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1,5-Diaminonaphthalene Hydrochloride Assisted Laser Desorption/ Ionization Mass Spectrometry Imaging of Small Molecules in Tissues Following Focal Cerebral Ischemia Huihui Liu,†,∥ Rui Chen,‡ Jiyun Wang,†,∥ Suming Chen,†,∥ Caiqiao Xiong,†,∥ Jianing Wang,†,∥ Jian Hou,†,∥ Qing He,†,∥ Ning Zhang,†,∥ Zongxiu Nie,*,†,§,∥ and Lanqun Mao*,†,§,∥ †
Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry Chinese Academy of Sciences, Beijing 100190, China ∥ Beijing National Laboratory for Molecular Sciences, Beijing 100190, China ‡ College of Chemistry and Chemical Engineering, Yunnan Normal University, Kunming 650500, Yunnan, China § Beijing Center for Mass Spectrometry, Beijing 100190, China S Supporting Information *
ABSTRACT: A sensitive analytical technique for visualizing small endogenous molecules simultaneously is of great significance for clearly elucidating metabolic mechanisms during pathological progression. In the present study, 1,5naphthalenediamine (1,5-DAN) hydrochloride was prepared for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) of small molecules in liver, brain, and kidneys from mice. Furthermore, 1,5-DAN hydrochloride assisted LDI MSI of small molecules in brain tissue of rats subjected to middle cerebral artery occlusion (MCAO) was carried out to investigate the altered metabolic pathways and mechanisms underlying the development of ischemic brain damage. Our results suggested that the newly prepared matrix possessed brilliant features including low cost, strong ultraviolet absorption, high salt tolerance capacity, and fewer background signals especially in the low mass range (typically m/z < 500), which permitted us to visualize the spatial distribution of a broad range of small molecule metabolites including metal ions, amino acids, carboxylic acids, nucleotide derivatives, peptide, and lipids simultaneously. Nineteen endogenous metabolites involved in metabolic networks such as ATP metabolism, tricarboxylic acid (TCA) cycle, glutamate-glutamine cycle, and malate-aspartate shuttle, together with metal ions and phospholipids as well as antioxidants underwent relatively obvious changes after 24 h of MCAO. The results were highly consistent with the data obtained by MRM MS analysis. These findings highlighted the promising potential of the organic salt matrix for application in the field of biomedical research.
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to now. Considering its prevalence and severe consequences, elucidation of the metabolic mechanisms underlying cerebral ischemia is of great significance for minimizing the severity of ischemic damage. Rodent animal models are commonly used in the investigations of mechanisms of ischemic cerebral injury and the development of novel anti-ischemic drugs. MCAO using the intraluminal suture in rodents is a widely accepted and wellstandardized animal model to study the pathogenesis of focal cerebral ischemia.14 Various techniques such as high-performance liquid chromatography (HPLC),15 nuclear magnetic resonance (NMR),5,8,15 online electrochemical monitoring,16,17 and positron emission tomography (PET)18 have been applied for investigation of metabolic changes in rat brain following
he human brain is very sensitive to ischemia as it consumes about one-fifth of the oxygen and glucose.1,2 According to the American Heart Association, stroke ranks only behind cancer and cardiac disease as one of the leading causes of death and a major cause of disability. Among the most frequently occurring types of stroke, ischemic stroke accounts for almost 80% of the total cases in China and Western countries.3 In the case of cerebral ischemia, restricted delivery of oxygen and glucose would affect the synthesis and metabolism of ATP, glycolysis, TCA cycle, and its related metabolic pathways such as glutamate-glutamine cycle and malate-aspartate shuttle.4−7 Furthermore, disturbance of influx and efflux of metal ions8 as well as alterations of antioxidant substances5,9 also occurred. Accompanied with these events are the ischemic cascades such as acidosis,10 inflammation,11 excessive generation of reactive oxygen species,12 necrosis, apoptosis, and eventually neuronal death.13 Pathophysiological mechanisms of cerebral ischemia are extremely complicated, and our ability to prevent and cure strokes remains limited up © 2014 American Chemical Society
Received: May 21, 2014 Accepted: September 23, 2014 Published: September 23, 2014 10114
dx.doi.org/10.1021/ac5034566 | Anal. Chem. 2014, 86, 10114−10121
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Article
brain tissues of a MCAO rat model permitted us to visualize the spatial distribution and alteration of a broad range of small molecule metabolites including metal ions, amino acids, carboxylic acids, nucleotide derivatives, peptide, and lipids simultaneously. These findings suggest that the newly prepared organic salt matrix has potential application in small molecule in situ MSI and in the field of biomedical research.
MCAO. Nevertheless, information regarding the spatial localization of metabolites which is indispensable for accurately understanding complex biological processes cannot be obtained by using HPLC, NMR, and electrochemical techniques. Except for analytical techniques mentioned above, MALDITOF MS has also been used for investigation of metabolic changes in rat brain following MCAO. Experiments by Wang et al.19 directly profiled the changes of phospholipids and lysophospholipids in rat brain extracts postischemia using MALDI-TOF MS. A previous study by our group20 monitored level changes of glucose in rat brain microdialysates after MCAO by MALDI-TOF MS. In addition, a remarkable new technology, MALDI MSI, which was introduced by Caprioli in 199721 and emerged as a blooming field among the numerous imaging applications, has also been utilized for investigation of metabolic alterations during the ischemic period. Comparing with conventional imaging techniques, MALDI-MSI has demonstrated its unique features, namely, no necessity of labeling, high sensitivity, high throughout, molecule-specific, and the capacity of in situ localizing a wide range of biomolecules simultaneously from a tissue specimen in one single run. Accompanied with technological and methodological improvements in the instrumentation, sample preparation, and data acquisition and handling, MALDI-MSI has currently become one of the most powerful technologies for its promising potential in the field of biomedical research and has found application prospects in disease diagnosis and prognosis,22 biomarker discovery,23 and drug development.24 Koizumi et al. performed MALDI-MSI on rat brain sections 24 h after MCAO and revealed the production of lysophosphatidylcholine in the injured ischemic rat brain.25 Whitehead et al.26 examined the spatial profile of ganglioside species using MALDI MSI following MCAO reperfusion injury in the mouse. Another study by MALDI MSI identified 11 upregulated phospholipids and 7 other downregulated phospholipids in ischemic-damaged regions.27 MALDI-MSI with 9-aminoacridine (9-AA) visualized changes in the spatiotemporal distributions of eight cerebral metabolites involved in glycosis, TCA cycle, glutamate-glutamine cycle, and malate-aspartate shuttle in response to pathological perturbation.4 By combining MSI with LC-MS, Irie et al.6 observed a region-specific metabolic behavior in amino acid and nucleotide metabolism as well as in the TCA cycle in the ischemic hemisphere. It is generally believed that the detection of molecules in MALDI analysis is to a great extent dependent on the choice of matrix. Many novel promising matrices, such as porous silicon,28 nanomaterials,29 and organic matrices,20,30−32 have sprung up in recent years. Although those previous MALDI MSI studies provided spatial information and changes of metabolites during the process of ischemia, the range of the metabolites is still limited and insufficient to understand the complex disease processes. In order to obtain as much information simultaneously about altered metabolic pathways and networks during the progress of ischemia, 1,5-DAN hydrochloride was prepared as a matrix in the present study from its precursor 1,5-DAN, an organic matrix for MALDI analysis.32−34 Like other organic salts for a matrix springing up in recent years,20,35,36 the newly prepared matrix possessed brilliant features including low cost, strong ultraviolet absorption, high salt tolerance capacity, and fewer background signals especially in the low mass range (typically m/z < 500). 1,5-DAN hydrochloride assisted LDI MSI of small molecules in
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EXPERIMENTAL SECTION Chemicals and Reagents. 1,5-DAN, 9-AA, and 1,8bis(dimethylamino)naphthalene (DMAN) for matrix preparation, 2,3,5-triphenyltetrazolium chloride (TTC) for tissue staining, and chloral hydrate for anesthesia were purchased from Sigma-Aldrich (St. Louis, MO). Standards including aspartate, taurine, glutamate, glutamine, hypoxanthine, xanthine, N-acetylaspartate, ascorbic acid, citric acid, creatine, glucose, glutathione, adenosine, inosine, AMP, ADP, ATP, maltose, maltotriose, and peptide standard mixture were also purchased from Sigma-Aldrich (St. Louis, MO). Artificial cerebrospinal fluid (ACSF) II was bought from Huzhou InnoReagents Co., Ltd. (Zhejiang, China). Animals. Three male Kunming mice (20−22 g) and ten Sprague−Dawley rats (250−260 g) were provided by the Experimental Animal Center of Peking University. The animal experiments were performed according to the NIH Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication, No. 3040-2, revised 1999, Bethesda, MD) and were approved by the Animal Care and Use Committee of the Chinese Academy of Sciences. Establishment of the MCAO Model. The MCAO was carried out as described by Longa et al.37 with little modification. In brief, animals (n = 7) were anesthetized by intraperitoneal injection of chloral hydrate (350 mg/kg), and a short cervical incision was made on the rats. The right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were surgically exposed and separated from adjacent nerves and surrounding tissues. The CCA and ECA were permanently ligated, and a silk suture was loosely tied around the ICA. A nylon monofilament of 40 mm length with the tip coated with poly-L-lysine (final tip diameter of 0.36 ± 0.02 mm) (Beijing Sunbio Biotech Co., Ltd., Beijing, China) was inserted gently through the V-notch cut between carotid bifurcation and the ligation of CCA and impelled nearly 18−20 mm. The nylon thread was fixed to the CCA at the notch, and the cervical incision was sutured. The shamoperated control group rats (n = 3) underwent the same surgical operation except for the insertion of nylon monofilament. The body temperature of the animals was maintained at 37 ± 1 °C throughout the whole surgery. Neurological evaluation was performed according to a 5-points scale as described in the literature.37 Furthermore, the brain tissue sections were stained with TTC (Supporting Information) in order to identify regions of necrosis. Sample Preparation. Preparation of Matrixes and Standard Solutions. Solutions of DMAN, 9-AA, 1,5-DAN, and 1,5-DAN hydrochloride for matrix and mixture standard solutions containing small molecules are prepared according to the methods described in the Supporting Information. Tissue Dissecting. Sprague−Dawley rats were euthanized with chloral hydrate (350 mg/kg, i.p.) at 24 h post MCAO, and then brain tissues were removed and snap-frozen in liquid nitrogen. Liver, brain, and kidneys tissues from normal Kunming mice were dissected and flash-frozen as the same 10115
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method. All tissues were stored at −80 °C until further preparation. Tissue Sectioning. Tissues were fixed atop a drop of saline on the cutting stage. All tissues were sectioned at 10 μm thickness using a Leica CM1950 cryostat (Leica Microsystems GmbH, Wetzlar, Germany) at −17 ◦C and thaw mounted onto indium tin oxide (ITO) coated glass slides. The glass slides were then placed into a vacuum desiccator for approximately 1 h before matrix application. Plasma and Tissue Homogenate Preparation. Preparation of plasma sample and tissue homogenates are according to the methods described in the Supporting Information. Quantitative Analysis of Metabolites in Tissue Homogenates with MRM MS. Quantitative analysis of metabolites in brain tissue homogenates of rat subjected to MCAO was performed on an AB Sciex QTrap 4500 mass spectrometer using scheduled multiple reaction monitoring (MRM) via an electrospray ionization (ESI) source in both positive and negative modes. A 5 μL aliquot of the sample was injected directly through a bipass into MS without chromatography separation. Nitrogen was used for the collision gas. Curtain gas (CUR), nebulizer gas (GS1), and turbo-gas (GS2) were set at 25, 45, and 50 psi, respectively. The electrospray voltage was set at 5.5 kV, and the turbo ion spray source temperature was maintained at 500 °C. The declustering potentials (DP) and collision energies (CE) were optimized for each analyte (Table S1). Peak area was recorded for each analyte, and data analysis was performed using Analyst and Multiquant software (Applied Biosystems). Two-tailed Student’s t test was performed to compare relative amount of metabolites between the ischemic hemisphere and contralateral hemisphere. P-values ≤0.05 were considered statistically significant. MALDI Analysis. Evaluation of New Matrix Candidate. For MALDI analysis, the dried-droplet sample preparation method was used as follows: 1 μL of sample solution was mixed with 1 μL of matrix solution, and 1 μL of the mixture was then pipetted on the stainless steel target probe, following by drying under a stream of nitrogen gas at room temperature. Mass Spectrometry Imaging. For MSI, the matrix solution, 1,5-DAN in 50% ethanol/water prepared as described above, was sprayed onto the tissue sections mounted onto ITO coated glass slides using an automatic matrix sprayer (ImagePrep, Bruker Daltonics) and made sure to have homogeneous matrix coverage over the entire tissue surface. An Ultraflextreme MALDI-TOF/TOF MS (Bruker Daltonics, Billerica, MA) equipped with a smartbeam Nd: YAG 355 nm laser was utilized for MALDI analysis. The laser is fired at a repetition rate of 2000 Hz, and the analyzer was operated in negative reflectron mode. The negative-ion mass spectra in the reflector mode were collected with a pulsed ion extraction time of 80 ns, an accelerating voltage of 20.00 kV, an extraction voltage of 17.90 kV, a lens voltage of 5.85 kV, and a reflector voltage of 21.15 kV. The laser spot size was set at medium focus (∼50 μm laser spot diameter), and laser power was optimized at the start of each run and then fixed for the whole experiment. The mass spectra data were acquired over a mass range of m/z 0−1000 Da. Mass calibration was performed with external standards prior to data acquisition. For MSI analysis, imaging spatial resolution was set to 200 μm for tissues from mice, 50 and 100 μm for brain tissues from rats. Each spectrum consists of 200 laser shots. Regions of interest were manually defined in the imaging software using both the optical image and MSI data
image. MALDI mass spectra were processed with the total ion current (TIC) normalization, and the signal intensity of each imaging data was represented as the normalized intensity. Further Detailed Structural Confirmation. MS/MS fragmentations using the LIFT technique on the Ultraflextreme MALDI-TOF/TOF MS together with Fourier transform ion cyclotron resonance (FTICR) MS as well as Obitrap MS were used for further confirmation of the identified metabolites. Detailed information is described in the Supporting Information. Metabolites were identified or predicted by comparing MS or MS/MS spectra with those of standard compounds or referring to databases (METLIN, http://metlin.scripps.edu/; MassBank, http://www.massbank.jp/; Human Metabolome Database, http://www.hmdb.ca/; and LIPID MAPS, http:// www.lipidmaps.org/).
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RESULTS AND DISCUSSION Evaluation of the New Matrix Candidate. Figure S1-g and Figure S2 presented the MALDI mass spectra of 1,5-DAN hydrochloride acquired in negative and positive ion modes, respectively. The positive ion spectrum showed only a single peak at m/z 158.09 which corresponded to the 1,5-DAN radical cation. The negative ion spectrum exhibited prominent peaks arising from [Cl]− and [HCl2]− at m/z 34.8 and 70.8, accompanied by weak chloride-adducted clusters of Fe(2+) ([FeCl3]− and [FeCl4]− at m/z 162.8 and 199.8). Compared with the mass spectra of 1,5-DAN (Figure S1-e), 1,5-DAN hydrochloride exhibited much simpler mass spectrum and few matrix derived background signals especially at the m/z range of
