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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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ABSTRACT

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

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

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(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

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

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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

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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

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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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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

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

and

gene

repair.3

Folic

acid

receptor

(FR)

is

a

glycosyl

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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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

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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

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

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

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

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

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

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

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

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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

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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

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

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

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

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

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

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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

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

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

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

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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

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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

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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

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

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

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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

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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

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

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

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

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

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Information).

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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.

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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

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

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

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

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

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

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

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

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

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

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

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

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

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

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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

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

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

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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]+,

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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

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

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

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

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

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

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

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

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

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

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characteristic signals of FA.

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

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

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

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

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Hz.

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Direct Analysis of FA in Tissue by LDPI-MS

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

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

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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

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

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

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

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

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

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

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

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

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

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

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

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administered.

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Signal Linearity and Limit of Detection

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There is a tradeoff between achieving desirable sensitivity and reasonable spectral

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

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

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

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

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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

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

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

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

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

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

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

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

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

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

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

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

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

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the plume was ca. 110 m/s.

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To investigate the relationship between signal intensity and FA concentration,

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

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

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

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

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

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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

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

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

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

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

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

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

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reached the level of pmol.

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LDPI-MS Imaging of FA-dosed tissues

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

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

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

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

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

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

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

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

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

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

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

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characterize the regions of cancerous and normal tissue.

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

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

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

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

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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

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

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

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

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R2013a, Mathwork, USA).

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CONCLUSION

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

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

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

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

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

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

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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

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potentially useful in rapid clinical epithelial cancer diagnosis and immediate

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

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

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

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comparison to established commercial MS instrumentation including low sensitivity and

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

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

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

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ACKNOWLEGEMENT

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

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grants (No. S2013010016551) for financial assistance.

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Notes

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The authors declare no competing financial interest.

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FIGURE LEGENDS

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

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desorption postionization mass spectrometer imaging apparatus and the sample hold with

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

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

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

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energy (no LD).

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

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

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

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

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

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

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

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

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265.

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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for TOC only

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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)

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