3,4-Dimethoxycinnamic acid (DMCA) as a novel matrix for enhanced

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3,4-Dimethoxycinnamic acid (DMCA) as a novel matrix for enhanced in situ detection and imaging of lowMW compounds in biological tissues by MALDI-MSI Huixin He, Liang Qin, Yawen Zhang, Manman Han, Jinming Li, Yaqin Liu, Kaidi Qiu, Xiaoyan Dai, Yanyan Li, Maomao Zeng, Huihong Guo, Yijun Zhou, and Xiaodong Wang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b03522 • Publication Date (Web): 14 Jan 2019 Downloaded from http://pubs.acs.org on January 14, 2019

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Analytical Chemistry

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3,4-Dimethoxycinnamic acid (DMCA) as a novel matrix for enhanced

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in situ detection and imaging of low-MW compounds in biological

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tissues by MALDI-MSI

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Huixin He,†,‡,#, Liang Qin, †,‡,# Yawen Zhang,†,‡ Manman Han,†,‡ Jinming Li,†,‡ Yaqin Liu,†,‡

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Kaidi Qiu,†,‡ Xiaoyan Dai,†,‡ Yanyan Li,§ Maomao Zeng,⊥,∥ Huihong Guo,○ Yijun Zhou,‡

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and Xiaodong Wang*,†,‡

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†

Centre for Imaging & Systems Biology, Minzu University of China, Beijing 100081, China.

‡

College of Life and Environmental Sciences, Minzu University of China, Beijing 100081, China.

§

The Hospital of Minzu University of China, Minzu University of China, Beijing 100081, China.

⊥

State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China.

∥

International Joint Laboratory on Food Safety, Jiangnan University, Wuxi 214122, China.

○ #

College of Biological Sciences and Biotechnology, Beijing Forestry University, Beijing 100083, China

These authors contributed equally to this work.

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*Corresponding author:

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Prof. Xiaodong Wang, Ph.D

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Centre for Imaging & Systems Biology, College of Life and Environmental Sciences, Minzu

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University of China

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#27 Zhongguancun South Avenue

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Beijing, 100081, China

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Email: [email protected]

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Tel.: +86-10-68932922; Fax: +86-10-68936927 1 ACS Paragon Plus Environment

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ABSTRACT

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Low molecular weight (low-MW) compounds have many essential functions in biological

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processes, and the molecular imaging of as many low-MW compounds as possible is critical

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for

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desorption/ionization mass spectrometry imaging (MALDI-MSI) is an emerging molecular-

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imaging technology which enables determination of the spatial distributions and the relative

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abundances of diverse endogenous compounds in tissues. New matrices suitable for the

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imaging of low-MW compounds by MALDI-MSI are important for the technological

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advancement of tissue imaging. In this study, 3,4-dimethoxycinnamic acid (DMCA) was

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evaluated as a new matrix for enhanced low-MW compound detection by MALDI-MSI due to

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its strong ultraviolet absorption, low matrix-ion related interferences below m/z 500, and the

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high ionization efficiency for the analysis of low-MW compounds. DMCA was successfully

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used for improved in-situ detection of low molecular-weight metabolites (m/z < 500) and lipids

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in both rat liver, rat brain and germinating Chinese-yew seed tissue sections. The use of DMCA

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led to the successful in-situ detection of 303, 200, and 248 low-MW compound ion signals

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from these three tissues, respectively. Both MALDI-MS/MS and LC-MS/MS were used to

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identify these ion signals, leading to the identification of 115 low-MW compounds from rat

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liver (including 53 lipids, 29 oligopeptides, and 33 metabolites), 130 low-MW compounds

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from rat brain (including 104 lipids, 5 oligopeptides, and 21 metabolites), and 111 low-MW

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compounds from germinating Chinese-yew seeds (including 77 lipids, 22 oligopeptides, 8

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flavonoids, and 4 alkaloids). A larger number of low-MW compounds could be detected and

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imaged when DMCA was used as the MALDI matrix than with other commonly-used MALDI

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matrices such as 2,5-dihydroxybenzoic acid, α-cyano-4-hydroxycinnamic acid, 2-

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mercaptobenzothiazole, graphene oxide, and silver nanoparticles. Our work provides a new

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and powerful matrix for enhanced MALDI-MS profiling of low-MW compounds in both

understanding

the

complex

biological

processes.

Matrix-assisted

laser

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animal and plant tissues.

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INTRODUCTION

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Endogenous low molecular weight (low-MW) compounds, especially low molecular-weight

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metabolites (m/z < 500) and lipids, perform multiple essential functions in multitudinous

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biological processes, including energy transformation, signal transduction and regulation,

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nutrient transportation, cytoskeleton and cytomembrane construction, etc.1,2 Previous studies

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have shown that abnormal levels of low-MW compounds are often associated with cancer,3

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neurological diseases,4 and systemic diseases.5 The comprehensive in-situ detection and

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imaging of endogenous low-MW compounds has been a very important aspect in both

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metabolomics and lipidomics for biomarker discovery and pathogenic mechanism

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elucidation.6,7

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Over the past two decades, many mass spectrometry (MS)-based methods have been developed

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and widely used for the detection of low-MW compounds.8,9 Among the various MS-based

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methods, matrix-assisted laser desorption/ionization MS imaging (MALDI-MSI) has proven

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to be a powerful molecular imaging approach that enables simultaneous detection and

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characterization of the spatial distributions and relative abundances of a wide range of

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compounds (for example, proteins, peptides, lipids, nucleotides, and drugs) directly from the

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surface of biological tissue sections.10,11 MALDI-MSI provides distinct advantages in terms of

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high sensitivity, high throughput, and molecular specificity derived from the use of mass

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spectrometers to detect in-situ multiple ionized biomolecules within a tissue section.12,13 Thus

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far, MALDI-MSI has been applied to diverse areas of biology, including zoology,14 basic

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medical science,3,15 pharmaceutical science,16 microbiology,17,18 and plant science.19,20

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In a typical MALDI‐MS experiment, the MALDI matrix plays a key role during the ionization 3 ACS Paragon Plus Environment

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process.7 The proper choice and application of MALDI matrix for efficient desorption and soft

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ionization of analytes on the surface of biological tissue sections depends on its strong laser-

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energy absorption and high-efficiency analyte ionization in the matrix plume.21 For the in-situ

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detection of endogenous low-MW compounds, the appropriate selection of a MALDI matrix

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is an even more a critical aspect for optimal MALDI-MSI, as it can reduce or avoid excessive

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interferences from matrix-related ion production in the low-mass range and result in significant

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enhancement in detection of analytes in biological tissue samples.22,23 Currently, 2,5-

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dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid (CHCA) are the most

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commonly-used MALDI matrices for low-MW compound imaging (especially for lipid) in the

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positive-ion mode and several classes of glycerophospholipids, including sphingomyelins

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(SMs), phosphatidylcholines (PCs), phosphatidic acids (PAs), phosphatidylethanolamine (PEs),

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and phosphatidylinositol (PIs), have been successfully detected.22,24 However, DHB often

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causes molecular delocalization and poor spot-to-spot reproducibility, due to its large crystal

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size and inhomogeneous crystallization, and the high abundance of matrix-related background

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signals from CHCA also limits its wide application for the in-situ detection of low-MW

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compounds. In addition, many aromatic compounds, such as 9-aminoacridine (9-AA)25 and N-

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(1-naphthyl) ethylenediamine dihydrochloride (NEDC),24 have been widely used as MALDI

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matrices for low molecular-weight compound detection in the negative-ion mode, but their use

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is still often affected by the interfering matrix-ion clusters. Moreover, 1,8-bis (dimethyl-amino)

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naphthalene (DMAN), useful for the analysis of acidic low-MW compound analysis,26 is not

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suitable for the MSI analysis of large tissue sections due to its high volatility under high-

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vacuum MALDI source conditions.27 Recently, hydroxyflavones,22 dithranol,28 quercetin,29

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and curcumin30 have been screened and optimized as powerful MALDI matrices for the tissue

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imaging of low-MW compounds, especially for lipid and drug MS imaging. However, many

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high-abundance matrix-related ions are still observed in the low-mass region of the spectra, 4 ACS Paragon Plus Environment


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and these could potentially interfere with the imaging of low-MW analytes. Many

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nanomaterials, such as graphene oxide (GO),23,31 silver nanoparticles (AgNPs),32 and gold

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nanoparticles,33,34 have been used as MALDI matrices for the detection of various low-MW

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compounds due to their efficient laser energy absorption and transfer, with low background

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interference. However, these nanomaterial matrices are not suitable for long-term and high-

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frequency use in MALDI-MSI, if the safety of the instrument is a concern. Because most of

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these nanomaterials possess high electrical conductivity and chemical stability, they have the

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potential to cause some unexpected failure or damage to the mass spectrometers, such as a short

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circuit. The continuous screening and discovery of new MALDI matrices with low or no

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background ions is still an important aspect of mass spectrometric imaging for low-MW

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compound analysis.10,35

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In this work, 3,4-dimethoxycinnamic acid (DMCA), a derivative of cinnamic acid, is reported

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as a new MALDI matrix for the in-situ detection and imaging of endogenous low-MW

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compounds directly on thinly cut rat liver, rat brain, and germinating Chinese-yew seed tissue

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sections by MALDI-TOF/TOF MS using a 355-nm Nd:YAG UV laser. Our results show that

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the use of DMCA significantly enhances MALDI-MS imaging of endogenous molecules in the

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low mass range (< 500 Da) in both animal and plant tissue sections with a relatively clean

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background.

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EXPERIMENTAL SECTION

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Reagents and Materials. DMCA, cinnamamide, 4-methylcinnamic acid, trans-4-

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hydroxycinnamic acid, arginine (Arg), tyrosine (Tyr), and phenylalanine (Phe) were purchased

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from J&K Scientific (Beijing, China). LC/MS-grade acetonitrile, trifluoroacetic acid (TFA),

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hematoxylin and eosin (H&E) stain, alanine (Ala), aspartic acid (Asp), asparagine (Asn), 5 ACS Paragon Plus Environment

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cysteine (Cys), glutamic acid (Glu), glutamine (Gln), glycine (Gly), histidine (His), isoleucine

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(Ile), leucine (Leu), lysine (Lys), methionine (Met), serine (Ser), threonine (Thr), tryptophan

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(Trp), hydroxyproline (Hyp), proline (Pro), valine (Val), N-(Carboxymethyl) lysine (CML),

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purine, niacinamide, nicotinic acid, pyroglutamic acid, homoserine, homocysteine, ornithine,

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citrulline, carnitine, bradykinin (1-7), graphene oxide (GO), and silver nanoparticles (AgNPs)

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were purchased from Sigma-Aldrich (St. Louis, MO). Ultrapure water used throughout the

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experiments was obtained from a Milli-Q system (Millipore USA). Germinating Chinese-yew

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(Taxus chinensis var. mairei) seeds were collected from Jiujiang city in November 2016

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(Jiangxi Province, China). The rat brain and liver were derived from an 8-week-old Adult male

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Sprague-Dawley rat (Shanghai Super-B&K Laboratory Animal Corp. Ltd, Shanghai, China).

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All tissue samples were flash-frozen by slowly immersing them in liquid nitrogen to avoid

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shattering after harvest. The use of animal organs for this study was approved by the Ethics

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Committee of College of Life and Environmental Sciences, Minzu University of China.

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UV-vis Absorption Spectroscopy. The absorption spectra of the DMCA solutions were

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measured on a UV-vis spectrometer (V-550, Jasco, Japan). For preparation of these solutions,

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a 2×10-5 M solution of DMCA was dissolved in 70% aqueous acetonitrile which contained 0%,

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0.1%, 0.2%, or 0.3% TFA. The wavelength scanned were from 200 to 500 nm. The UV-Vis

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absorption spectrum of solid-phase DMCA was acquired on a U-3900 spectrophotometer

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(Hitachi, Japan). Spectra were acquired from 200 to 500 nm.

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Optimization of Matrix Solution Composition. Orthogonal array testing with three variables

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including DMCA concentration, % ACN, and % TFA was performed to establish the optimal

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DMCA matrix solution. Solution compositions of 70%, 80%, and 90% ACN in water, DMCA

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concentrations of 8, 10, 12 mg/mL, and 0.1%, 0.2 %, and 0.3% TFA were chosen for the

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optimization matrix. Serial homogeneous rat liver tissue sections (12 μm thick) were used for

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this optimization. 6 ACS Paragon Plus Environment


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Microscopy visualization. The microscopic images of the dried DMCA matrix particles

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formed by spraying at concentrations of 8, 10, or 12 mg/mL onto glass slides were captured

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with a BX53 microscope (Olympus Life Science, Tokyo, Japan). The matrix solutions were

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coated on 2.5×2.5 cm glass slides with an air-brush sprayer (model 200, Badger Air-brush,

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Franklin Park, IL) and then viewed with an Olympus BX53 microscope under 10×, 20×, and

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40× magnification.

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Quantitative Determination of Bradykinin (1-7) Peptide Standard using DMCA as a

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MALDI Matrix. For quantitative determination, a series of concentrations of bradykinin (1-7)

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peptide standard (0.0, 0.0002, 0.0004, 0.0006, 0.0008, 0.001, 0.002, 0.004, 0.006, 0.008, 0.01,

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0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 4.0, 6.0, 8.0, and 10.0

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μg/mL) were prepared in distilled deionized water containing 0.1% TFA. DMCA was dissolved

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at the optimal concentration of 10 mg/mL in 90:10 ACN:water containing 0.1% TFA. One μL

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of each of the bradykinin (1-7) peptide standard solutions were spotted onto a Bruker's

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Anchorchip target plate, and air-dried 1 μL of DMCA matrix solution was added. Linear

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regression plots were constructed using Microsoft Excel 2016, by plotting the peak intensities

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of the target compounds versus the concentrations of the standard solutions.

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Preparation of Low-MW Standard Compounds. Standard solutions of each amino acid and

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other low-MW compounds were prepared at a concentration of 1 mM in deionized water for

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MALDI-TOF MS detection. For preparation of the mixed standard solution containing 21 low-

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MW compounds, Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Met, Phe, Pro, Ser, Thr,

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Tyr, Trp, Val, homocysteine, citrulline, and carnitine, were first dissolved in deionized water at

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a concentration of 10 mM, followed by dilution with deionized water to the desired

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concentrations. One μL of each standard solution was then spotted onto an Anchorchip target

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plate for MALDI-TOF MS detection.

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Tissue Sectioning. The frozen rat liver, rat brain, and germinating Chinese-yew seed tissues

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were cryo-sectioned at -20 oC into 12-μm thick slices in a Leica CM1860 cryostat (Leica

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Microsystems Inc., Wetzlar, Germany). The serial tissue slices were then immediately thaw-

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mounted on the conductive side of indium tin oxide (ITO) coated microscopic glass slides

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(Bruker Daltonics, Bremen, Germany).

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Matrix Coating. DMCA was prepared at a concentration of 10 mg/ml in ACN:water:TFA

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(90:10:0.1, v/v/v). 2-MBT at a concentration of 12 mg/mL was dissolved in 80:20

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methanol:water, containing 2% FA. DHB was prepared at 20 mg/mL in 80:20 methanol:water,

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containing 0.1% TFA. CHCA was prepared at 1.7 mg/mL in 50:50 ACN:water, containing 0.1%

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TFA. In addition, 0.5 mg/mL of GO in 50:50 methanol:water and 0.1 mg/mL of AgNPs in 50:50

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methanol:water were also prepared as two other matrix solutions. Matrix coating was carried

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out with an air-brush sprayer (model 200, Badger Air-brush, Franklin Park, IL). The matrix

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solutions were sprayed five cycles (5 s spray, and 60 s drying time) on the surfaces of the rat

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liver, rat brain, and germinating Chinese-yew seed tissue sections to pre-seed a thin layer of

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matrix. After air-drying in a vented fume hood, the matrix solution was evenly sprayed with

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forty more cycles on the same tissue sections. An Epson Perfection V550 Photo Scanner was

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used to take optical images of the tissue sections.

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Histological Staining. Hematoxylin and eosin (H&E) staining was performed using a

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previously reported procedure to obtain standard histological optical images.36

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MALDI-MS. All of the profiling and imaging experiments were performed using a Bruker

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Autoflex Speed MALDI time-of-fight (TOF)/TOF mass spectrometer (Bruker Daltonics,

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Billerica, MA). The MALDI source was equipped with a 2000-Hz solid-state Smartbeam

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Nd:YAG UV laser (355 nm) (Azura Laser AG, Berlin, Germany). All the mass spectra were

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acquired over a mass range of m/z 150 to 3500 in the positive ion mode with broadband 8 ACS Paragon Plus Environment


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detection. For the acquisition of MALDI-MS profiling data, the mass spectra were recorded

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from an accumulation of 20 laser scans and each scan was accumulated from 500 laser shots.

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For imaging data acquisition, 100-μm and 50-μm laser raster step-sizes were used for the in

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situ detection of endogenous low-MW compounds in the rat brain and in the germinating

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Chinese-yew seed tissue sections, respectively, and each scan (pixel) was accumulated from

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500 laser shots. Based on the use of the Bruker's FlexImaging 4.1 software, a correction pen

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was used to mark the “teaching points” (generally, three points) around a tissue section for the

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correct positioning of the UV laser for the spectral acquisition. The m/z values of the ions of

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the following compounds -- His [(M + H)+, m/z 156.0768], bradykinin 1-7 [(M + H)+, m/z

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757.3992], angiotensin II [(M + H)+, m/z 1046.5418], angiotensin I [(M + H)+, m/z 1296.6848],

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substance P [(M + H)+, m/z 1347.7354], bombesin [(M + H)+, m/z 1619.8223], ACTH clip 1-17

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[(M + H)+, m/z 2093.0862], ACTH clip 18-39 [(M + H)+, m/z 2465.1983], and somatostain 28

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[(M + H)+, m/z 3147.4710] -- were used for external mass calibration. His [(M + H)+, m/z

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156.0768], bradykinin 1-7 [(M + H)+, m/z 757.3992], angiotensin II [(M + H)+, m/z 1046.5418]

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and angiotensin I [(M + H)+, m/z 1296.6848] ions were chosen, in combination with the matrix

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ions DMCA[(M+H)+, m/z 209.0814], DMCA[(M-H2O+H)+, m/z 191.0708] or 2-MBT[(M+H)+,

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m/z 167.9942], CHCA[(M+H)+, m/z 190.0504], DHB[(M+H)+, m/z 155.0344], for internal

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mass calibration. The cubic enhanced mode was selected for calibration.

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Data Analysis. For MS profiling data analysis, the Bruker FlexAnalysis 3.4 software was used

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for the preliminary mass spectral viewing and processing. After the monoisotopic peak list had

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been generated and exported, two metabolome databases, i.e., METLIN37 and LIPID

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MAPS,38,39 were used to search the measured m/z values against possible metabolite identities,

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within an allowable mass error of ±10 ppm. For database searching, three ion adduct forms

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(i.e., [M+H]+, [M+Na]+, and [M+K]+) were considered. Reconstruction of the ion maps of

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detected low-MW compounds was performed by using the Bruker FlexImaging 4.1 software. 9 ACS Paragon Plus Environment

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Low-MW Compound Extraction and Identification by LC-MS/MS. Low-MW compounds

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were extracted from the same rat liver, rat brain, and germinating Chinese-yew seeds,

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respectively, for LC-MS/MS analysis. The details of the low-MW compounds extraction and

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LC-MS/MS analysis strategy for low-MW compound identification and structure confirmation

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can be found in the Supporting Information.

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RESULTS AND DISCUSSION

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Properties of DMCA as a MALDI Matrix. In order to serve as an effective MALDI matrix

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in MSI testing, a potential matrix compounds must exhibit a strong absorption of photons at

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the operational wavelength of the laser.22,40,41 Many types of UV lasers are currently used as

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the laser sources for desorption and ionization on most MALDI mass spectrometers,42

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including nitrogen lasers (337 nm), frequency-tripled Nd:YLF lasers (349 nm), and frequency-

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tripled or quadrupled Nd:YAG lasers (355 or 266 nm, respectively). The MALDI-TOF/TOF

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MS used in this work was equipped with a Nd:YAG UV laser (355 nm). Considering that UV

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absorption is a prerequisite for any MALDI matrix, UV-vis absorption spectra were first

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measured to investigate the laser absorption of DMCA. Figure 1A and 1B show the chemical

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structure and UV-vis absorption spectra of DMCA dissolved in 70% acetonitrile (ACN) in

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water, containing 0%, 0.1%, 0.2% and 0.3% TFA. As shown, a chemical structure similar to

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the commonly-used matrix CHCA is found in DMCA. A broad UV absorbance range from 300

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nm to 400 nm, similar to that reported for CHCA, was also observed in DMCA, covering the

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355-nm wavelength generated by the Nd:YAG UV laser used in this study. For MALDI-MSI

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sample preparation, matrix solution needs to be deposited on the tissue section to form a solid

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phase matrix layer. This matrix layer play a key role as a direct medium for energy absorption

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from the UV laser in a MALDI source. Therefore, the UV absorption spectrum was also 10 ACS Paragon Plus Environment


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acquired from solid-phase DMCA, which had been directly deposited on a quartz microscopic

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glass slide. As shown in the Figure 1C, solid-state DMCA also presented a strong absorbance

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at 355 nm. These results indicated that DMCA has great potential for use as a new MALDI

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matrix.

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In addition, three other compounds from 157 available cinnamic acid derivatives, including

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cinnamamide, 4-methylcinnamic acid, and trans-4-hydroxycinnamic acid were randomly

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chosen as potential MALDI matrices for UV absorbance detection, and compared with DMCA.

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As shown in Supporting Information Figure S1, DMCA shows the strongest 355-nm UV

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absorption, and weaker 355-nm UV absorption was found for trans-4-hydroxycinnamic acid.

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In contrast, it was clearly observed that cinnamamide and 4-methylcinnamic acid did not

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absorb UV laser energy at 355 nm, making them unsuitable as matrix compounds for the

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detection of low-MW compounds from rat brain and germinating Chinese-yew seed tissue

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sections (Supporting Information Figure S2). Interestingly, trans-4-hydroxycinnamic acid

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was also found to be unsuitable for the in-situ detection of low-MW compounds in tissue

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sections, according to its poor UV absorption at 355 nm, but the reason for this is still unclear.

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Additional cinnamic compounds with similar structures should be investigated to see if any

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others perform as well as DMCA.

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The major limitation for MALDI-MS analysis of endogenous low-MW compounds in

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biological samples is the presence of strong background signals in the low mass region of the

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mass spectrum which are produced by the matrix and related processes.22 Meanwhile, the

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strong UV absorbance of a potential new matrix may also give rise to abundant matrix-related

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cluster-ion signals during the laser desorption/ionization process, resulting in excessive

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background interference. Thus, we also investigated the background MALDI-MS signals from

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DMCA. The matrix background from DMCA in the positive-ion mode is presented in

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Supporting Information Figure S3. Our experiments showed that a very low laser power, i.e., 11 ACS Paragon Plus Environment

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20% of the full scale of the Nd:YAG UV laser power, which is ca. 2 mJ per pulse according to

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the product specification of laser source, is strong enough for efficient MS signal detection

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with DMCA as the MALDI matrix. Moreover, the matrix background spectrum from DMCA

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was extremely clean compared to several commonly used MALDI matrices, such as DHB,

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CHCA, and 2-MBT, and only 8 matrix- or matrix-related ions (S/N>3) were detected. As shown

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in Supporting Information Figure S3A, the most abundant peak is the ion at m/z 328.30 we

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believe to be an impurity, clusters, or adducts of DMCA, and the peaks at m/z 191.91 and

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209.08 which correspond to DMCA adduct ions of [M-H2O+H]+ and [M+H]+, respectively. In

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comparison to commonly-used matrices, the number of the matrix-related ions using DMCA

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was significantly lower than with DHB (78 matrix ions), CHCA (127 matrix ions), or 2-MBT

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(104 matrix ions) (Supporting Information Figure S3B-D). Recently, nanomaterials, such as

282

GO, AgNPs, gold nanoparticles, etc., have been reported as MALDI matrices for the detection

283

of various low-MW compounds in tissue samples with very low background interference.

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However, most of these nanomaterials possess high electrical conductivity and chemical

285

stability and thus have potential to cause unexpected failure or damage to the mass spectrometer,

286

such as short circuits or component breakdown, in the case of long-term and high frequency

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use of these conductive nanomaterial matrices.

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As mentioned above, the use of DMCA as a MALDI matrix means fewer matrix-related

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background signal ions in the low mass range, especially m/z

3,4-Dimethoxycinnamic acid (DMCA) as a novel matrix for enhanced

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