3-Aminophthalhydrazide (Luminol) As a Matrix for Dual-Polarity

May 31, 2019 - ... cerebral artery occlusion (MCAO) model as described previously. ... The UV absorption bands of many widely applied matrixes such as...
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Cite This: Anal. Chem. 2019, 91, 8221−8228

3‑Aminophthalhydrazide (Luminol) As a Matrix for Dual-Polarity MALDI MS Imaging Bin Li,*,∥,‡,† Ruiyang Sun,∥,‡,† Andrew Gordon,∥,‡ Junyue Ge,∥,‡ Ying Zhang,∥,‡ Ping Li,*,∥,‡ and Hua Yang*,∥,‡ ∥

State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, Jiangsu 210009, China School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu 211198, China

‡

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ABSTRACT: In many aspects of the matrix-assisted laser desorption/ ionization mass spectrometry imaging (MALDI MSI) technique, the discovery of new MALDI matrixes has been a major task for the improvement of ionization efficiency, signal intensity, and molecular coverage. In this work, five analog compounds, including phthalhydrazide, 3-aminophthalhydrazide (3-APH or luminol) and its sodium salt, 4-aminophthalhydrazide (4-APH), and 3-nitrophthalhydrazide (3NPH) were evaluated as potential matrixes for MALDI Fouriertransform ion cyclotron resonance (FTICR) MSI of metabolites in mouse brain tissue. The five candidate MALDI matrixes were mainly evaluated according to the solid-state ultraviolet absorption, the ion yields and species, and the dual-polarity detection. Among the five candidate matrixes, 3-APH and its sodium salt enabled the detection of endogenous metabolites better than the three other candidates in dual polarities. The best results were observed with 3-APH. Compared with commonly used MALDI matrixes such as 2,5dihydroxybenzoic acid, α-cyano-4-hydroxycinnamic acid, and 9-aminoacridine, 3-APH exhibited superior performance in dual polarity MALDI MSI, higher sensitivity, broader molecular coverage, and lower background noise. The use of 3-APH led to ontissue MALDI FTICR MSI of 159 and 207 mouse brain metabolites in the positive and negative ion modes, respectively. Among these metabolites, nucleotides, fatty acids, glycerolipids, glycerophospholipids, sphingolipids, and saccharolipids are included. 3-APH was further used for MALDI FTICR MSI of metabolic responses to ischemia-induced disturbances in mouse brain subjected to middle cerebral artery occlusion (MCAO), thus revealing the alteration of 105 metabolites in the ipsilateral hemispheres. This further emphasizes the great potential of 3-APH as a matrix for the localization of biomarkers in brain diseases. Until now, 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4hydroxycinnamic acid (CHCA) have been the most popular positive ion MALDI matrixes, which are extensively used for the profiling and imaging of lipids, peptides, and various secondary metabolites. Sinapinic acid (SA) is the first choice for MALDI analysis of protein molecule weight above approximately 4 kDa.12 In negative ion MALDI MS, 9-aminoacridine (9-AA), a moderately strong base, has been successfully used for the analysis of small molecules such as nucleotides and glucose 6phosphate.13,14 However, some limitations of conventional matrixes are observed in MALDI MS imaging. These may include high background signals of the matrix ions below m/z 500, one polarity, and a very broad and uneven crystal size distribution. In the past decade, efforts have been made to search for new organic MALDI matrixes with good performances such as low

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ass spectrometry imaging (MSI) techniques have become an important tissue imaging tool for precise localization of thousands of biological molecules in a single experiment. It is a label-free, in situ, and untargeted spatiochemical imaging technique with a high degree of specificity. With its unparalleled and unique capabilities, MSI has become a popular visualization tool and has been broadly applied in biology, medicine, and pharmacology.1−3 Among various MSI techniques, matrix-assisted laser desorption/ionization (MALDI) imaging is extensively applied to the imaging analysis of various biomolecules such as proteins, peptides, lipids, small metabolites, and drug substances.4−7 Unlike the two other popular MSI techniques, i.e., desorption electrospray ionization (DESI) imaging8 and secondary ionization mass spectrometry (SIMS) imaging,9 the detection of molecules by UV-MALDI MS imaging is believed to be largely dependent on the choice of matrix.10 Applying an appropriate UV absorbing matrix is one of the pivotal factors in obtaining satisfactory signal-to-noise ratio (S/N), high coverage of molecular species, and high-quality ion images.11,12 © 2019 American Chemical Society

Received: February 12, 2019 Accepted: May 31, 2019 Published: May 31, 2019 8221

DOI: 10.1021/acs.analchem.9b00803 Anal. Chem. 2019, 91, 8221−8228

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transform ion cyclotron resonance (FTICR) MS measurements of endogenous metabolites in sections of mouse brain tissue surrogates. Five candidate matrixes were mainly evaluated on the basis of the following characteristics: matrix ultraviolet optical absorption, ion yields, and tissue imaging in positive and negative ion modes. 3-APH and its sodium salt exhibited great potential for use as dual-polarity MALDI matrixes, with the best results obtained using the 3-APH matrix. We therefore present 3-APH as a novel MALDI matrix for the analysis and imaging of the variation of brain metabolites in mouse models subjected to middle cerebral artery occlusion (MCAO) in both positive and negative ion modes.

background interferences, high salt-tolerance potential, low volatility in the high vacuum, availability for dual-polarity detection, and capability of forming uniform matrix crystals at ∼10 μm for high spatial resolution MALDI MSI.11 For example, dithranol,15 quercertin,16 and curcumin17 were investigated for tissue imaging of lipids in positive ion mode. In negative ion mode, 4-phenyl-α-cyanocinnamic acid amide,18 1,8-bis(dimethylamino) naphthalene (DMAN),19 1,8-di(piperidinyl)naphthalene (DPN),20 maleic anhydride proton sponge,21 1,6diphenyl-1,3,5-hexatriene (DPH),22 and N-phenyl-2-naphthylamine (PNA)23 proved to be suitable for imaging small molecules such as amino acids, fatty acids, and lipids. Additionally, 1,5-diaminonapthalene (1,5-DAN) exhibited high efficiency by sublimation coating for the imaging of lipids in both positive and negative ion modes.24 Furthermore, chemical synthesis and structure modification are alternatives to develop new matrixes with desirable physicochemical properties such as optical absorption, crystallinity, and vacuum stability.18,21 Another significant trend in laser desorption/ ionization (LDI) MSI is the development of nonorganic materials, such as metallic,25,26 carbon-27 and silicon-based nanomaterials,28 metal oxides,29 and corresponding modified materials.30 These provide a clearer background with limited interference peaks and are particularly suitable for tissue imaging of small molecules. Although the mechanism of MALDI is still debated, in the positive ion mode, cation transfer from the matrix such as weak organic acids (e.g., DHB and CHCA) to analyte molecules is found to be the predominant process for gas-phase cationization.31 In the negative ion mode, abstraction of a proton by the matrix such as strong bases (e.g., 9-AA and DMAN) may be mainly associated with the formation of deprotonated anions.13 In terms of dual-polarity MALDI MSI, often used matrixes for positive ion MALDI MSI such as DHB and CHCA could also be used in negative ion mode. However, significant differences in ion yields and species are generally observed in the two ionization modes. For example, DHB and CHCA generate high ion yields in positive ion mode but low ion yields in negative ion mode, thus resulting in the loss of spatial information on molecules readily ionized in the negative ion mode such as fatty acids, nucleotides, cardiolipins, and gangliosides. Matrixes with both positive and negative polarities have attracted intense interest because they can significantly broaden the molecular coverage and acquire two ion images from a tissue section. Generally, several criteria need to be considered when evaluating an organic compound as a potential UV-MALDI matrix. Matching the UV absorption of the matrix with the usual MALDI nitrogen laser or frequency-tripled Nd:YAG laser operating at 337 nm and 355 nm, respectively, is one necessary significant property. The abilities to cocrystallize with the analytes and ionize while generating minimal matrix signals are also necessary characteristics of an ideal MALDI matrix. Moreover, since the process of vacuum MALDI MS imaging of tissue sections typically last for hours, high vacuum stability of the matrix is therefore essential for consistent acquisition of a series of mass spectra across the tissues. As aforementioned, several reported MALDI matrixes such as 1,5-DAN, 9-AA, and PNA are nitrogen substituted aromatic compounds. Therefore, in this work, we evaluated phthalhydrazide and its analogs including 3-aminophthalhydrazide (3APH, commonly known as luminol) and its sodium salt, 4aminophthalhydrazide (4-APH, commonly known as isoluminol), and 3-nitrophthalhydrazide (3-NPH) for MALDI Fourier-

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MATERIALS AND METHODS Chemicals. Phthalhydrazide, 3-aminophthalhydrazide (3APH), 3-APH sodium salt, 4-aminophthalhydrazide (4-APH), 3-nitrophthalhydrazide (3-NPH), 2,5-dihydroxybenzoic acid (DHB), α-cyano-4-hydroxycinnamic acid (CHCA), and 9aminoacridine (9-AA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). LC-MS grade methanol, acetonitrile, and ammonia hydroxide (NH4OH) were purchased from Merck (Darmstadt, Germany). Deionized water was prepared by a Milli-Q water purification system (Millipore, Billerica, MA, USA). UV Spectroscopy of Candidate Matrixes. All UV absorption spectra of candidate matrixes in solid state were recorded on a UV−vis spectrophotometer (U-3900 HITACHI, Japan) in the range of 200−400 nm. Candidate matrixes dissolved in pure methanol containing 1.2% NH4OH were deposited on quartz substrates using a home-built automatic spray for solid-state absorption measurements. Animals and MCAO Model. Male 8-week-old C57BL/6J mice were purchased from SIPPR-BK (Shanghai, China) and kept in an environmentally controlled breeding room for at least 1 week before the experiment. Animal experiments were carried out in accordance with the Guidelines for Animal Experimentation of China Pharmaceutical University (Nanjing, China), with the protocol approved by the Animal Ethics Committee of the institution. Mice weighing 21−23 g were used in this study. An experimental stroke was induced using a middle cerebral artery occlusion (MCAO) model as described previously.32 Briefly, the mice were anesthetized with a mixture of chloral hydrate (3%); 6−0 nylon monofilament coated with poly L-lysine was introduced into the internal carotid artery to block the middle cerebral artery at its origin for 1 h. At 24 h after MCAO, mice were sacrificed by decapitation, and the brain was quickly dissected, frozen on dry ice, and stored at −80 °C until use. The brain tissue sections were stained with cresyl violet (Nissl staining) after MALDI MS imaging and removal of the matrix with pure ethanol to identify regions of necrosis. A total of 6 mice were used for the creation of the MCAO model. Three MCAO mice with a similar infarct size determined by Nissl staining and three non-MCAO (control) mice were used for the MALDI MS imaging. Sample Preparation for the Evaluation of Candidate Matrixes. To maintain consistency, we applied tissue surrogates to critically evaluate the performance of the different candidate matrixes. Brain dissected from normal mouse was rinsed in saline. To the rinsed section, 0.2 mL of cold saline was added and homogenized with a ball mill (JXFSTPRP-24, Shanghai Jingxin Experimental Technology, Shanghai, China) for 2 min, immediately frozen, and stored at −80 °C. 8222

DOI: 10.1021/acs.analchem.9b00803 Anal. Chem. 2019, 91, 8221−8228

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Analytical Chemistry For the evaluation of candidate matrixes, 12 μm thick sections of tissue surrogates were prepared at −20 °C using a cryostat (3050S, Leica, Germany) and thaw-mounted onto indium− tin−oxide (ITO)-coated glass slides (Bruker Daltonics, USA). Repeatability measurements were performed on three sections of tissue surrogates coated with the five candidate matrixes for three consecutive days. The matrix application system and coating procedure were similar to that in Tissue Sample Preparation for MALDI MS Imaging. Tissue Sample Preparation for MALDI MS Imaging. In all cases, 12 μm thick horizontal (non-MCAO mice) and coronal tissue sections (MCAO mice) were prepared at −20 °C using a cryostat and thaw-mounted onto ITO-coated glass slides. For MALDI MS imaging of endogenous metabolite in non-MCAO mouse brain, the horizontal sections were applied due to better display of internal structures. For MALDI MS imaging of MCAO mouse brain, the coronal sections were applied because the infarcts generated by MCAO are mainly located at the striatum and the dorsolateral cortex. A laboratory-constructed electrospray was used for the uniform application of matrix solution. The matrix application system and coating procedure were similar to previously published work with some modifications.33 Briefly, for MALDI MS imaging experiments, all candidate matrixes were dissolved in methanol containing 1.2% NH4OH at a concentration of 8.7 mg mL−1. Additionally, 30 mg mL−1 DHB (MeOH/H2O, 7:3, v/v), 10 mg mL−1 CHCA (ACN/H2O, 7:3, v/v), and 10 mg mL−19-AA (MeOH/H2O, 7:3, v/v) were prepared for the comparative analysis. For homogeneous deposition of matrix solution onto the brain tissues, a voltage of 5.9 kV was applied to the spray nozzle with the ITO slide held at ground. The emitter-to-tissue distance was approximately 13 cm. The flow rate was set to 1 mL h−1 and gas pressure to 50 psi to deliver and nebulize matrix solution, respectively. MALDI FTICR MS Imaging Instrumentation. All measurements were performed using a 9.4T solariX FTICR mass spectrometer (Bruker Daltonics, USA) equipped with a dual ion source (ESI and MALDI) and a Smartbeam II laser (1 kHz). An m/z range of 150−2000 was acquired in positive ion mode, and m/z 150−3000 was acquired in negative ion mode. Single-scan spectra consisted of 100 accumulated laser shots at 1 kHz with a laser focus set to “medium”. Laser power was 40% for both positive and negative ion mode. MALDI images were acquired at a 150 μm spatial resolution for normal brain tissues and 225 μm for ischemic mouse brain tissues. Mass spectrometer calibration was performed externally in dual polarities using DHB matrix peaks and a Peptide Calibration Standard Kit II (Bruker Daltonics, USA). Calibration of the m/z scale of the MALDI FTICR MS in both positive and negative ion modes is an important step in obtaining an accurate mass. Data was analyzed using Data Analysis version 4.0 and flexImaging version 4.1 software (Bruker Daltonics, USA).

well with solid-state and N2 gas laser sources.37 Many studies demonstrated significant differences between the solution and solid-phase UV absorbance spectra of matrix compounds.37 Usually, the UV absorption profile of a target compound in a solution is measured to evaluate its matrix potential. However, such acquired measurements are potentially problematic due to solvent dependent effects since the UV-MALDI matrix is typically used in the solid state.35 Therefore, acquiring the UV absorption profiles of the five candidate matrixes in the solid state was preferred for the initial assessment. In Figure 1,

Figure 1. Chemical structures of five candidate matrix compounds and their solid-state UV absorption spectra measured on dry and crystallized matrixes.

phthalhydrazide (purple line) and 3-NPH (green line) have three absorption peaks at around 237, 266, and 300 nm and 233, 267, and 313 nm, respectively. 3-APH (red line) exhibits three distinct maxima at around 233, 304, and 355 nm, respectively, while its sodium salts (black line) exhibits one distinct maxima at around 233 nm and a broad absorption band in the range 325355 nm. When the peak intensity at 355 nm is compared, the absorption of 3-APH sodium salts is lower than 3-APH. Compared to 3-APH, no absorption maximum at 355 nm was observed for 3-NPH and phthalhydrazide. The auxochrome −NH2 directly conjugated with the π-system of the phthalhydrazide contributes to the variation of the absorption profile and intensity. 4-APH (blue line) exhibits only two distinct maxima at 229 and 267 nm, respectively. Although 4-APH displayed the strongest absorption band among the five candidate matrixes, the change in position of the −NH2 on the phthalazine ring caused a large decrease of absorption at 355 nm. Therefore, the maximum UV absorption of 3-APH and its sodium salts in the solid state matched well with the MALDI FTICR MS equipped with a 355 nm Nd:YAG UV laser. The maximum UV absorption of the potential matrix compound matching the operational laser wavelength is one of many properties that have been considered as a critical requirement for the UV-MALDI MSI matrix. The ion yields in UV-MALDI FTICR MS were evaluated next by spraying each

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RESULTS AND DISCUSSION Evaluation of Phthalhydrazide and Its Homologues as a Matrix for UV-MALDI FTICR MS. In UV-MALDI MS, high optical absorption at the operational laser wavelength is considered to be one of the crucial advantageous properties of the UV-MALDI matrix.34−36 Currently, solid-state laser and gas laser operating at 355 and 337 nm, respectively, are the predominant laser sources equipped in most commercial MALDI MS instruments. The UV absorption bands of many widely applied matrixes such as DHB, CHCA, and 9-AA match 8223

DOI: 10.1021/acs.analchem.9b00803 Anal. Chem. 2019, 91, 8221−8228

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Figure 2. MALDI FTICR mass spectra acquired from sections of tissue surrogates coated with five candidate matrixes in the positive (left) and negative (right) ion modes.

results. While the total ion intensity obtained using CHCA was higher than 3-APH, the chemical entities acquired from a single mass spectrum using CHCA (71) was less than 3-APH (79). As shown in Figure S4, high laser power could result in the high ion intensity. However, mass measurement errors caused by frequency perturbations in FTICR MS were observed at 50% laser power. This could be attributed mainly to space charge effects resulting from an excess of ions trapped in the ICR cell. In the negative ion mode, intense MALDI signals and a wide range of chemical entities were observed using 3-APH compared with 9-AA at 40% laser power. Finally, laser power set at 40% was adopted because it produced the highest ion intensities with acceptable mass measurement errors in both positive and negative ion modes. Compared with most often used matrixes, 3-APH resulted in very rich metabolite signatures and was suitable for MALDI FTICR imaging in two polarities with good sensitivity. Besides the general properties of the ideal MALDI matrix, the maximum absorption at the operational laser wavelength, and the ability to promote analyte desorption and ionization, 3-APH met more criteria for the MALDI MS imaging measurements. Figure S6 shows the morphology of the matrix crystal layer coated on mouse brain tissues obtained by optical microscopy. The 3-APH coating exhibits a homogeneous sample coverage with the crystal sizes at micrometer scale, which can mitigate the possible analyte delocalization, improve spot-to-spot reproducibility, and provide the potential for high spatial resolution MALDI MS imaging. Moreover, other characteristics of 3-APH used as the MALDI MS imaging matrix, such as its high vacuum stability (Figure S7), the low yield of matrix-related ions (Figure S2), and low matrix concentrations (8.7 mg mL−1), were also demonstrated. Optimization of the Matrix Solvent. It has been demonstrated that the matrix solvent composition directly influences matrix−analyte interaction, matrix crystal size, and ion yields. These must be optimized to obtain high sensitivity detection of analytes.14 We examined multiple combinations of

candidate matrix onto three sections of tissue surrogates and measured consistently for day-to-day repeatability (Figure S1). The respective MALDI FTICR mass spectra were acquired in both positive and negative ion modes for the individual candidate matrixes (Figure 2). In Figure 2, intense MALDI signals mainly consisting of lipids were observed in the two polarities for 3-APH and its sodium salt in comparison with 3NPH, 4-APH, and phthalhydrazide. As shown, in dual-polarity detection, there were almost no endogenous compound signals detected with phthalhydrazide, and very low signal intensity was observed when 3-NPH and 4-APH were used as the matrix. Additionally, in the negative ion mode, high background signals within the range of m/z 400−600 were observed with 3-NPH as the matrix (Figures 2 and S2). We therefore compared the performance of 3-APH with its sodium salt. As shown in Figure 2, minor differences were observed in 3-APH and its sodium salt in positive ion mode. However, fewer ions in the mass range of m/z 1000−3000 were detected when using 3-APH sodium salt in negative ion mode. Further comparison of 3-APH with its sodium salt was made by considering the molecular coverage in MALDI FTICR MS imaging. As demonstrated in Figure S3, the use of 3-APH and its sodium salt led to on-tissue MALDI FTICR MS detection and imaging of 159 and 133 endogenous compounds from a mouse brain section in the positive ion mode, respectively, and 207 and 149 compounds in the negative ion mode, respectively. Therefore, MALDI MS results suggested that 3-APH is superior to its sodium salts particularly for negative ion MALDI FTICR MS with a 355 nm laser. Next, we compared the performance of 3-APH with three commonly used MALDI matrixes, DHB, CHCA, and 9-AA. Initially, optimization of laser power was performed (Figure S4). As shown in Figures S4 and S5, although the observed single mass spectral profiles from sections of tissue surrogates were similar across 3-APH and DHB matrixes in the positive ion mode, the total ion intensity obtained by using 3-APH was higher than DHB, consistent with laser power optimization 8224

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Figure 3. Venn diagram showing the species and numbers of putatively identified endogenous metabolites detected using MALDI FTICR MS imaging with the 3-APH matrix in positive and negative ion modes.

matrix solvents to identify a set of optimized conditions for subsequent MALDI MSI experiments. 3-APH is comparatively insoluble in water and less soluble in commonly used organic solutions such as methanol but is soluble in base. To ascertain which solvent combination will be suitable, three solvent systems were used, and each was spiked with NH4OH. The three solvent combinations include solution A (pure MeOH containing 1.2% NH4OH), solution B (MeOH/H2O (1:1, v/v) containing 1.2% NH4OH), and solution C (MeOH/H2O (4:1, v/v) containing 1.2% NH4OH). As shown in Figure S8, the mean peak intensities of most selected lipids detected in positive ion mode were improved with solution A. However, in the negative ion mode, the three solutions did not appear to provide a universal advantage in promoting intensities of selected lipids. For example, selected lipid ions at m/z 699.50, m/z 806.51, m/z 885.55, m/z 888.62, m/z 945.55, and m/z 973.58 have higher intensities with solution A, and ions at m/z 856.51 and m/z 883.53 were better detected with solution C compared with A. However, for m/z 1857.95 and m/z 2215.07, no statistically significant difference was observed using the three solvent system. This is likely related to the different degree of analyte− matrix interactions for each solvent toward the different lipid species. Solution A was consequently used in the subsequent dual-polarity MALDI FTICR MSI experiments. Characterization of Metabolites in Normal Mouse Brain with 3-APH MALDI FTICR MS. After 3-APH was ascertained as a suitable MALDI matrix for the detection of endogenous metabolites particularly for lipid species in both positive and negative ion modes, its application in MALDI FTICR MS imaging of endogenous metabolites was illustrated using the brain tissue sections of the normal adult mouse. As shown in Figure 3 and Tables S1 and S2, 159 and 207 endogenous entities were detected from mouse brain tissue with 3-APH in positive and negative polarities, respectively. In positive ion mode, intense peaks were putatively attributed to phosphatidylcholines (PCs), sphingomyelin (SMs), and phosphatidylethanolamines (PEs) identified by accurate mass and/or tandem mass spectra matching in the LIPID MAPS database (www.lipidmaps.org) and several published reports (Tables S1 and S2). Moreover, other lipid species including glycerolipids (GLs), ceramides (CERs), glycosphingolipids (GSLs), and sterols were also detected (Table S1). In negative ion mode, the major metabolites detected corresponded to phosphatidic acids (PAs), phosphatidylinositol (PIs), phosphatidylserine (PSs), cardiolipin (CLs), PEs, and GSLs. Other molecules were also detected with 3-APH such as phosphati-

dylglycerol (PGs), cyclic phosphatidic acids (cPAs), phosphtatidylinositol phosphates (PIPs), fatty acids (FAs), and nucleotides (Table S2). The identification workflow of ions detected in the negative ion mode is similar to those used for positive ions. The different kinds of adduct ions were only counted as one compound. Furthermore, in situ tandem MS on the intense lipid signals was conducted in order to evaluate the fragmentation patterns using 3-APH as a matrix. For example, in positive ion mode (Figure S9), MS/MS of m/z 756.5513 produced a characteristic tandem mass spectrum corresponding to the PC(32:0) with characteristic loss of 59.07 (N(CH3)3) and 183.06 (phosphocholine). MS/MS of m/z 830.5079 produced a characteristic tandem mass spectrum corresponding to the PE(40:6) with characteristic loss of 43.04 (C2H5N) and 141.02 (PE headgroup). In negative ion mode, as demonstrated in Figure S10, m/z 1544.8694 and 1572.9007 corresponding to monoisotopic masses of GM1(d36:1) and GM1(d38:1) were subjected to tandem MS analysis, respectively. The fragmentation patterns of these two gangliosides are consistent with previous research.38 For example, the tandem mass spectrum of GM1(d36:1) ([M − H]−, m/z 1544.8694) exhibits product ions at 888.64 and 726.59, which are responsible for [M − H − NeuAc − Hex − HexNAc]− and [M − H − NeuAc − Hex − HexNAc − Hex]−, respectively (Figure S10). Another characteristic fragment ion at m/z 290.08 was observed, confirming the existence of a sialic acid moiety.39 MALDI FTICR MSI of Spatial Distribution of Metabolites in Mouse Brain. The negative and positive ion MALDI FTICR MS imaging was serially performed on one brain tissue section using 3-APH with a spatial resolution of 150 μm. In this case, the negative grid array was aligned with an offset of 100 μm in both x and y dimensions with respect to the array defined for positive data acquisition. In Figure 4, the Nissl-stained brain section shows histologically distinguishable structures, including cerebellum white matter and granular layers, inferior colliculus (IC), superior colliculus (SC), hippocampus (HIP), thalamic nucleus (TN), fimbria, lateral ventricle (LV), striatum, corpus callosum (CC), and cerebral cortex (CTX). As shown in Figure 4, 3-APH yields a significant amount of spatial information on endogenous chemical entities by serially imaging the same tissue section with the dual polarities. Different species of metabolites exhibit tissue-specific distribution across the mouse horizontal brain tissue section. Compared to commonly used 9-AA, the 3APH matrix exhibits a distinct advantage over 9-AA in the detection sensitivity and species coverage of lipids in negative 8225

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expressed less in the cerebellum region. However, in the HIP region, GM1(d36:1) is widely distributed, but GM1(d38:1) is confined in the dentate gyrus molecular layer and the stratum lacunosum moleculare. This observation is consistent with previous high resolution MALDI MS imaging results.43 Furthermore, the spatial distribution patterns of some small molecules such as fatty acids and nucleosides were also visualized (Figure 4) and consistent with previous data.33,40 For example, FA (22:6) was mainly found in the cerebellum granular layers, HIP, TN, striatum, and CTX.40 In positive ion MALDI MS imaging, 3-APH also enabled the revelation of spatial distribution of various lipid species. For example, some lipids from the same species exhibited heterogeneous distribution patterns. [SM(d36:1) + K]+ is mainly observed in the cerebellum granular layers, HIP, lateral septal nucleus (LSN), and TN, while [SM(d42:2) + K]+ is observed in the complementary regions including cerebellum white layers, IC, SC, fimbria, and striatum. In addition, high abundances of [DG(40:8) + Na]+, [Cer(42:2) + K]+, and [SM(d38:1) + K]+ were found in LV and ventral hippocampal commissure (VHC) areas. Besides being the building blocks of the outer and inner cell membrane, lipids are critically important for brain function and regulate plenty of physiological and pathological processes.6,44 Tissue-specific accumulation of lipids can improve our understanding of the distinct function of individual lipids or their classes, aiding to explain their roles and functions in various diseases. For example, [Cer(42:2) + K]+ is highly confined in the LV, the largest cavities of the ventricular system containing the cerebrospinal fluid (CSF), and its presence may be involved in brain protection or may cause neurological diseases if the ceramide homeostasis is interrupted.45,46 Visualization of Abnormal Metabolism in Mouse Brain Subjected to MCAO. To investigate its versatility as a matrix, 3-APH was also employed for MALDI FTICR MSI of pathological specimens. Tests were performed using the ischemic stroke (IS) model; i.e., mice subjected to MCAO. Ischemic strokes occur due to the sudden loss of fresh blood circulation to a region of the brain, resulting in a corresponding neurologic dysfunction.47 Abnormal metabolism of endogenous chemicals has been implicated in the pathogenesis of IS.23,48−50 The use of 3-APH for endogenous metabolite MALDI MS imaging may provide additional information for the understanding of complex mechanistic insights associated with IS or for the discovery of novel biomarkers. As shown in Figures S11 and S12, repeatability measurements and statistical analysis of abnormal metabolism in mouse brain subjected to MCAO were conducted with three replicates. The ratios of the ion intensity of selected metabolites in the left/contralateral hemisphere to the ion intensity of the same metabolites in the right/ischemic hemisphere were calculated. A bar chart displays the fold change in selected ions (Figure S12). As shown in Figures 5 and S11 and Table S3, many molecular species exhibit significant changes in coronal sections of the infarcted mouse brain. Interestingly, three different adduct ions of LPC(16:0), i.e., [LPC(16:0) + H]+, [LPC(16:0) + Na]+, and [LPC(16:0) + K]+, presented an inconsistent change in ischemic mouse brain. In the ischemic region, IS elevated [LPC(16:0) + Na]+ signals and lowered [LPC(16:0) + K]+ signals, but [LPC(16:0) + H]+ presented relative homogeneous distribution across ipsilateral and contralateral hemispheres. This observation is consistent with previous studies that showed the Na+/K+ homeostasis in the brain after IS was altered, leading to the alteration of the

Figure 4. MALDI FTICR MS images of selected metabolites acquired in the positive and negative ion modes from a mouse horizontal brain section coated with 3-APH. Ion images are correlated to the same brain section stained with cresyl violet after MALDI MS imaging and removal of 3-APH. Ion images were recorded with a step size of 150 μm with a 100 μm offset between the positive and negative grid arrays.

ion mode. For example, using 3-APH, [PE(38:5) − H]− was detected but not with 9-AA, and the S/N ratios of some lipids (e.g., [PE(38:4) − H]−, [PI(36:6) − H]−, and [ST(d40:1) − H]−) were significantly increased using 3-APH as matrix as compared with 9-AA, which are consistent with previous studies.18,40 One of the major advantages of the 3-APH matrix lies in its superior detection of many species of GSLs, a subclass of sphingolipids in negative ion mode, including neutral, acidic, and amphoteric GSLs (Table S2). GSLs present rich and various chemical structures with biological functions and have attracted intense interest to study these types of molecules. By matching accurate mass and/or tandem MS obtained by FTICR MS with online databases and the literature, the identity of GSLs were putatively assigned and listed in Table S2. In Figure 4, tissuespecific spatial distributions of gangliosides containing one or more sialic acids, such as GMs, GDs, and GTs, are clearly revealed with 3-APH matrix, which is consistent with previous data.41,42 For example, [GM1(d36:1) − H]− (m/z 1544.8694) and [GM1(d38:1) − H]− (m/z 1572.9007) present similar and different tissue-specific distribution patterns. These two gangliosides are mainly distributed in HIP, CTX, and striatum and 8226

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Analytical Chemistry

sodium salts provided more endogenous chemical entities in both positive and negative ion detection and were associated with low background interferences, high molecular coverage, and high vacuum stability. The best matrix performance was observed with the use of 3-APH, particularly in negative polarity. In mouse brain, 159 and 207 endogenous entities were detected with 3-APH in positive and negative polarities, respectively (Tables S1 and S2). To further demonstrate the application of 3-APH as a new MALDI matrix, mouse brain sections subjected to MCAO were analyzed with 3-APH MALDI FTICR MS imaging. The complex metabolite alterations such as nucleotides, fatty acids, phospholipids, and sphingolipids were visualized in the ipsilateral and contralateral hemispheres. The alteration in Na+ and K+ homeostasis induced by ischemic brain injury was also visualized by the alkali metal adduct ions for lipids. In total, 57 and 48 endogenous metabolites exhibited a large change in the ipsilateral hemispheres when detected in positive and negative ion modes, respectively (Table S3). This demonstrates the paramount importance for a comprehensive illustration of molecular mechanisms at sites of tissue injury induced by different diseases. The MSI results generally agreed with previous findings concerning the role of endogenous metabolite species in an ischemic injury. However, more work is further required to validate the observed metabolite alterations in brain by MALDI MSI.

Figure 5. MALDI FTICR MS images of selected metabolites acquired in the positive and negative ion modes from a non-MCAO (control) and MCAO mice coronal brain section coated with 3-APH. Ion images are correlated to the same brain sections stained with cresyl violet after MALDI MS imaging and removal of 3-APH. According to the cresyl violet stained images, the left side of the MCAO mouse brain is the contralateral hemisphere, and the right side shows ischemic damage. Ion images were recorded with a step size of 225 μm with a 150 μm offset between the positive and negative grid arrays.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.9b00803. Repeatability of MALDI FTICR MS experiments; candidate matrix background mass spectra; Venn diagram; optimization of laser power; MALDI FTICR mass spectra; optical image of 3-APH crystals; measurements of vacuum stability; optimization of the matrix solutions; MS/MS spectra; comparison of selected metabolite changes (PDF) Metabolite identity putatively assigned in mouse brain tissues by positive and negative ion MALDI FTICR MSI with 3-APH as matrix; metabolite identity putatively assigned in necrotic areas of ischemic mouse brain by dual-polarity MALDI FTICR MSI with 3-APH as matrix (PDF)

cationization profile of the brain lipids. 49,50 However, [PC(32:0) + H]+ and [PC(32:0) + K]+ presented a consistent change in the ischemic mouse brain; both were decreased in the ischemic hemisphere. In negative ion mode, several small molecules such as AMP, ADP, GMP, GSH, and FAs exhibited a noticeable decrease in signals within the ischemic region. This observation is in agreement with previous research.23,48 Here, ATP signal was undetectable, mainly due to its very quick postmortem degradation.48 IS also changed ganglioside signals in the ischemic region in contrast to the contralateral hemisphere. Gangliosides are considered to be intimately involved in the development of various brain diseases.42,51,52 MALDI MSI results showed that [GM1(d38:1) − H]− decreased but [GM2(d36:1) − H]− increased within the MCAO-induced infarcted hemisphere, and no large difference in [GM3(d36:1) − H]− signal was observed in the brain section (Figure S12). In a previous study, the highest level of GM1, GM2, and GM3 change was observed in the MCAO mouse model at the 3 day reperfusion.51

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (B.L.). *E-mail: [email protected] (P.L.). *E-mail: [email protected] (H.Y.).

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CONCLUSIONS UV-MALDI MS is a complex multiple-step process where matrix factor plays a significant role in the determination of the final imaging quality. Visualization of the spatial distribution of endogenous metabolites in dual polarities with one matrix significantly broadens the molecular coverage. In this work, we comprehensively evaluated commercially available 3-APH and related analogs for their application as UV-MALDI matrix in terms of optical absorption, ion yields, and tissue imaging. In general, MALDI MS results were consistent with the UV absorption properties of individual matrix compounds in the solid state. Among the five candidate matrixes, 3-APH and its

ORCID

Bin Li: 0000-0002-7713-159X Author Contributions †

B.L. and R.S. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (No. 81730104, No. 81773873, and No. 8227

DOI: 10.1021/acs.analchem.9b00803 Anal. Chem. 2019, 91, 8221−8228

Article

Analytical Chemistry

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81722048), the National Standardization Program for Chinese Medicine (ZYBZH-C-JS-35), the National Science and Technology Major Projects for “Major New Drugs Innovation and Development” (No. 2017ZX09301012003), the 111 Project (No. B16046), and the “Double First-Class” University Project (CPU2018GY09). The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies.

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DOI: 10.1021/acs.analchem.9b00803 Anal. Chem. 2019, 91, 8221−8228