Laser Desorption Postionization Mass Spectrometry Imaging of Folic

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Laser Desorption Postionization Mass Spectrometry Imaging of Folic Acid Molecules in Tumor Tissue Qiao Lu, Yongjun Hu, Jiaxin Chen, and Shan Jin Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b00140 • Publication Date (Web): 12 Jul 2017 Downloaded from http://pubs.acs.org on July 13, 2017

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Analytical Chemistry

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Laser Desorption Postionization Mass Spectrometry Imaging

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of Folic Acid Molecules in Tumor Tissue

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Qiao Lu, Yongjun Hu*, Jiaxin Chen and Shan Jin

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MOE Key laboratory of Laser Life Science & Institute of Laser Life Science, College of

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Biophotonics, South China Normal University, Guangzhou 510631, P. R. China

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* Corresponding author. Tel: 86-20-85217070; fax: 86-20-85216052

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Email: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT

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Mass spectrometry imaging (MSI) is an innovative and powerful tool in biomedical

3

research. It is well known that folic acid (FA) has a high affinity for folic acid receptor

4

(FR) which is overexpressing in epithelial cancer. Herein, we propose a novel method to

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diagnose cancer through direct mapping of the label-free FA spatial distribution in tissue

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sections by state-of-the-art laser desorption postionization mass spectrometry imaging

7

(LDPI-MSI). Compared with other tumor imaging methods, such as fluorescence

8

imaging, photoacoustic imaging (PAI), magnetic resonance imaging (MRI) and

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micro-SPECT/CT, complicated synthesis and labeling processes are not required. The

10

LDPI-MSI was performed on 30 µm thick sections from a murine model of breast cancer

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(inoculation of 4T1 cells) that were pre-dosed with 20 mg/kg of FA. The image obtained

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from the characteristic mass spectrometric signature of FA at m/z 265 illustrated that FA

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was concentrated primarily in tumor tissue and displayed somewhat lower retention in

14

adjacent normal controls. The results suggest that the proposed method could be used

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potentially in cancer diagnosis.

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KEYWORDS: folic acid, folic acid receptor, tumor, diagnosis, laser desorption

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postionization mass spectrometry imaging

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2


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INTRODUCTION

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With ever increasing incidence and mortality, cancer has become one of the leading

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causes of death and a major threat to public health in recent years.1 In the context of

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cancer diagnosis, the need for detecting tumor biomarkers in biopsy specimens has,

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therefore, become an urgent matter. In this respect, tumor tissue samples are extremely

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significant because they may reveal the complex nature and heterogeneity of the disease2

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Analytical tools that provide molecular information associated with cancers are thus

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critical to both diagnosis and targeted treatment of the disease.

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As is well known, folic acid (FA) or folate, as a water-soluble B vitamin, is a typical

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antibody for active tumor targeting because FA is vital for cell metabolism, DNA

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synthesis

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phosphatidylinositol-anchored membrane protein that binds FA with a high affinity for

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mediate cellular uptake of FA.4 Although barely expressed in normal tissues, FR is

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expressed at high levels and widely distributed in tumors to satisfy the abundant FA

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requirement.5 Thus, the administration of FA and FA conjugated drugs has been exploited

16

for diagnosis and chemotherapy of many epithelial neoplasms.6-8

and

gene

repair.3

Folic

acid

receptor

(FR)

is

a

glycosyl

17

Recently, a variety of FA receptor-targeted imaging agents have been developed for

18

carcinoma diagnosis by fluorescence imaging, photoacoustic imaging (PAI), magnetic

19

resonance imaging (MRI) and micro-SPECT/CT. For instance, Kennedy et al.

20

demonstrated that their proposed method had the ability to visualize folic acid

21

receptor-expressing cancer tissues in various peritoneal, subcutaneous, and metastatic 3



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1

murine tumor models following intravenous administration of a folic acid-fluorescein

2

conjugate.9 Zhang et al. used FA-targeted iron oxide (Fe3O4) nanoparticles (NPs) as a

3

T2-negative contrast agent for MRI to accurately detect ovarian cancer tissues in an

4

intraperitoneal xenograft tumor model.10 Guo et al. synthesized 99mTc-Labeled dimeric FA

5

for FR-Targeting and from the in vivo micro-SPECT/CT imaging results, good tumor

6

uptake of 99mTc-HYNIC-D1-FA2 was observed in KB tumor-bearing mice.11 Very recently,

7

a FA ligand conjugated lipid-polyaniliane hybrid NP (FA-lipid-PANI NP) was used for

8

PAI guided photothermal therapy of cancer.12 These FA-based targeting diagnostic

9

strategies have advantages in several aspects, especially for in vivo imaging. However,

10

the drawbacks include the need for complex synthesis and a tedious labeling process.

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Mass spectrometry (MS) provides a powerful platform for detecting small molecular

12

compounds, metal elements and macromolecules such as proteins and nucleic acids.

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Attractive features of the technique include very high sensitivity, high throughput and the

14

ability to provide accurate atomic/molecular weight and structural information.13

15

Nowadays, matrix-assisted laser desorption/ionization-mass spectrometry imaging

16

(MALDI-MSI) has become a powerful tool for analyzing the spatial distribution of

17

thermally labile large molecules, such as peptides and proteins within intact tissue.14 The

18

past decade has also witnessed the huge progress of MALDI-MSI in the cancer research

19

pipeline, especially for the identification of the potential cancer biomarkers.15-17 Despite

20

its popularity and numerous advantages, MALDI has several limitations associated with

21

its utilization in MSI. A major drawback of MALDI relates to the capability to analyze 4


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low molecular weight compounds (97% pure) was purchased from Sigma-Aldrich (St. Louis,

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MO) and used without further purification. Alcohol (C2H5OH), sodium hydroxide

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(NaOH), sodium chloride (NaCl), dipotassium phosphate (K2HPO4) and sodium

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hydrogen phosphate (Na2HPO4) were obtained from Guangzhou Chemical Reagent

21

Factory (Guangzhou, China). The buffer solution used in the experiment was 7



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phosphate-buffered saline (PBS，pH 7.4) which contained 100 mM NaCl and 10 mM

2

Na2HPO4/K2HPO4. The embedding material, optimum cutting temperature (OCT), was

3

obtained from Yi Sheng Biotechnology Co., Ltd (Shanghai, China). All reagents were of

4

analytical grade or higher and all aqueous solutions were prepared using high-purity

5

water (≥18 MΩ/cm2) obtained by passing distilled and deionized through the ELGA

6

water purification system. Standard solutions (0.5-10 nmol/mL FA) were prepared by

7

appropriate dilution of the stock FA solution (see Supporting Information).

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Cell Culture

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The 4T1 mammary cancer cells were maintained as monolayer cultures in RPMI 1640

11

medium (Gibco, Invitrogen) supplemented with 10% fetal bovine serum (Gibco,

12

Invitrogen), 100 µg/mL streptomycin, 100 U/mL penicillin and 0.25 µg/mL amphotericin

13

in 5% CO2 and 95% air at 37 °C. The cells were removed from culture flasks by adding

14

0.05% trypsin solution when 80% confluence was reached. For inoculation in animals,

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the cell suspension was centrifuged and then resuspended in sterile PBS to obtain a

16

solution containing 5 × 105 cells/mL.

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Animal Tumor Model

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In this research, we used the murine model of mammary cancer (inoculation of 4T1 cells).

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All animal experiments were conducted in accordance with the guidelines of the

21

Institutional Animal Care and Use Committee (IACUC) of Guangdong Province. Female 8


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5-week old Balb/c mice (Guangdong Medical Laboratory Animal Center, Foshan),

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weighing approximately 20 g were used in this study. Food and water intake were

3

assessed. The dorsal region of the mouse was shaved and inoculated subcutaneously with

4

5 × 104 4T1 cells per animal. Within 20 days after inoculation of cells, the tumors were

5

measured to be ca. 1 cm3; subsequently, 20 mg/kg of FA (see Supporting Information)

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was administered intravenously to each mouse.

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Tissue Preparation

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The mice were humanely sacrificed by the inhalation of carbon dioxide and cervical

10

dislocation one hour after dosing. Then, the tumor tissues and adjacent normal controls

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were excised within 15 min. The tissues were quickly collected and frozen in dry

12

ice-chilled isopentane for 30 s, prior to storage at -80 °C in liquid nitrogen. Tissue

13

sections of thickness 30 µm were obtained using a cryo-microtome (CM1850, Leica,

14

Germany) at -20 °C with sections being deposited on a flat corundum substrate for the

15

LDPI-MS analysis. The experimental procedures are outlined in Figure S-2 (Supporting

16

Information).

17 18

RESULTS AND DISCUSSION

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Direct Analysis of Neat FA by LDPI-MS

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Figure 2 displays the LDPI-MS spectra of neat FA, which was coated on the sample plate.

21

First, the sample was vaporized by the desorption laser and a gaseous plume was formed 9



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above the sample plate, the plume being formed by the species which had emanated from

2

the sample. Thereafter, the species were ionized by the 118 nm laser beam. This process

3

is different to surface assisted laser desorption/ionization (SALDI) where the substrate

4

acts as a source of thermal energy to promote the laser desorption/ionization process.32

5

VUV single photon ionization (VUV SPI) is photon energy dependent and occurs for

6

molecules whose ionization energy (IE) are below the energy of irradiating photons. The

7

IEs of most organic species are below 10.5 eV, ranging typically from 7 to 10 eV.25,33 The

8

molecular structure of FA is shown in Figure S-3 (Supporting Information). In an attempt

9

to estimate the IE of FA, several theoretical calculations were performed with the

10

GAUSSIAN 03 and GAUSSIAN 09 program packages34,35 before commencement of

11

experiments. The molecule structure was optimized by using the MP2 theory at the

12

6-31+G(d,p) level. By the theoretical calculations, the IE of FA was estimated ca. 9.07 eV.

13

Hence, it was reasonable for FA to be ionized under 118 nm radiation.

14

Due to the internal energy36,37 which was deposited within the FA molecules upon

15

transferring to the gas phase from the sample plate surface, molecular fragmentation

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would be occurred during this desorption process. With respect to the mass spectra, two

17

prominent peaks for FA, corresponding to m/z 265 and m/z 295, can be seen in Figure 2a,

18

demonstrating that the LDPI-MS technique can detect FA directly. According to the

19

molecular structure of FA and the molecular weight (MW) of the two fragment ions, it

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could be predicted that the two fragment ions were [C12H13N2O5]+ and [C14H11N6O2]+,

21

respectively. To further confirm the accurate mass of these two characteristic fragments, a 10


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higher resolution electrospray ionization mass spectrometry (ESI-MS) was used for

2

analysis of FA. The ESI-MS (Figure S-4 of the Supporting Information) of the fragment

3

at m/z 289.0787 displayed similar structure to it observed in LDPI-MS at m/z 265, except

4

the former was protonated and adducted with Na+, and the fragment at m/z 295 in

5

LDPI-MS was also observed in the ESI-MS at m/z 295.0929. It can be seen from the

6

Figure 2(b) and Figure 2(c) that no distinct mass spectral signals were observed when

7

only desorption laser or ionization laser is employed on FA, which indicates that a single

8

desorption laser or ionization laser is unable to generate a meaningful signal contribution

9

in this experiment. Thus, the signals convincingly result from the LDPI process. In

10

subsequent experiments, the fragments at m/z 265 and m/z 295 were taken as the

11

characteristic signals of FA.

12

The analysis of the mass spectra was carried out by a code developed with the

13

scientific software Origin 9.0 (Origin lab, North Hampton, HA) which allows for mass

14

calibration, spectra analysis and peak identification. Each saved spectrum was the

15

average of 16 laser shots acquired by using an oscilloscope, with a laser frequency of 10

16

Hz.

17 18

Direct Analysis of FA in Tissue by LDPI-MS

19

It was the primary question that whether LDPI-MS could produce any significant ion

20

signals from the intact tissue, especially in the mass range of the mass signal related to

21

FA. Thus, experiments were taken to verify its feasibility. Figure 3(b) shows the 11



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LDPI-MS spectrum of the control tissue from the harvested tissues of mice which had not

2

been dosed with FA. It can clearly be seen that abundant peaks were less than m/z 100,

3

which suggests the signals might be related to neutral fragmentation or vaporization of

4

endogenous compounds in the tissues, such as small peptides, aromatic amines, or other

5

biological species.25,38 These neutral fragments could be ionized via SPI under 118 nm

6

VUV irradiation. Meanwhile, no significant signal could be observed in the high mass

7

regions for any tissues obtained from the mice, no matter how high the laser energy

8

irradiated allowed by our experimental set-up. Figure 3(c) and Figure 3(d) represent

9

conditions for laser desorption (LD) without any VUV postionization (PI) and 10.5 eV

10

VUV ionization without LD, respectively: neither of these control spectra show

11

significant signal contributions. In contrast, Figure 3(a) displays the LDPI mass spectrum

12

of mice tissues where a pharmacologically relevant dose of FA 20 mg/kg, was

13

administered.

14 15

Signal Linearity and Limit of Detection

16

There is a tradeoff between achieving desirable sensitivity and reasonable spectral

17

resolution. In the interests of achieving high ion yields and to optimize the analyte

18

response, the distance ∆Z between the sample surface and the VUV beam and the delay

19

time τ between the desorption laser and the ionization laser were both investigated. It was

20

decided that a distance of ~ 2 mm, corresponding to the delay time of ~ 18 µs, was

21

appropriate for achieving an acceptable signal response. Some ions might be generated 12


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by the desorption laser and then removed by a pulsed extraction field during the delay

2

time between the two pulses, while the ions produced by the 118 nm beam were

3

accelerated to the flight tube and strike the MCP.39 Meanwhile, the sensitivity in VUV

4

SPI depends on the ionization yield, which is governed by both the light intensity and the

5

interception volume of the VUV light with the desorption plume. If the ionizing laser

6

fires too early and too close to the sample surface, the interception volume would be

7

small. Thus, the flux of ionized neutral species would be low with a resultant decreased

8

detection sensitivity. In the current set up (∆Z = 2.0 mm and τ = 18 µs), the spot size of

9

the ionization laser was appropriate for ionizing a sufficient quantity of neutral FA

10

molecules and a sufficiently high sensitivity was attained. Figure 4 was plotted as the

11

signal intensity of m/z 265. The average velocity of the gas plume, calculated as v = ∆Z/τ,

12

with ∆Z = 2.0 mm and τ = 18 µs, was 110 m/s. The translational temperature of the plume

13

was estimated to be about 320 K while the average translational velocity of molecules in

14

the plume was ca. 110 m/s.

15

To investigate the relationship between signal intensity and FA concentration,

16

systematical experiments were performed. Figure S-5a and Figure S-5b (Supporting

17

Information) show the mass spectra and the calibration curve for FA by LDPI-MS,

18

respectively. The FA concentrations on the tissue section were 0.5-10 pmol/mm2. Surface

19

concentrations were calculated on the basis of a 20 mm2 area being covered by 20 µL

20

droplet of standard solution. Each concentration value was estimated from the average of

21

at least three spots. The linear regression equation was Y = 0.05067 + 6.952X with a 13



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correlation coefficient of 0.9891, where Y and X were the relative intensity of m/z 265

2

and the concentration of FA deposited on each tissue section, respectively. There is also a

3

tradeoff between sensitivity and resolution: a lager desorption laser spot size would

4

increase the sensitivity with more neutral species being vaporized and then ionized, but

5

good spatial resolution would be more difficult to achieve. In the present set up, the

6

analyte signal could still be observed in the mass spectrum even when the concentration

7

of FA was as low as 0.3 pmol/mm2. This demonstrated that the LOD (S/N = 3) has

8

reached the level of pmol.

9 10

LDPI-MS Imaging of FA-dosed tissues

11

In this experiment, a pair of adjacent tissue sections corresponding to the excised tumor

12

tissues and proximal normal controls was used for histological analysis and LDPI-MSI

13

analysis, respectively. For histological processing, a slice of the tissue section was stained

14

using hematoxylin and eosin (H&E), as shown in Figure 5(a). The colored regions in

15

Figure 5(b) show the spatial distribution of the FA fragment at m/z 265 and the black area

16

shows the background. From this MS image, it can be clearly seen that the signal in

17

certain regions is more intense than in other areas. A strong correlation could be observed

18

between the MS image and the pathologist annotations from the H&E staining image.

19

LDPI-MS imaging experiments validated that the uptake of FA was concentrated

20

primarily in tumor tissues and displayed somewhat lower retention in normal tissues. An

14


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interesting aspect of the FA LDPI-MS imaging experiment is the possibility to directly

2

characterize the regions of cancerous and normal tissue.

3

The data in Figure 5(b) were acquired by continuously moving the sample stage in a

4

back and forth raster pattern, shifting vertically at the end of each lane scan and then

5

moving back. The sample plate’s moving rate in the y-dimension (parallel to the VUV

6

beam) was 250 µm/s, with respect to the desorption laser running at 10 Hz, for collecting

7

the spectrum at each corresponding position, and the step size in the x-dimension was

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200 µm. The intensities of the mass spectra for each point were integrated and then used

9

to create a 6 × 6 mm image (Figure 5(b)). Customized software (Labview 2012, National

10

Instruments, Austin, TX) was used for image data acquisition and control of the X-Y-Z

11

translation stage. The data analysis was performed with the scientific software (MATLAB

12

R2013a, Mathwork, USA).

13 14

CONCLUSION

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This protocol based on LDPI-MS technique is matrix-free and rapid analysis, without

16

complicated sample preparation and labelling process. It has the superiority in detecting

17

exogenous compounds in tissues as the interference of the background signal could be

18

minimized. The results of LDPI-MS imaging display that FA has a higher concentration

19

in FR overexpressing tumor tissues than the adjacent normal controls. That is, LDPI-MS

20

is suitable for direct detection and imaging of FA in biological tissues and is able to

21

differentiate and characterize the regions of tumor and normal tissues. Therefore, FA, as a 15



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tumor-targeting agent of epithelial cancer, united with LDPI-MS imaging technique is

2

potentially useful in rapid clinical epithelial cancer diagnosis and immediate

3

image-guided surgery. Clearly, LDPI-MS is a powerful and promising approach to impart

4

important information with respect to active drug targeting and adjuvant therapy.

5

This technique based on present home-built LDPI-MS has some shortcomings in

6

comparison to established commercial MS instrumentation including low sensitivity and

7

spatial resolution. Improvements in spatial resolution and signal intensity can be achieved

8

through optimization of the spot size of the desorption laser and using a commercial

9

reflectron TOF-MS instead of a linear mode TOF-MS.

10 11

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ACKNOWLEGEMENT

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The authors thank the NSFC (No. 21273083, U1332132) grants and Guangdong-NSF

21

grants (No. S2013010016551) for financial assistance.

19



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1 2

Notes

3

The authors declare no competing financial interest.

4

FIGURE LEGENDS

5

Figure 1. Schematic drawing of the LDPI-MSI. Schematic diagram of the laser

6

desorption postionization mass spectrometer imaging apparatus and the sample hold with

7

an X-Y-Z stage. The lower panel illustrates the laser trigger sequence.

8

Figure 2. Mass spectra of FA by LDPI-MS: (a) neat FA, (b) direct ionization by

9

desorption laser without VUV radiation, (c) only 118 nm VUV beam at 10.5 eV photon

10

energy (no LD).

11

Figure 3. Mass spectra of tumor tissue by LDPI-MS: (a) Mass spectrum of FA-dosed

12

tissue. (b) Mass spectrum of tissue not dosed with FA. (c) Only desorption laser emitted

13

without VUV radiation. (d) Only 118 nm VUV emitted without desorption laser.

14

Figure 4. Parameters affecting the LDPI ion yields. The heat map illustrates the

15

affecting of the parameters on the LDPI ion yields: the proper distance between the

16

sample surface and VUV beam ∆Z and laser pulse delay τ.

17

Figure 5. LDPI-MS images of the tissue sections. The image (a) shows the H&E

18

staining of the tissue section and (b) depicts spatial distribution of FA dominate ion at m/z

19

265.

20 21

20


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1 2 3 4

Figure 1

5 6

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Figure 2

2 3

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Figure 3

2 3

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Figure 4

2 3

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Figure 5

2 3 4

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1

for TOC only

2

26


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Figure 3. Mass spectra of tumor tissue by LDPI-MS: (a) Mass spectrum of FA-dosed tissue. (b) Mass spectrum of tissue not dosed with FA. (c) Only desorption laser emitted without VUV radiation. (d) Only 118 nm VUV emitted without desorption laser. 98x120mm (300 x 300 DPI)



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Figure 4. Parameters affecting the LDPI ion yields. The heat map illustrates the affecting of the parameters on the LDPI ion yields: the proper distance between the sample surface and VUV beam ∆Z and laser pulse delay τ. 71x64mm (300 x 300 DPI)


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Figure 5. LDPI-MS images of the tissue sections. The image (a) shows the H&E staining of the tissue section and (b) depicts spatial distribution of FA dominate ion at m/z 265. 46x26mm (600 x 600 DPI)


Laser Desorption Postionization Mass Spectrometry Imaging of Folic

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