Research Article Received: 21 May 2014
Revised: 21 July 2014
Accepted: 22 July 2014
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2014, 28, 2084–2088 (wileyonlinelibrary.com) DOI: 10.1002/rcm.6993
Imaging of whole zebra fish (Danio rerio) by desorption electrospray ionization mass spectrometry Alexander Chramow1, Tanam S. Hamid1, Livia S. Eberlin2, Marion Girod3 and Demian R. Ifa1* 1
York University, Toronto, Ontario, Canada Stanford University, Stanford, CA, USA 3 ISA CNRS University of Lyon 1, Villeurbanne, Cedex, France 2
RATIONALE: To demonstrate the potential use of zebra fish (Danio rerio) as a model vertebrate organism by producing two-dimensional ion images of the whole zebra fish, and being able to distinguish particular areas of interest such as the brain, spinal cord, and stomach region using a desorption electrospray ionization (DESI) ion source coupled to a linear ion trap. METHODS: Imaging experiments are performed on 45 μm sagittal slices of zebra fish (Danio rerio), which are thaw-mounted onto microscope glass slides. The slides are then analyzed using a solvent of acetonitrile/dimethylformamide (50:50) (ACN/ DMF), with a solvent flow rate of 1.5 μL/min; data are acquired in negative ion mode. Raw mass spectrum data files are converted into a readable file for Biomap. The images produced are then analyzed for ion distributions. RESULTS: We are able to create clear, distinct, chemical intensity images of the brain, spinal cord, and stomach based on lipid content as well as bile salt. The identities of these compounds were confirmed by tandem mass spectrometric (MS/MS) experiments and comparisons with literature. CONCLUSIONS: Imaging of whole zebra fish is possible using ambient ionization techniques such as DESI. Analyses are fast and reliable. For most of the compounds observed, the identification by MS/MS can be performed directly from the fish tissue sample. Copyright © 2014 John Wiley & Sons, Ltd.
Mass spectrometry imaging (MSI) is a well-established tool allowing two-dimensional visualization of the spatial distribution of multiple analytes by their mass-to-charge ratios (m/z). Due to high sensitivity, high speed of analysis and high chemical specificity, MSI has been accepted worldwide as an effective system to detect and identify a broad range of complex molecules in biological samples. Traditional MSI methods such as matrix-assisted laser desorption/ionization (MALDI)[1] and secondary ion mass spectrometry (SIMS)[2] require sample pretreatment and analysis to be performed in vacuum. On the other hand, ambient ionization techniques such as desorption electrospray ionization (DESI)[3] require no sample pretreatment; sampling and ionization occur outside a vacuum system at atmospheric pressure, and the methods have demonstrated spatial resolutions of 40–500 μm.[4–7] The ionization process in DESI produces gas-phase ions from the condensed-phase analyte by creating charged microdroplets. These microdroplets are generated via a ’droplet pick up’ mechanism and proceed to the inlet of the mass spectrometer. This spray-based ionization is a soft technique, resulting in little to no fragmentation. Ambient techniques are useful for the analysis of low molecular weight compounds since they lack the interference caused by matrix
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* Correspondence to: D. R. Ifa, York University, Toronto, Ontario, Canada. E-mail: [email protected]
Rapid Commun. Mass Spectrom. 2014, 28, 2084–2088
ions from techniques such as MALDI. Ambient ionization sources can also analyze larger compounds such as bio and synthetic polymers.[8] DESI has already seen a wide variety of applications including molecular imaging of lipids, proteins, fatty acids, hormones and other compounds.[9] DESI has also been extensively used to characterize biological tissues such as rat brain,[4] human bladder,[10] rat spinal cord,[11] human liver,[12] etc. Previous studies using DESI involved mapping the distribution of clozapine and metabolites in rat brain, lung, kidney and testies.[5] Another study focused on creating ion images of propanolol in whole mouse tissue sections.[13] Lately, zebra fish have been receiving increased attention as they exhibit similar gene sequences and organ systems to humans.[14] This is why zebra fish are being studied and researched as an ideal vertebrate model organism. Zebra fish embryos have even been used as a model system for studying diseases and drug discovery due to their optical clarity.[14] The use of zebra fish in various laboratory experiments has been increasing due to low space requirement, low maintenance cost, rapid generation cycle, and rapid development.[15] Several biomolecules have specific distributions in organs, such as bile salts which are derivatives of cholesterol and are mostly synthesized in the liver. They perform various important functions in the liver and the intestines including elimination of cholesterol from liver into bile and promoting the absorption of lipids and lipid soluble vitamins from intestine.[16] The structure of bile salt found in zebra fish differs a little from the structure of bile found in mammals
Copyright © 2014 John Wiley & Sons, Ltd.
Imaging of whole zebra fish by DESI-MS and is composed of four steroid rings and a sulfate functional group.[17] The zebra fish bile salt has a trans configuration at the A/B ring juncture, resulting in a planar conformation. It is also considered to be evolutionarily primitive compared to more advanced bile salts which have a bent conformation due to a cis A/B ring juncture. Lipids have numerous important roles in biological systems, such as cell signalling, membrane structure, protein modifications, and cell-cell and cell-protein interactions, to name but a few.[19] Previous studies using DESI have successfully imaged deprotonated forms of various lipids in rat spinal cord such as phosphatidylinositol (PI), phosphatidylserine (PS), sulfatide (ST), etc.[11,18] It is also possible to detect deprotonated forms of fatty acids such as oleic acid, stearic acid, palmitic acid, etc., in the m/z 200–400 region. The goal of this research is to demonstrate the potential of using the zebra fish as a vertebrate model organism for further biological experimentation, by being able to create twodimensional ion maps representing specific areas of interest. These areas include the nervous system (brain, and spinal cord), gastrointestinal system, and body fat. Assessing the lipid and bile salt profiles on the same tissue section will aid the process of drawing correlation between molecular information and morphology of various organs of the zebra fish. This can facilitate the understanding of biochemical processes which could potentially be valuable for toxicological research.
EXPERIMENTAL Sample and chemicals Male adult zebra fish were kindly donated by Dr. Chun Peng’s research group (Biology Department, York University). Tricaine mesylate was purchased from Sigma-Aldrich Canada Co. (Oakville, ON, Canada) to euthanize the fish. Carboxymethyl cellulose, used to create the moulds, was also purchased from Sigma-Aldrich Canada Co. Solvents used were acetonitrile (ACN) (Optima LC/MS) from Fisher Scientific Company
(Ottawa, ON, Canada), and N,N-dimethylformamide (DMF) (HPLC grade) from Sigma-Aldrich Canada Co. Zebra fish tissue preparation/sectioning The workflow for tissue preparation and analysis is shown in Fig. 1. The zebra fish were first euthanized by placing them in a small aquarium and adding a solution of tricaine mesylate. They were then placed in disposable plastic rectangular frame/mould in order to protect them from deformation and damage during cryosectioning. These moulds were filled with carboxymethyl cellulose (CMC) solution and placed immediately in the refrigerator at 8°C for chilling overnight. A solution of carboxymethyl cellulose was made by mixing CMC and distilled water until it formed a saturated paste. The frozen CMC blocks were removed from the plastic frame and continuously sectioned into 20–60 μm thick sagittal sections at 17°C using a Shandon cryotome FE and FSE (Thermo Fischer Scientific, Nepean, ON, Canada). The frozen fish slices were thaw mounted on microscope glass slides and kept in the freezer until use. The fish slides were then dried in the lab in the open air for 30 min prior to analysis by DESI-MSI.
DESI imaging experiments Imaging was carried out using a lab-built prototype DESI ion source. In order to desorb the desired analytes, a morphologically friendly solvent system composed of ACN/DMF (50:50) was used.[8] The DESI spray tip was positioned approximately 2.6 mm from the tissue sample at an incident angle of 52°. The inlet of the mass spectrometer was approximately 2 mm from the tissue sample surface with a collection angle of approximately 10°. The spray tip-to-inlet distance was 5 mm. Experiments were conducted in negative ion mode with spray voltage of 5 kV, injection time 250 ms, 3 microscans averaged, solvent flow rate of 1.5 μL/min, and nebulizing gas (N2) pressure of 100 psi. The mass spectrometer used was a LTQ linear ion trap controlled by
Rapid Commun. Mass Spectrom. 2014, 28, 2084–2088
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Figure 1. Overview of zebra fish tissue preparation and analysis by DESI-MSI.
A. Chramow et al. Xcalibur 2.0 software (Thermo Fisher Scientific, San Jose, CA, USA). The two-dimensional (2D) imaging experiments were performed by using a moving stage scanning an area of 2.5 cm in the x-direction and 0.6 cm in the y-direction. The velocity for the surface moving stage was 208.3 μm/s acquiring mass spectra ranging from m/z 150 to 900. The 2D images acquired consisted of arrays of 125 × 30 pixels, each pixel covering an area of 200 × 200 μm2. Each pixel required 0.96 s to be scanned making the total scan time for the 2D image to be approximately 60 min (0.96 s /scan × 125 scans × 30 lines). An in-house program allowed the conversion of the Xcalibur 2.0 mass spectra files (.raw) into a format compatible with Biomap,[20] creating single ion images as well as overlays of two different ion images. In order to adjust the colour contrast of the images, the rainbow colour gradient was selected and the signal intensity of the ions was normalized for all images used. The color intensities for the ions were all normalized to the same level so that one ion might not appear deceivingly more abundant than another. The fatty acid, bile salt and lipid species were identified by comparing the generated product ion spectra with literature data and from the MS/MS data generated through collisioninduced dissociation (CID) tandem MS experiments. CID experiments were performed directly on a tissue slice by moving the sample stage in order to position an area of interest under the spray tip. The CID energy was optimized by manually ramping up the energy from 10–30 units and monitoring the ion fragments. Optimization was attained when a stable signal with multiple ion fragments were produced. The m/z range for the ion selection window was 1.
RESULTS AND DISCUSSION The analysis of the zebra fish slice surface provided a diverse collection of ions with fatty acids dominating the lower m/z range (m/z 600). The images created from one 45 μm thick sagittal slice of a zebra fish are shown in Figs. 2(b)–2(f). Slices were taken at this thickness due to the ease of reproducibility with the chosen embedding medium. A selection of embedding media has previously been studied by Nelson et al.[21] for the purpose of freezing and cryosectioning zebra fish. They determined that
a more optimal medium consisting of 5% CMC and 10% gelatine mixture can be used to achieve reproducible slices of 16 μm thickness. The sample was analyzed in negative ion mode, since it is known that most phospholipids and sphingolipids ionize easily in this mode.[22] Analysis was also performed on the samples in positive ion mode. We were able to create an ion image of an ion m/z 265, located in the eye of the fish, which is thought to be thiamine. Reproducibility of this ion was poor, possibly due to the structure of the eye, or perhaps damage during cryosectioning. The ion images are set next to an optical image (Fig. 2(a)) of another sliced zebra fish frozen in a CMC mold, adhered to the mounting plate of a cryotome. In the optical image there is distinction between different tissue types: digestive, neuronal, muscular. Figure 2(b) represents the chemical intensity map of ion m/z 281, deprotonated oleic acid. Since it is found in the majority of the tissue it serves as a good background for making comparisons with other ions of interest (m/z 531, 834, 885, 890). Figs. 2(c)–2(f) show images of the chemical distribution of ions of interests shown in a colour gradient. All images were normalized to the same intensity gain. The ion of m/z 834 (Fig. 2(c)) appears localized in the area representing the brain of the zebra fish. This ion is phosphatidylserine (PS 40:6); its structure is confirmed by comparing MS/MS spectra (MS/MS: m/z 834, 747, 437, 419, 327, 283) with the literature.[23] The most intense peak at m/z 747 [M–H–87] corresponds to the loss of the serine head group (87 Da), which confirms the identification as a phosphatidylserine (PS). It is reasonable to find this ion localized within the brain since the main species of lipids detected in this organ are phosphatidylserines (PS), phosphatidylinositols (PI), and sulfatides (ST). In a previous study[4] analyzing rat brains using DESI, the ion m/z 834 (PS 40:6) was found with high intensity in areas correlating to grey matter, rather than white matter. This indicates that this phospholipid resides mostly within the cell body, or soma, of neurons. Phosphatidylinositol (PI 38:4) is represented as ion m/z 885 (Fig. 2(d)). This assignment is confirmed by the presence of inositolphosphate (m/z 259) in the MS/MS spectra (MS/MS: m/z 885, 599, 581, 419, 303, 283, 259). Phosphatidylinositols (PI) play a crucial role in intracellular communication,[23] therefore it would be logical to see this ion in neuronal tissue, with a much greater accumulation and intensity in the area representing the spinal cord, where a lot of information is cross-linked and carried through.
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Figure 2. (a) Optical image of zebra fish in CMC mould. (b–f) Images of ion distribution produced from DESI analysis. (b) Image of deprotonated oleic acid m/z 281, (c) phosphatidylserine (PS 40:6) m/z 834, (d) phosphatidylinositol (PI 38:4) m/z 885, (e) bile salt 5α-cyprinol 27-sulfate m/z 531, and (f) sulfatide (ST 24:0) m/z 890.
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Rapid Commun. Mass Spectrom. 2014, 28, 2084–2088
Imaging of whole zebra fish by DESI-MS
Figure 3. DESI( ) mass spectrum from the intestinal system of the zebra fish showing: m/z 498.5, C24 bile acid-(OH)2-taurine; m/z 515.5, C27 bile alcohol(OH)4-SO4; and m/z 531.5, 5α-cyprinol 27-sulfate. Another lipid appears as an ion with m/z 890 (Fig. 2(f)), and is identified as a sulfatide (ST 24:0). This is confirmed by the presence of the fatty acid amide containing ion m/z 392 in the MS/MS spectra[24] (MS/MS: m/z 890, 872, 652, 522, 392, 258). Sulfatides (ST) are very important sphingolipids since they are present in the myelin sheath and have a role in cell differentiation and adhesion.[24] The areas of the zebra fish highlighted by the distribution and intensity of the ion at m/z 890 could possibly represent the white matter of the nervous system. A pattern containing sulfate-conjugated polyhydroxy bile alcohols, m/z 498, 515 and 531 (Fig. 3), was specifically localized within the area corresponding to the stomach, or intestinal system of the zebra fish (Fig. 2(e)). This pattern was previously reported in a study on the evolution of bile salts which identified that at least 98% of the bile salts in zebra fish are from 5α-cyprinol 27-sulfate.[17] The mass spectra recorded for the areas containing bile salt are comparable to those reported for purified extracts of zebra fish bile.[17] The ions formed during MS/MS experiments of bile salts were scarce and unstable thus restricting structural information.
CONCLUSIONS
Rapid Commun. Mass Spectrom. 2014, 28, 2084–2088
REFERENCES [1] E. H. Seeley, K. Schwamborn, R. M. Caprioli. Imaging of intact tissue sections: moving beyond the microscope. J. Biol. Chem. 2011, 286, 25459. [2] M. K. Passarelli, N. Winograd. Lipid imaging with timeof-flight secondary ion mass spectrometry. Biochim. Biophys. Acta 2011, 1811, 976. [3] A. L. Dill, L. S. Eberlin, D. R. Ifa, R. G. Cooks. Perspectives in imaging using mass spectrometry. Chem. Commun. 2011, 47, 2741. [4] J. M. Wiseman, D. R. Ifa, Q. Song, R. G. Cooks. Tissue imaging at atmospheric pressure using desorption electrospray ionization (DESI) mass spectrometry. Angew. Chem. Int. Ed. 2006, 45, 7188. [5] J. M. Wiseman, D. R. Ifa, Y. Zhu, C. B. Kissinger, N. E. Manicke, P. T. Kisinger, R. G. Cooks. Desorption electrospray ionization mass spectrometry: Imaging drugs and metabolites in tissues. Natl. Acad. Sci. USA 2008, 105, 18120. [6] V. Kertesz, G. J. Van Berkel. Improved imaging resolution in desorption electrospray ionization mass spectrometry. Rapid Commun. Mass Spectrom. 2008, 22, 2639. [7] L. S. Eberlin, D. R. Ifa, C. Wu, R. G. Cooks. Three-dimensional vizualization of mouse brain by lipid analysis using ambient ionization mass spectrometry. Angew. Chem. Int. Ed. 2010, 49, 873. [8] C. Wu, A. L. Dill, L. S. Eberlin, R. G. Cooks, D. R. Ifa. Mass spectrometry imaging under ambient conditions. Mass Spectrom. Rev. 2013, 32, 218. [9] R. G. Cooks, N. E. Manicke, A. L. Dill, D. R. Ifa, L. S. Eberlin, A. B. Costa, H. Wang, G. Huang, Z. Ouyang. New ionization methods and miniature mass spectrometers for biomedicine: DESI imaging for cancer diagnostics and paper spray ionization for therapeutic drug monitoring. Faraday Discuss. 2011, 149, 247. [10] A. L. Dill, L. S. Ebrelin, A. B. Costa, C. Zheng, D. R. Ifa, L. Cheng, T. A. Masterson, M. O. Koch, O. Vitek, R. G. Cooks. Multivariate statistical identification of human bladder carcinomas using ambient ionization imaging mass spectrometry. Chem. Eur. J. 2011, 17, 2897. [11] M. Girod, Y. Shi, J. Cheng, R. G. Cooks. Mapping lipid alterations in traumatically injured rat spinal cord by desorption electrospray ionization imaging mass spectrometry. Anal. Chem. 2011, 83, 207.
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In this study, the use of DESI-MSI to investigate zebra fish as a vertebrate model organism is determined to be possible. It is demonstrated that certain organs and regions of interest are visible and identifiable by having the ability to showcase the chemical intensity distribution of selected ions. Areas of interest, such as the brain, spinal cord, and stomach, can be identified by their lipid species or bile salt make-up. Most of these markers can be further characterized and their structures elucidated through MS/MS experiments, which can be acquired directly from a tissue sample. The use of ambient ionization techniques greatly facilitates the study of biological tissues and small organisms by reducing time needed for sample preparation or extensive extractions in order to characterize specific ions. The ability to analyze whole organisms could allow for future research on bioaccumulation and toxicological studies by exposing the fish to toxins, ionic liquids, detergents, etc. Previous studies on the zebra fish have included determining the LD50 (lethal dose causing 50% death in a population) for various toxins such as metribuzin,[25] xinjunan,[26] and endosulfan.[27]
By applying the information discovered by these studies, the zebra fish could potentially be monitored for the distribution of drugs or toxins as well as their metabolites within the whole body, using DESI-MSI.
A. Chramow et al. [12] S. Gerbig, O. Golf, J. Balog, J. Denes, Z. Baranyai, A. Zarand, E. Raso, J. Timar, Z. Takats. Analysis of colorectal adenocarcinoma tissue by desorption electrospray ionization mass spectrometric imaging. Anal. Bioanal. Chem. 2012, 403, 2315. [13] V. Kertesz, G. J. Van Berkel, M. Vavrek, K. A. Koeplinger, B. B. Schneider, T. R. Covey. Comparison of drug distribution images from whole-body thin tissue sections obtained using desorption electrospray ionization tandem mass spectrometry and autoradiography. Anal. Chem. 2008, 80, 5168. [14] D. R. Love, F. B. Pichler, A. Dodd, B. R. Copp, D. R. Greenwood. Technology for high-throughput screens: the present and future using zebrafish. Curr. Opin. Biotechnol. 2004, 15, 564. [15] H. M. Stern, L. I. Zon. Cancer genetics and drug discovery in the zebrafish. Nat. Rev. Cancer. 2003, 3, 533. [16] P. Song, Y. Zhang, C. D. Klaassen. Dose-response of five bile acids on serum and liver bile acid concentrations and hepatotoxicty in mice. Toxicol. Sci. 2011, 123, 359. [17] E. J. Reschly, N. Ai, S. Ekins, W. J. Welsh, L. R. Hagey, A. F. Hofmann, M. D. Krasowski. Evolution of the bile salt nuclear receptor FXR in vertebrates. J. Lipid Res. 2008, 49, 1577. [18] M. Girod, Y. Shi, J. Cheng, R. G. Cooks. Desorption electrospray ionization imaging mass spectrometry of lipids in rat spinal cord. J. Am. Soc. Mass Spectrom. 2010, 21, 1177. [19] A. D. Watson. Thematic review series: systems biology approaches to metabolic and cardiovascular disorders. Lipidomics: a global approach to lipid analysis in biological systems. J. Lipid Res. 2006, 47, 2101.
[20] Biomap freeware. Available: http://www.maldi-msi.org. [21] K. A. Nelson, G. J. Daniels, J. W. Fournie, M. J. Hemmer. Optimization of whole-body zebrafish sectioning methods for mass spectrometry imaging. J. Biomol. Technol. 2013, 24, 119. [22] N. E. Manicke, J. M. Wisemann, D. R. Ifa, R. G. Cooks. Desorption electrospray ionization (DESI) mass spectrometry and tandem mass spectrometry (MS/MS) of phospholipids and sphingolipids: ionization, adduct formation, and fragmentation. J. Am. Soc. Mass Spectrom. 2008, 19, 531. [23] S. N. Jackson, H. J. Wang, A. S. Woods. In situ structural characterization of glycerophospholipids and sulfatides in brain tissue using MALDI-MS/MS. J. Am. Soc. Mass Spectrom. 2007, 18, 17. [24] F. F. Hsu, A. Bohrer, J. Turk. Electrospray ionization tandem mass spectrometric analysis of sulfatide. Determination of fragmentation patterns and characterization of molecular species expressed in brain and in pancreatic islets. Biochem. Biophys. Acta 1998, 1392, 202. [25] L. Plhalova, S. Stepanova, E. Praskova, L. Chromcova, L. Zelnickova, L. Divisova, M. Skoric, V. Pistekova, I. Bedanova, Z. Svobodova. The effects of subchronic exposure to metribuzin on Danio rerio. Sci. World J. 2012, 728189. [26] Q. Li, S. Chen, S. Zhang, C. Li, J. Zhou, X. Ma, X. Li. Bioconcentration study of Xinjunan in zebrafish. Environ. Monit. Assess. 2011, 183, 113. [27] C. M. Jonsson, M. C. F. Toledo. Acute toxicity of endosulfan to the fish Hyphessobrycon bifasciatus and Brachydanio rerio. Arch. Environ. Contam. Toxicol. 1993, 24, 151.
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Copyright © 2014 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2014, 28, 2084–2088
Revised: 21 July 2014
Accepted: 22 July 2014
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2014, 28, 2084–2088 (wileyonlinelibrary.com) DOI: 10.1002/rcm.6993
Imaging of whole zebra fish (Danio rerio) by desorption electrospray ionization mass spectrometry Alexander Chramow1, Tanam S. Hamid1, Livia S. Eberlin2, Marion Girod3 and Demian R. Ifa1* 1
York University, Toronto, Ontario, Canada Stanford University, Stanford, CA, USA 3 ISA CNRS University of Lyon 1, Villeurbanne, Cedex, France 2
RATIONALE: To demonstrate the potential use of zebra fish (Danio rerio) as a model vertebrate organism by producing two-dimensional ion images of the whole zebra fish, and being able to distinguish particular areas of interest such as the brain, spinal cord, and stomach region using a desorption electrospray ionization (DESI) ion source coupled to a linear ion trap. METHODS: Imaging experiments are performed on 45 μm sagittal slices of zebra fish (Danio rerio), which are thaw-mounted onto microscope glass slides. The slides are then analyzed using a solvent of acetonitrile/dimethylformamide (50:50) (ACN/ DMF), with a solvent flow rate of 1.5 μL/min; data are acquired in negative ion mode. Raw mass spectrum data files are converted into a readable file for Biomap. The images produced are then analyzed for ion distributions. RESULTS: We are able to create clear, distinct, chemical intensity images of the brain, spinal cord, and stomach based on lipid content as well as bile salt. The identities of these compounds were confirmed by tandem mass spectrometric (MS/MS) experiments and comparisons with literature. CONCLUSIONS: Imaging of whole zebra fish is possible using ambient ionization techniques such as DESI. Analyses are fast and reliable. For most of the compounds observed, the identification by MS/MS can be performed directly from the fish tissue sample. Copyright © 2014 John Wiley & Sons, Ltd.
Mass spectrometry imaging (MSI) is a well-established tool allowing two-dimensional visualization of the spatial distribution of multiple analytes by their mass-to-charge ratios (m/z). Due to high sensitivity, high speed of analysis and high chemical specificity, MSI has been accepted worldwide as an effective system to detect and identify a broad range of complex molecules in biological samples. Traditional MSI methods such as matrix-assisted laser desorption/ionization (MALDI)[1] and secondary ion mass spectrometry (SIMS)[2] require sample pretreatment and analysis to be performed in vacuum. On the other hand, ambient ionization techniques such as desorption electrospray ionization (DESI)[3] require no sample pretreatment; sampling and ionization occur outside a vacuum system at atmospheric pressure, and the methods have demonstrated spatial resolutions of 40–500 μm.[4–7] The ionization process in DESI produces gas-phase ions from the condensed-phase analyte by creating charged microdroplets. These microdroplets are generated via a ’droplet pick up’ mechanism and proceed to the inlet of the mass spectrometer. This spray-based ionization is a soft technique, resulting in little to no fragmentation. Ambient techniques are useful for the analysis of low molecular weight compounds since they lack the interference caused by matrix
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* Correspondence to: D. R. Ifa, York University, Toronto, Ontario, Canada. E-mail: [email protected]
Rapid Commun. Mass Spectrom. 2014, 28, 2084–2088
ions from techniques such as MALDI. Ambient ionization sources can also analyze larger compounds such as bio and synthetic polymers.[8] DESI has already seen a wide variety of applications including molecular imaging of lipids, proteins, fatty acids, hormones and other compounds.[9] DESI has also been extensively used to characterize biological tissues such as rat brain,[4] human bladder,[10] rat spinal cord,[11] human liver,[12] etc. Previous studies using DESI involved mapping the distribution of clozapine and metabolites in rat brain, lung, kidney and testies.[5] Another study focused on creating ion images of propanolol in whole mouse tissue sections.[13] Lately, zebra fish have been receiving increased attention as they exhibit similar gene sequences and organ systems to humans.[14] This is why zebra fish are being studied and researched as an ideal vertebrate model organism. Zebra fish embryos have even been used as a model system for studying diseases and drug discovery due to their optical clarity.[14] The use of zebra fish in various laboratory experiments has been increasing due to low space requirement, low maintenance cost, rapid generation cycle, and rapid development.[15] Several biomolecules have specific distributions in organs, such as bile salts which are derivatives of cholesterol and are mostly synthesized in the liver. They perform various important functions in the liver and the intestines including elimination of cholesterol from liver into bile and promoting the absorption of lipids and lipid soluble vitamins from intestine.[16] The structure of bile salt found in zebra fish differs a little from the structure of bile found in mammals
Copyright © 2014 John Wiley & Sons, Ltd.
Imaging of whole zebra fish by DESI-MS and is composed of four steroid rings and a sulfate functional group.[17] The zebra fish bile salt has a trans configuration at the A/B ring juncture, resulting in a planar conformation. It is also considered to be evolutionarily primitive compared to more advanced bile salts which have a bent conformation due to a cis A/B ring juncture. Lipids have numerous important roles in biological systems, such as cell signalling, membrane structure, protein modifications, and cell-cell and cell-protein interactions, to name but a few.[19] Previous studies using DESI have successfully imaged deprotonated forms of various lipids in rat spinal cord such as phosphatidylinositol (PI), phosphatidylserine (PS), sulfatide (ST), etc.[11,18] It is also possible to detect deprotonated forms of fatty acids such as oleic acid, stearic acid, palmitic acid, etc., in the m/z 200–400 region. The goal of this research is to demonstrate the potential of using the zebra fish as a vertebrate model organism for further biological experimentation, by being able to create twodimensional ion maps representing specific areas of interest. These areas include the nervous system (brain, and spinal cord), gastrointestinal system, and body fat. Assessing the lipid and bile salt profiles on the same tissue section will aid the process of drawing correlation between molecular information and morphology of various organs of the zebra fish. This can facilitate the understanding of biochemical processes which could potentially be valuable for toxicological research.
EXPERIMENTAL Sample and chemicals Male adult zebra fish were kindly donated by Dr. Chun Peng’s research group (Biology Department, York University). Tricaine mesylate was purchased from Sigma-Aldrich Canada Co. (Oakville, ON, Canada) to euthanize the fish. Carboxymethyl cellulose, used to create the moulds, was also purchased from Sigma-Aldrich Canada Co. Solvents used were acetonitrile (ACN) (Optima LC/MS) from Fisher Scientific Company
(Ottawa, ON, Canada), and N,N-dimethylformamide (DMF) (HPLC grade) from Sigma-Aldrich Canada Co. Zebra fish tissue preparation/sectioning The workflow for tissue preparation and analysis is shown in Fig. 1. The zebra fish were first euthanized by placing them in a small aquarium and adding a solution of tricaine mesylate. They were then placed in disposable plastic rectangular frame/mould in order to protect them from deformation and damage during cryosectioning. These moulds were filled with carboxymethyl cellulose (CMC) solution and placed immediately in the refrigerator at 8°C for chilling overnight. A solution of carboxymethyl cellulose was made by mixing CMC and distilled water until it formed a saturated paste. The frozen CMC blocks were removed from the plastic frame and continuously sectioned into 20–60 μm thick sagittal sections at 17°C using a Shandon cryotome FE and FSE (Thermo Fischer Scientific, Nepean, ON, Canada). The frozen fish slices were thaw mounted on microscope glass slides and kept in the freezer until use. The fish slides were then dried in the lab in the open air for 30 min prior to analysis by DESI-MSI.
DESI imaging experiments Imaging was carried out using a lab-built prototype DESI ion source. In order to desorb the desired analytes, a morphologically friendly solvent system composed of ACN/DMF (50:50) was used.[8] The DESI spray tip was positioned approximately 2.6 mm from the tissue sample at an incident angle of 52°. The inlet of the mass spectrometer was approximately 2 mm from the tissue sample surface with a collection angle of approximately 10°. The spray tip-to-inlet distance was 5 mm. Experiments were conducted in negative ion mode with spray voltage of 5 kV, injection time 250 ms, 3 microscans averaged, solvent flow rate of 1.5 μL/min, and nebulizing gas (N2) pressure of 100 psi. The mass spectrometer used was a LTQ linear ion trap controlled by
Rapid Commun. Mass Spectrom. 2014, 28, 2084–2088
Copyright © 2014 John Wiley & Sons, Ltd.
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Figure 1. Overview of zebra fish tissue preparation and analysis by DESI-MSI.
A. Chramow et al. Xcalibur 2.0 software (Thermo Fisher Scientific, San Jose, CA, USA). The two-dimensional (2D) imaging experiments were performed by using a moving stage scanning an area of 2.5 cm in the x-direction and 0.6 cm in the y-direction. The velocity for the surface moving stage was 208.3 μm/s acquiring mass spectra ranging from m/z 150 to 900. The 2D images acquired consisted of arrays of 125 × 30 pixels, each pixel covering an area of 200 × 200 μm2. Each pixel required 0.96 s to be scanned making the total scan time for the 2D image to be approximately 60 min (0.96 s /scan × 125 scans × 30 lines). An in-house program allowed the conversion of the Xcalibur 2.0 mass spectra files (.raw) into a format compatible with Biomap,[20] creating single ion images as well as overlays of two different ion images. In order to adjust the colour contrast of the images, the rainbow colour gradient was selected and the signal intensity of the ions was normalized for all images used. The color intensities for the ions were all normalized to the same level so that one ion might not appear deceivingly more abundant than another. The fatty acid, bile salt and lipid species were identified by comparing the generated product ion spectra with literature data and from the MS/MS data generated through collisioninduced dissociation (CID) tandem MS experiments. CID experiments were performed directly on a tissue slice by moving the sample stage in order to position an area of interest under the spray tip. The CID energy was optimized by manually ramping up the energy from 10–30 units and monitoring the ion fragments. Optimization was attained when a stable signal with multiple ion fragments were produced. The m/z range for the ion selection window was 1.
RESULTS AND DISCUSSION The analysis of the zebra fish slice surface provided a diverse collection of ions with fatty acids dominating the lower m/z range (m/z 600). The images created from one 45 μm thick sagittal slice of a zebra fish are shown in Figs. 2(b)–2(f). Slices were taken at this thickness due to the ease of reproducibility with the chosen embedding medium. A selection of embedding media has previously been studied by Nelson et al.[21] for the purpose of freezing and cryosectioning zebra fish. They determined that
a more optimal medium consisting of 5% CMC and 10% gelatine mixture can be used to achieve reproducible slices of 16 μm thickness. The sample was analyzed in negative ion mode, since it is known that most phospholipids and sphingolipids ionize easily in this mode.[22] Analysis was also performed on the samples in positive ion mode. We were able to create an ion image of an ion m/z 265, located in the eye of the fish, which is thought to be thiamine. Reproducibility of this ion was poor, possibly due to the structure of the eye, or perhaps damage during cryosectioning. The ion images are set next to an optical image (Fig. 2(a)) of another sliced zebra fish frozen in a CMC mold, adhered to the mounting plate of a cryotome. In the optical image there is distinction between different tissue types: digestive, neuronal, muscular. Figure 2(b) represents the chemical intensity map of ion m/z 281, deprotonated oleic acid. Since it is found in the majority of the tissue it serves as a good background for making comparisons with other ions of interest (m/z 531, 834, 885, 890). Figs. 2(c)–2(f) show images of the chemical distribution of ions of interests shown in a colour gradient. All images were normalized to the same intensity gain. The ion of m/z 834 (Fig. 2(c)) appears localized in the area representing the brain of the zebra fish. This ion is phosphatidylserine (PS 40:6); its structure is confirmed by comparing MS/MS spectra (MS/MS: m/z 834, 747, 437, 419, 327, 283) with the literature.[23] The most intense peak at m/z 747 [M–H–87] corresponds to the loss of the serine head group (87 Da), which confirms the identification as a phosphatidylserine (PS). It is reasonable to find this ion localized within the brain since the main species of lipids detected in this organ are phosphatidylserines (PS), phosphatidylinositols (PI), and sulfatides (ST). In a previous study[4] analyzing rat brains using DESI, the ion m/z 834 (PS 40:6) was found with high intensity in areas correlating to grey matter, rather than white matter. This indicates that this phospholipid resides mostly within the cell body, or soma, of neurons. Phosphatidylinositol (PI 38:4) is represented as ion m/z 885 (Fig. 2(d)). This assignment is confirmed by the presence of inositolphosphate (m/z 259) in the MS/MS spectra (MS/MS: m/z 885, 599, 581, 419, 303, 283, 259). Phosphatidylinositols (PI) play a crucial role in intracellular communication,[23] therefore it would be logical to see this ion in neuronal tissue, with a much greater accumulation and intensity in the area representing the spinal cord, where a lot of information is cross-linked and carried through.
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Figure 2. (a) Optical image of zebra fish in CMC mould. (b–f) Images of ion distribution produced from DESI analysis. (b) Image of deprotonated oleic acid m/z 281, (c) phosphatidylserine (PS 40:6) m/z 834, (d) phosphatidylinositol (PI 38:4) m/z 885, (e) bile salt 5α-cyprinol 27-sulfate m/z 531, and (f) sulfatide (ST 24:0) m/z 890.
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Imaging of whole zebra fish by DESI-MS
Figure 3. DESI( ) mass spectrum from the intestinal system of the zebra fish showing: m/z 498.5, C24 bile acid-(OH)2-taurine; m/z 515.5, C27 bile alcohol(OH)4-SO4; and m/z 531.5, 5α-cyprinol 27-sulfate. Another lipid appears as an ion with m/z 890 (Fig. 2(f)), and is identified as a sulfatide (ST 24:0). This is confirmed by the presence of the fatty acid amide containing ion m/z 392 in the MS/MS spectra[24] (MS/MS: m/z 890, 872, 652, 522, 392, 258). Sulfatides (ST) are very important sphingolipids since they are present in the myelin sheath and have a role in cell differentiation and adhesion.[24] The areas of the zebra fish highlighted by the distribution and intensity of the ion at m/z 890 could possibly represent the white matter of the nervous system. A pattern containing sulfate-conjugated polyhydroxy bile alcohols, m/z 498, 515 and 531 (Fig. 3), was specifically localized within the area corresponding to the stomach, or intestinal system of the zebra fish (Fig. 2(e)). This pattern was previously reported in a study on the evolution of bile salts which identified that at least 98% of the bile salts in zebra fish are from 5α-cyprinol 27-sulfate.[17] The mass spectra recorded for the areas containing bile salt are comparable to those reported for purified extracts of zebra fish bile.[17] The ions formed during MS/MS experiments of bile salts were scarce and unstable thus restricting structural information.
CONCLUSIONS
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REFERENCES [1] E. H. Seeley, K. Schwamborn, R. M. Caprioli. Imaging of intact tissue sections: moving beyond the microscope. J. Biol. Chem. 2011, 286, 25459. [2] M. K. Passarelli, N. Winograd. Lipid imaging with timeof-flight secondary ion mass spectrometry. Biochim. Biophys. Acta 2011, 1811, 976. [3] A. L. Dill, L. S. Eberlin, D. R. Ifa, R. G. Cooks. Perspectives in imaging using mass spectrometry. Chem. Commun. 2011, 47, 2741. [4] J. M. Wiseman, D. R. Ifa, Q. Song, R. G. Cooks. Tissue imaging at atmospheric pressure using desorption electrospray ionization (DESI) mass spectrometry. Angew. Chem. Int. Ed. 2006, 45, 7188. [5] J. M. Wiseman, D. R. Ifa, Y. Zhu, C. B. Kissinger, N. E. Manicke, P. T. Kisinger, R. G. Cooks. Desorption electrospray ionization mass spectrometry: Imaging drugs and metabolites in tissues. Natl. Acad. Sci. USA 2008, 105, 18120. [6] V. Kertesz, G. J. Van Berkel. Improved imaging resolution in desorption electrospray ionization mass spectrometry. Rapid Commun. Mass Spectrom. 2008, 22, 2639. [7] L. S. Eberlin, D. R. Ifa, C. Wu, R. G. Cooks. Three-dimensional vizualization of mouse brain by lipid analysis using ambient ionization mass spectrometry. Angew. Chem. Int. Ed. 2010, 49, 873. [8] C. Wu, A. L. Dill, L. S. Eberlin, R. G. Cooks, D. R. Ifa. Mass spectrometry imaging under ambient conditions. Mass Spectrom. Rev. 2013, 32, 218. [9] R. G. Cooks, N. E. Manicke, A. L. Dill, D. R. Ifa, L. S. Eberlin, A. B. Costa, H. Wang, G. Huang, Z. Ouyang. New ionization methods and miniature mass spectrometers for biomedicine: DESI imaging for cancer diagnostics and paper spray ionization for therapeutic drug monitoring. Faraday Discuss. 2011, 149, 247. [10] A. L. Dill, L. S. Ebrelin, A. B. Costa, C. Zheng, D. R. Ifa, L. Cheng, T. A. Masterson, M. O. Koch, O. Vitek, R. G. Cooks. Multivariate statistical identification of human bladder carcinomas using ambient ionization imaging mass spectrometry. Chem. Eur. J. 2011, 17, 2897. [11] M. Girod, Y. Shi, J. Cheng, R. G. Cooks. Mapping lipid alterations in traumatically injured rat spinal cord by desorption electrospray ionization imaging mass spectrometry. Anal. Chem. 2011, 83, 207.
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In this study, the use of DESI-MSI to investigate zebra fish as a vertebrate model organism is determined to be possible. It is demonstrated that certain organs and regions of interest are visible and identifiable by having the ability to showcase the chemical intensity distribution of selected ions. Areas of interest, such as the brain, spinal cord, and stomach, can be identified by their lipid species or bile salt make-up. Most of these markers can be further characterized and their structures elucidated through MS/MS experiments, which can be acquired directly from a tissue sample. The use of ambient ionization techniques greatly facilitates the study of biological tissues and small organisms by reducing time needed for sample preparation or extensive extractions in order to characterize specific ions. The ability to analyze whole organisms could allow for future research on bioaccumulation and toxicological studies by exposing the fish to toxins, ionic liquids, detergents, etc. Previous studies on the zebra fish have included determining the LD50 (lethal dose causing 50% death in a population) for various toxins such as metribuzin,[25] xinjunan,[26] and endosulfan.[27]
By applying the information discovered by these studies, the zebra fish could potentially be monitored for the distribution of drugs or toxins as well as their metabolites within the whole body, using DESI-MSI.
A. Chramow et al. [12] S. Gerbig, O. Golf, J. Balog, J. Denes, Z. Baranyai, A. Zarand, E. Raso, J. Timar, Z. Takats. Analysis of colorectal adenocarcinoma tissue by desorption electrospray ionization mass spectrometric imaging. Anal. Bioanal. Chem. 2012, 403, 2315. [13] V. Kertesz, G. J. Van Berkel, M. Vavrek, K. A. Koeplinger, B. B. Schneider, T. R. Covey. Comparison of drug distribution images from whole-body thin tissue sections obtained using desorption electrospray ionization tandem mass spectrometry and autoradiography. Anal. Chem. 2008, 80, 5168. [14] D. R. Love, F. B. Pichler, A. Dodd, B. R. Copp, D. R. Greenwood. Technology for high-throughput screens: the present and future using zebrafish. Curr. Opin. Biotechnol. 2004, 15, 564. [15] H. M. Stern, L. I. Zon. Cancer genetics and drug discovery in the zebrafish. Nat. Rev. Cancer. 2003, 3, 533. [16] P. Song, Y. Zhang, C. D. Klaassen. Dose-response of five bile acids on serum and liver bile acid concentrations and hepatotoxicty in mice. Toxicol. Sci. 2011, 123, 359. [17] E. J. Reschly, N. Ai, S. Ekins, W. J. Welsh, L. R. Hagey, A. F. Hofmann, M. D. Krasowski. Evolution of the bile salt nuclear receptor FXR in vertebrates. J. Lipid Res. 2008, 49, 1577. [18] M. Girod, Y. Shi, J. Cheng, R. G. Cooks. Desorption electrospray ionization imaging mass spectrometry of lipids in rat spinal cord. J. Am. Soc. Mass Spectrom. 2010, 21, 1177. [19] A. D. Watson. Thematic review series: systems biology approaches to metabolic and cardiovascular disorders. Lipidomics: a global approach to lipid analysis in biological systems. J. Lipid Res. 2006, 47, 2101.
[20] Biomap freeware. Available: http://www.maldi-msi.org. [21] K. A. Nelson, G. J. Daniels, J. W. Fournie, M. J. Hemmer. Optimization of whole-body zebrafish sectioning methods for mass spectrometry imaging. J. Biomol. Technol. 2013, 24, 119. [22] N. E. Manicke, J. M. Wisemann, D. R. Ifa, R. G. Cooks. Desorption electrospray ionization (DESI) mass spectrometry and tandem mass spectrometry (MS/MS) of phospholipids and sphingolipids: ionization, adduct formation, and fragmentation. J. Am. Soc. Mass Spectrom. 2008, 19, 531. [23] S. N. Jackson, H. J. Wang, A. S. Woods. In situ structural characterization of glycerophospholipids and sulfatides in brain tissue using MALDI-MS/MS. J. Am. Soc. Mass Spectrom. 2007, 18, 17. [24] F. F. Hsu, A. Bohrer, J. Turk. Electrospray ionization tandem mass spectrometric analysis of sulfatide. Determination of fragmentation patterns and characterization of molecular species expressed in brain and in pancreatic islets. Biochem. Biophys. Acta 1998, 1392, 202. [25] L. Plhalova, S. Stepanova, E. Praskova, L. Chromcova, L. Zelnickova, L. Divisova, M. Skoric, V. Pistekova, I. Bedanova, Z. Svobodova. The effects of subchronic exposure to metribuzin on Danio rerio. Sci. World J. 2012, 728189. [26] Q. Li, S. Chen, S. Zhang, C. Li, J. Zhou, X. Ma, X. Li. Bioconcentration study of Xinjunan in zebrafish. Environ. Monit. Assess. 2011, 183, 113. [27] C. M. Jonsson, M. C. F. Toledo. Acute toxicity of endosulfan to the fish Hyphessobrycon bifasciatus and Brachydanio rerio. Arch. Environ. Contam. Toxicol. 1993, 24, 151.
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