental settings were similar to those for normal MALDI
analysis.
19
Results and discussion
The DI mass spectrum obtained from a fresh spinach leaf with
methanol–water (1/1) as the added solvent is shown in Fig. 2a. The
spectrum was dominated with peaks that were identified as monog-
alactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol
(DGDG), the two most abundant membrane glycerolipids in higher
plant tissue,
20
based on their masses and MS/MS mass spectra
(Fig. S2 in the ESI†). These glycerolipids, however, could not be
detected by direct analysis of the spinach leaf (or the scratched
spinach leaf) using DESI, MALDI, paper spray (Fig. 2b–d) or
desorption atmospheric pressure chemical ionization (DAPCI).
21
DESI
9
and DAPCI
21–23
are usually used to detect analytes on surfaces
and DAPCI
21–23
is more suitable for analysis of relatively volatile
compounds. The endogenous glycerolipids may have strong affinity
inside the spinach leaf and thus could not be desorbed and ionized by
these two techniques. DAPCI has been reported to detect some
pigments from the spinach leaf. The undetectability of the glycer-
olipids by MALDI and paper spray was probably due to the signal
suppression. Analysis of the spinach leaf extract using MALDI,
paper spray (data not shown) or nano-electrospray ionization (nano-
ESI) (Fig. 2e) was also not able to detect the glycerolipids. These
results reveal that DI analysis of tissue is more straightforward and
allows detection of some compounds that cannot be detected by
other techniques of direct analysis. MGDG and DGDG play
important roles in plant photosynthesis.
24
Our results indicated that
DI could be a simple and rapid method for detection and monitoring
of these glycerolipids in plant growth and development.
Other chemical constituents, mainly including plant pigments,
commonly detected for spinach using LC-MS
25–28
were not observed
with DI. The spinach extract, obtained using extraction solvents of
methanol–H
2
O (1/1), the same solvent system used in DI, was
analyzed by LC-MS for a comparison in this study. In addition to
those glycerolipids observed with DI, more glycerolipids and a range
of plant pigments, such as phenophytin a and pyropheophorbide a,
were observed by LC-MS (Fig. S3 in ESI†), indicating that LC-MS
analysis gave more complete information about the composition of
spinach leaf. The reason why glycerolipids were predominately
detected in the DI spectrum could be that in response to cutting, the
glycerolipids significantly accumulated at the wounded region of the
leaf,
29
where the ionization occurred. Further investigation is required
for more detailed understanding of the DI mechanism.
Coptis chinensis Franch is a herbal medicine with biological effects
such as detoxification and prevention of sepsis and its complica-
tions.
30,31
A piece of dried Coptis chinensis Franch root was analyzed
by the DI method. The spectrum obtained (Fig. 3a) was very similar
to that obtained by analysis of Coptis chinensis Franch tissue using
MALDI,
32
DESI and paper spray, respectively (Fig. 3b and c), and
was in good agreement with the results obtained by analysis of the
Coptis chinensis Franch extract using LC-MS
33
and nano-ESI
(Fig. 3d) respectively. The predominant peaks at m/z320, 336, and
352 correspond to alkaloids coptisine, berberine/epiberberine, and
palmatine, respectively,
32,33
which can be easily ionized.
24
Our results
suggested that for dried tissue samples enriched with easily ionized
species, their DI spectra might be similar to those obtained by other
methods. The signal intensity of the major peaks obtained by DI was
comparable to those obtained by paper spray and nano-ESI, but
higher than that obtained by DESI (Fig. 3).
Both Schisandra sphenanthera and Schisandra chinensis are Fructus
Schisandrae.S. chinensis is mainly distributed in northern China, while
S. sphenanthera is mainly distributed in southern China.
31
The dried
ripe fruits of these two plants have long been used as herbal medicines,
but their quality is different due to their differences in contents of
lignans.
34
Differences in lignan constituents, e.g.,Schisandrin
and Schisandrol B, between Schisandra sphenanthera and Schisandra
chinensis could be easily observed by DI analysis of the dried fruits of
these plants (Fig. 4). The spectra obtained were very similar to those
Fig. 1 Experimental setup for DI analysis of biological tissue by MS.
3614 | Analyst, 2012, 137, 3613–3619 This journal is ªThe Royal Society of Chemistry 2012

previously obtained by analysis of methanol extracts of two herbs
using ESI-MS.
35
These lignan constituents, however, were not
observed in a recent DAPCI-MS study, in which only volatile
compounds, e.g., terpenoids, on the herb surface could be detected.
36
The rhizome of Polygonum multiflorum is another common herbal
medicine. The crude rhizome of P. multiflorum is toxic and needs to
be processed before it can be used as a medicine. The processing
involves hydrolysis of toxic glucoside compounds in the crude
rhizome of P. multiflorum into nontoxic deglycosylated
compounds.
37,38
DI mass spectra of crude and processed rhizomes of
P. multiflorum are shown in Fig. 5. Abundant peaks of 2,3,5,40-tet-
rahydroxystilbene-2-O-b-D-glucoside were observed in the spectrum
Fig. 2 Mass spectra of the fresh spinach leaf obtained by (a) DI, (b) DESI, (c) MALDI, (d) paper spray, and (e) nano-ESI (analysis of extract).
This journal is ªThe Royal Society of Chemistry 2012 Analyst, 2012, 137, 3613–3619 | 3615

of the crude medicinal herb. These peaks almost totally disappeared
in the spectrum for the processed rhizome of P. multiflorum,andthe
corresponding deglycosylated products could be observed. These
results demonstrated that DI is a simple, rapid and effective method
for detection of major constituents of various plant tissues and for
monitoring changes of these constituents, and can be used for
differentiation of plants, e.g.,herbalmedicines,from different sources
and different processing methods.
Some other common plant tissues were also investigated. As
shown in the preliminary results depicted in Fig. S4†, various
chemical constituents, such as sugars and amino acids, could be
detected in different plant materials. Further refinement of the
method will be performed to detect a broader range of chemicals in
plant materials of different texture and morphology.
DI analysis of animal tissue was also tested in this study. Animal
tissue is usually much softer than plant tissue and was held by
a stainless steel needle for DI analysis in this study (see Fig. S1b†).
Fig. 6a is the spectrum obtained for porcine heart using methanol–
water (1/1) as the added solvent. Similar to the results obtained by
DESI
11
or paper spray
16
for animal tissue analysis, phospholipids
such as phosphatidylcholine (PC) were predominantly observed in
this spectrum, and in the spectra of other animal tissues such as
porcine liver, porcine kidney, porcine spleen, porcine medulla,
porcine lung, bovine muscle, fish gill and fish heart (Fig. S5†). Lipids
are important compounds for energy storage and construction of cell
membranes, and are potential biomarkers for some diseases.
39,40
These results demonstrated that the DI method could be used for
rapid detection of lipids from various animal tissues.
Fig. 3 Mass spectra of Coptis chinensis Franch obtained by (a) DI, (b) DESI, (c) paper spray, and (d) nano-ESI (analysis of extract). Intensity of each
spectrum is labelled on the upper right corner.
3616 | Analyst, 2012, 137, 3613–3619 This journal is ªThe Royal Society of Chemistry 2012

Localization and detection of therapeutic drugs and their metab-
olites in animal tissue are important in pharmacokinetic studies.
16
DI
was attempted for such an analytical purpose in this study. One ng of
losartan, a drug for treatment of high blood pressure,
41
was spiked
onto 5 mg of porcine kidney. After solvent vaporization, the tissue
sample was analyzed by the DI approach with addition of 1 mLof
Fig. 4 DI mass spectra of S. sphenanthera and S. chinensis fruits, acquired on a triple-quadrupole mass spectrometer (Quattro Ultima, Waters, Milford,
MA), with methanol–water (1/1) as the added solvent. Intensity of each spectrum is labeled on the upper right corner.
Fig. 5 DI mass spectra of (a) crude and (b) processed rhizomes of P. multiflorum, obtained with methanol–water (1/1) as the added solvent.
This journal is ªThe Royal Society of Chemistry 2012 Analyst, 2012, 137, 3613–3619 | 3617

methanol. Protonated molecules and salt adducts of losartan were
clearly observed in the spectrum (Fig. 7a) and further confirmed by
the MS/MS study (data not shown). When a similar size of tissue
sample spiked with the same amount of losartan was extracted with
a minimal volume of methanol solvent, i.e.,20mL, and the extract
was analyzed by LC-ESI-MS, only protonated molecules of the
target compound were observed and the ion intensity obtained was
significantly lower than that in DI (Fig. 7b). These data suggested
that DI analysis was more straightforward and could offer higher
sensitivity than LC-MS. As shown in Fig. 7c, the signal intensity of
losartan was found to have a linear relationship with the spiked
amount of the pharmaceutical compound over two orders of
magnitude of sample concentrations. The precision of quantitative
data achieved was 30%, comparable to quantitative analysis using
DESI in some applications.
42
Although further investigation is still
needed for the real application and improvement of reproducibility
and linear dynamic range of the method, these preliminary results
indicated that the DI method has potential application in qualitative
and quantitative analyses of therapeutic drugs in animal tissue.
Detection of proteins directly from animal tissue by MS is of
significant interest due to the important roles of proteins in biological
processes. However, such detection can only be achieved by very few
mass spectrometric techniques
4
such as MALDI
12
and ELDI-MS.
14
In this study, by using methanol–acetone (1/1) containing 0.1% for-
mic acid as the added solvent, protein signals were successfully
observed in the DI mass spectrum of fresh porcine heart. As shown in
Fig. 6b, aand bsubunits of hemoglobin were observed, along with
abundant peaks of heme at the low mass region. The more hydro-
phobic solvents favor the observation of proteins, indicating that DI
analysis is a solvent-dependent extraction process. The detection of
proteins from animal tissue suggested that the DI technique could be
a potential tool for diagnosis of diseases such as hemoglobinopathy.
43
Conclusions
In summary, we have demonstrated that both plant and animal
tissues can be directly ionized and characteristic mass spectra can be
generated under ambient conditions. The experimental setup of this
new technique is very simple and analysis of one tissue sample can be
completed within one minute. Various plant and animal tissues have
been tested and compounds such as lipids, alkaloids, glucosides,
lignans, pharmaceuticals and proteins were observed in the spectra.
DI analysis can be considered as a complementary tool to tissue
imaging. In DI analysis, spray ionization directly occurs on the tissue
sample. The analysis is straightforward and allows us to observe
some compounds that cannot be detected by other direct MS tech-
niques. Although further investigation about the detailed mechanism
of this new technique is still required, our preliminary results indicate
that the directly applied high voltage, the added solvents, the textile
structure of tissue, and the properties and distribution of analytes
inside tissue are important factors for the spectra observed. Further
applications of this new technique are being studied.
Notes
The study in this paper was originally reported in an academic
meeting in May 2011.
44
During the submission of this paper, we
noticed two latest publications reporting direct mass spectrometric
analysis of plant tissue
45
and animal tissue.
46
Fig. 6 DI mass spectra of porcine heart. The two spectra were acquired
with (a) methanol–water (1/1) and (b) methanol–acetone (1/1) containing
0.1% formic acid as the added solvents respectively.
Fig. 7 Mass spectra of porcine kidney after spiking with losartan
obtained by (a) DI and (b) LC/MS. Intensity of each spectrum is labeled
on the upper right corner. (c) Linear relationship between signal intensity
of the losartan ion and concentration of the spiked losartan solution was
observed when 2 mL of losartan solution was spiked onto 5 mg of porcine
heart tissue. Signal intensity of the losartan ion was measured using
selected reaction monitoring (m/z423 > m/z207) on the triple-quadrupole
mass spectrometer.
3618 | Analyst, 2012, 137, 3613–3619 This journal is ªThe Royal Society of Chemistry 2012

Acknowledgements
This research was supported by The Hong Kong Polytechnic
University (grant no. A-PD1C, A-PH85, A-PK82 and A-PL48) and
NSFC (grant no. 20827007).
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