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In vitro analysis of metabolites from the untreated tissue of Torpedo californica electric organ by mid-infrared laser ablation electrospray ionization mass spectrometry

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Abstract

The neuromuscular junction (NMJ), where a motor neuron intercepts and activates a muscle fiber, is a highly versatile and complex subcellular region. Genomic and proteomic approaches using the large (>1 kg) electric organ of Torpedo californica have helped advancing our understanding of this minute (30–50 μm) electric synapse. However, the majority of these studies have focused on mRNA and proteins, therefore neglecting small signaling molecules involved in muscle-nerve ‘dialogue’. We developed a novel technique, mid-infrared laser ablation electrospray ionization (LAESI) mass spectrometry (MS), with the potential of detecting a diversity of small signaling molecules in vitro. LAESI uses the native water in the tissue as the matrix to couple the laser pulse energy into the target for the ablation process and enables its direct analysis essentially without sample preparation. Here, we report the detection of metabolites from the untreated frozen tissue of the Torpedo electric organ with LAESI MS at atmospheric pressure. A total of 24 metabolites were identified by accurate mass measurements, natural isotope patterns, and tandem mass spectrometry. Most of the identified metabolites were related to the cholinergic function of the electric synapse (acetylcholine and choline), fatty acid metabolism and acetyl transfer (carnitine and acetylcarnitine), the mitigation of osmotic stress (betaine and trimethylamine N-oxide), and energy production (creatine and creatinine). The biosynthetic precursors of these metabolites and their expected degradation products were also detected indicating that LAESI MS is well suited for tissue metabolomics with the ultimate goal of imaging and in vivo studies.

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References

  • Allen, D. L., Roy, R. R., & Edgerton, V. R. (1999). Myonuclear domains in muscle adaptation and disease. Muscle and Nerve, 22, 1350–1360. doi:10.1002/(SICI)1097-4598(199910)22:10<1350::AID-MUS3>3.0.CO;2-8.

    Article  PubMed  CAS  Google Scholar 

  • Anderson, D. C., King, S. C., & Parsons, S. M. (1982). Proton gradient linkage to active uptake of [3H]acetylcholine by Torpedo electric organ synaptic vesicles. Biochemistry, 21, 3037–3043. doi:10.1021/bi00256a001.

    Article  PubMed  CAS  Google Scholar 

  • Bain, M. A., Faull, R., Fornasini, G., et al. (2004). Quantifying trimethylamine and trimethylamine-N-oxide in human plasma: interference from endogenous quaternary ammonium compounds. Analytical Biochemistry, 334, 403–405. doi:10.1016/j.ab.2004.07.004.

    Article  PubMed  CAS  Google Scholar 

  • Bhuiyan, A. K. M. J., Jackson, S., Turnbull, D. M., et al. (1992). The measurement of carnitine and acyl-carnitines: Application to the investigation of patients with suspected inherited disorders of mitochondrial fatty acid oxidation. Clinica Chimica Acta, 207, 185–204. doi:10.1016/0009-8981(92)90118-A.

    Article  CAS  Google Scholar 

  • Bino, R. J., Hall, R. D., Fiehn, O., et al. (2004). Potential of metabolomics as a functional genomics tool. Trends in Plant Science, 9, 418–425. doi:10.1016/j.tplants.2004.07.004.

    Article  PubMed  CAS  Google Scholar 

  • Brass, E. P. (2002). Pivalate-generating prodrugs and carnitine homeostasis in man. Pharmacological Reviews, 54, 589–598. doi:10.1124/pr.54.4.589.

    Article  PubMed  CAS  Google Scholar 

  • Breer, H., Morris, S. J., & Whittaker, V. P. (1978). A structural model of cholinergic synaptic vesicles from the electric organ of Torpedo marmorata deduced from density measurements at different osmotic pressures. European Journal of Biochemistry, 87, 453–458. doi:10.1111/j.1432-1033.1978.tb12395.x.

    Article  PubMed  CAS  Google Scholar 

  • Bremer, J. (1983). Carnitine—metabolism and functions. Physiological Reviews, 63, 1420–1480.

    PubMed  CAS  Google Scholar 

  • Burg, M. B., & Ferraris, J. D. (2008). Intracellular organic osmolytes: Function and regulation. The Journal of Biological Chemistry, 283, 7309–7313. doi:10.1074/jbc.R700042200.

    Article  PubMed  CAS  Google Scholar 

  • Caldas, T., Demont-Caulet, N., Ghazi, A., & Richarme, G. (1999). Thermoprotection by glycine betaine and choline. Microbiology, 145, 2543–2548.

    PubMed  CAS  Google Scholar 

  • Cody, R. B., Laramee, J. A., & Durst, H. D. (2005). Versatile new ion source for the analysis of materials in open air under ambient conditions. Analytical Chemistry, 77, 2297–2302. doi:10.1021/ac050162j.

    Article  PubMed  CAS  Google Scholar 

  • Colmer, T. D., Corradini, F., Cawthray, G. R., & Otte, M. L. (2000). Analysis of dimethylsulphoniopropionate (DMSP), betaines and other organic solutes in plant tissue extracts using HPLC. Phytochemical Analysis, 11, 163–168. doi:10.1002/(SICI)1099-1565(200005/06)11:3<163::AID-PCA501>3.0.CO;2-0.

    Article  CAS  Google Scholar 

  • Corthay, J., Dunant, Y., Eder, L., & Loctin, F. (1985). Incorporation of acetate into acetylcholine, acetylcarnitine, and amino acids in the Torpedo electric organ. Journal of Neurochemistry, 45, 1809–1819. doi:10.1111/j.1471-4159.1985.tb10538.x.

    Article  PubMed  CAS  Google Scholar 

  • Dettmer, K., Aronov, P. A., & Hammock, B. D. (2007). Mass spectrometry-based metabolomics. Mass Spectrometry Reviews, 26, 51–78. doi:10.1002/mas.20108.

    Article  PubMed  CAS  Google Scholar 

  • Feldberg, W., & Fessard, A. (1942). The cholinergic nature of the nerves to the electric organ of the Torpedo (Torpedo marmorata). The Journal of Physiology, 101, 200–216.

    PubMed  CAS  Google Scholar 

  • Ghoshal, A. K., Guo, T., Soukhova, N., & Soldin, S. J. (2005). Rapid measurement of plasma acylcarnitines by liquid chromatography-tandem mass spectrometry without derivatization. Clinica Chimica Acta, 358, 104–112. doi:10.1016/j.cccn.2005.02.011.

    Article  CAS  Google Scholar 

  • Gillingwater, T. H., & Ribchester, R. R. (2003). The relationship of neuromuscular synapse elimination to synaptic degeneration and pathology: insights from WldS and other mutant mice. Journal of Neurocytology, 32, 863–881. doi:10.1023/B:NEUR.0000020629.51673.f5.

    Article  PubMed  CAS  Google Scholar 

  • Goldstein, L., Oppelt, W. W., & Maren, T. H. (1968). Osmotic regulation and urea metabolism in the lemon shark Negaprion brevirostris. The American Journal of Physiology, 215, 1493–1497.

    PubMed  CAS  Google Scholar 

  • Hanley-Jr., J., Bernasconi, A., Davis, R. et al. (2007). Quantitative analysis of acylcarnitines in plasma, serum and urine by liquid chromatography-tandem mass spectrometry. Proceedings of the 55th ASMS Conference on Mass Spectrometry and Allied Topics, Indianapolis, IN, 3–7 June, 2007.

  • Hayashi, Y., Katsumoto, Y., Oshige, I., Omori, S., & Yasuda, A. (2007). Comparative study of urea and betaine solutions by dielectric spectroscopy: Liquid structures of a protein denaturant and stabilizer. The Journal of Physical Chemistry B, 111, 11858–11863. doi:10.1021/jp073238j.

    Article  PubMed  CAS  Google Scholar 

  • Holm, P. I., Ueland, P. M., Kvalheim, G., & Lien, E. A. (2003). Determination of choline, betaine, and dimethylglycine in plasma by a high-throughput method based on normal-phase chromatography-tandem mass spectrometry. Clinical Chemistry, 49, 286–294. doi:10.1373/49.2.286.

    Article  PubMed  CAS  Google Scholar 

  • Israel, M., & Lesbats, B. (1981). Continuous determination by a chemi-luminescent method of acetylcholine-release and compartmentation in Torpedo electric organ synaptosomes. Journal of Neurochemistry, 37, 1475–1483. doi:10.1111/j.1471-4159.1981.tb06317.x.

    Article  PubMed  CAS  Google Scholar 

  • Jakobs, B. S., & Wanders, R. J. A. (1995). Fatty acid [beta]-oxidation in peroxisomes and mitochondria: The first, unequivocal evidence for the involvement of carnitine in shuttling propionyl-CoA from peroxisomes to mitochondria. Biochemical and Biophysical Research Communications, 213, 1035–1041. doi:10.1006/bbrc.1995.2232.

    Article  PubMed  CAS  Google Scholar 

  • Johnson, D. W. (2008). A flow injection electrospray ionization tandem mass spectrometric method for the simultaneous measurement of trimethylamine and trimethylamine N-oxide in urine. Journal of Mass Spectrometry, 43, 495–499. doi:10.1002/jms.1339.

    Article  PubMed  CAS  Google Scholar 

  • Kamimori, H., Hamashima, Y., & Konishi, M. (1994). Determination of carnitine and saturated-acyl group carnitines in human urine by high-performance liquid chromatography with fluorescence detection. Analytical Biochemistry, 218, 417–424. doi:10.1006/abio.1994.1201.

    Article  PubMed  CAS  Google Scholar 

  • Keller-Peck, C. R., Walsh, M. K., Gan, W. B., et al. (2001). Asynchronous synapse elimination in neonatal motor units: Studies using GFP transgenic mice. Neuron, 31, 381–394. doi:10.1016/S0896-6273(01)00383-X.

    Article  PubMed  CAS  Google Scholar 

  • Kent, G. C. (1992). Comparative anatomy of the vertebrates. St. Louis, MO: Mosby-Year Book.

    Google Scholar 

  • Keynes, R. D., Greeff, N. G., & Forster, I. C. (1992). Activation, inactivation and recovery in the sodium channels of the squid giant axon dialysed with different solutions. Philosophical Transactions of the Royal Society B Biological Sciences, 337, 471–484. doi:10.1098/rstb.1992.0122.

    Article  CAS  Google Scholar 

  • Kistler, J., & Stroud, R. M. (1981). Crystalline arrays of membrane-bound acetylcholine receptor. Proceedings of the National Academy of Sciences of the United States of America, 78, 3678–3682. doi:10.1073/pnas.78.6.3678.

  • Kistler, J., Stroud, R. M., Klymkowsky, M. W., Lalancette, R. A., & Fairclough, R. H. (1982). Structure and function of an acetylcholine receptor. Biophysical Journal, 37, 371–383.

    Article  PubMed  CAS  Google Scholar 

  • Li, Y., Shrestha, B., & Vertes, A. (2007). Atmospheric pressure molecular imaging by infrared MALDI mass spectrometry. Analytical Chemistry, 79, 523–532. doi:10.1021/ac061577n.

    Article  PubMed  CAS  Google Scholar 

  • Li, Y., Shrestha, B., & Vertes, A. (2008). Atmospheric pressure infrared MALDI imaging mass spectrometry for plant metabolomics. Analytical Chemistry, 80, 407–420. doi:10.1021/ac701703f.

    Article  PubMed  CAS  Google Scholar 

  • Mardones, C., Vizioli, N., Carducci, C., Rios, A., & Valcarcel, M. (1999). Separation and determination of carnitine and acyl-carnitines by capillary electrophoresis with indirect UV detection. Analytica Chimica Acta, 382, 23–31. doi:10.1016/S0003-2670(98)00790-9.

    Article  CAS  Google Scholar 

  • Mashego, M., Rumbold, K., De Mey, M., et al. (2007). Microbial metabolomics: Past, present and future methodologies. Biotechnology Letters, 29, 1–16. doi:10.1007/s10529-006-9218-0.

    Article  PubMed  CAS  Google Scholar 

  • McGarry, J. D., & Brown, N. F. (1997). The mitochondrial carnitine palmitoyltransferase system—From concept to molecular analysis. European Journal of Biochemistry, 244, 1–14. doi:10.1111/j.1432-1033.1997.00001.x.

    Article  PubMed  CAS  Google Scholar 

  • Mitra, A. K., McCarthy, M. P., & Stroud, R. M. (1989). Three-dimensional structure of the nicotinic acetylcholine receptor and location of the major associated 43-kD cytoskeletal protein, determined at 22 A by low dose electron microscopy and X-ray diffraction to 12.5 A. The Journal of Cell Biology, 109, 755–774. doi:10.1083/jcb.109.2.755. published erratum appears in The Journal of Cell Biology, 1989 Oct; 109 (4 Pt 1):1185.

    Article  PubMed  CAS  Google Scholar 

  • Morris, D., Bull, G., & Hebb, C. O. (1965). Acetylcholine in the electric organ of Torpedo. Nature, 207, 1295. doi:10.1038/2071295a0.

    Article  PubMed  CAS  Google Scholar 

  • Nazarian, J., Bouri, K., & Hoffman, E. P. (2005). Intracellular expression profiling by laser capture microdissection: Three novel components of the neuromuscular junction. Physiological Genomics, 21, 70–80. doi:10.1152/physiolgenomics.00227.2004.

    Article  PubMed  CAS  Google Scholar 

  • Nazarian, J., Hathout, Y., Vertes, A., & Hoffman, E. P. (2007). The proteome survey of an electricity-generating organ (Torpedo californica electric organ). Proteomics, 7, 617–627. doi:10.1002/pmic.200600686.

    Article  PubMed  CAS  Google Scholar 

  • Nemes, P., Barton, A. A., Li, Y., & Vertes, A. (2008). Ambient molecular imaging and depth profiling of live tissue by infrared laser ablation electrospray ionization mass spectrometry. Analytical Chemistry, 80, 4575–4582. doi:10.1021/ac8004082.

    Article  PubMed  CAS  Google Scholar 

  • Nemes, P., & Vertes, A. (2007). Laser ablation electrospray ionization for atmospheric pressure, in vivo, and imaging mass spectrometry. Analytical Chemistry, 79, 8098–8106. doi:10.1021/ac071181r.

    Article  PubMed  CAS  Google Scholar 

  • Park, E. I., & Garrow, T. A. (1999). Interaction between dietary methionine and methyl donor intake on rat liver betaine-homocysteine methyltransferase gene expression and organization of the human gene. The Journal of Biological Chemistry, 274, 7816–7824. doi:10.1074/jbc.274.12.7816.

    Article  PubMed  CAS  Google Scholar 

  • Pettegrew, J. W., Levine, J., & McClure, R. J. (2000). Acetyl-l-carnitine physical-chemical, metabolic, and therapeutic properties: Relevance for its mode of action in Alzheimer’s disease and geriatric depression. Molecular Psychiatry, 5, 616–632. doi:10.1038/sj.mp.4000805.

    Article  PubMed  CAS  Google Scholar 

  • Pierce, C. Y., Barr, J. R., Cody, R. B., et al. (2007). Ambient generation of fatty acid methyl ester ions from bacterial whole cells by direct analysis in real time (DART) mass spectrometry. Chemical Communications, 2007, 807–809. doi:10.1039/b613200f.

    Article  CAS  Google Scholar 

  • Randall, D. J., & Ip, Y. K. (2006). Ammonia as a respiratory gas in water and air-breathing fishes. Respiratory Physiology & Neurobiology, 154, 216–225. doi:10.1016/j.resp.2006.04.003.

    Article  CAS  Google Scholar 

  • Randall, D. J., Wood, C. M., Perry, S. F., et al. (1989). Urea excretion as a strategy for survival in a fish living in a very alkaline environment. Nature, 337, 165–166. doi:10.1038/337165a0.

    Article  PubMed  CAS  Google Scholar 

  • Rebouche, C. J., & Seim, H. (1998). Carnitine metabolism and its regulation in microorganisms and mammals. Annual Review of Nutrition, 18, 39–61. doi:10.1146/annurev.nutr.18.1.39.

    Article  PubMed  CAS  Google Scholar 

  • Rossi, S. G., Vazquez, A. E., Rotundo, R. L., et al. (2000). Local control of acetylcholinesterase gene expression in multinucleated skeletal muscle fibers: Individual nuclei respond to signals from the overlying plasma membrane; Myonuclear domains in muscle adaptation and disease; Postsynaptic signaling of new players at the neuromuscular junction. The Journal of Neuroscience, 20, 919–928.

    PubMed  CAS  Google Scholar 

  • Sanes, J. R., & Lichtman, J. W. (1999). Development of the vertebrate neuromuscular junction. Annual Review of Neuroscience, 22, 389–442. doi:10.1146/annurev.neuro.22.1.389.

    Article  PubMed  CAS  Google Scholar 

  • Sanes, J. R., & Lichtman, J. W. (2001). Induction, assembly, maturation and maintenance of a postsynaptic apparatus. Nature Reviews. Neuroscience, 2, 791–805. doi:10.1038/35097557.

    Article  PubMed  CAS  Google Scholar 

  • Shewan, J. M. (1953). The nitrogenous extractives from fresh fish muscle. II-Comparison of several gadoid and elasmobranch species. Journal of the Science of Food and Agriculture, 4, 565. doi:10.1002/jsfa.2740041202.

    Article  CAS  Google Scholar 

  • Shrestha, B., Li, Y., & Vertes, A. (2008). Rapid analysis of pharmaceuticals and excreted xenobiotic and endogenous metabolites with atmospheric pressure infrared MALDI mass spectrometry. Metabolomics, 4, 297–311. doi:10.1007/s11306-008-0120-8.

    Article  CAS  Google Scholar 

  • Smutna, M., Vorlova, L., & Svobodova, Z. (2002). Pathobiochemistry of ammonia in the internal environment of fish. Acta Veterinaria, 71, 169–181. Review.

    CAS  Google Scholar 

  • Stadler, H., & Fuldner, H. H. (1980). Proton NMR detection of acetylcholine status in synaptic vesicles. Nature, 286, 293–294. doi:10.1038/286293a0.

    Article  PubMed  CAS  Google Scholar 

  • Steiber, A., Kerner, J., & Hoppel, C. L. (2004). Carnitine: A nutritional, biosynthetic, and functional perspective. Molecular Aspects of Medicine, 25, 455–473. doi:10.1016/j.mam.2004.06.006.

    Article  PubMed  CAS  Google Scholar 

  • Takats, Z., Wiseman, J. M., Gologan, B., & Cooks, R. G. (2004). Mass spectrometry sampling under ambient conditions with desorption electrospray ionization. Science, 306, 471–473. doi:10.1126/science.1104404.

    Article  PubMed  CAS  Google Scholar 

  • Treberg, J. R., Speers-Roesch, B., Piermarini, P. M., et al. (2006). The accumulation of methylamine counteracting solutes in elasmobranchs with differing levels of urea: A comparison of marine and freshwater species. The Journal of Experimental Biology, 209, 860–870. doi:10.1242/jeb.02055.

    Article  PubMed  CAS  Google Scholar 

  • Vaz, F. M., Melegh, B., Bene, J., et al. (2002). Analysis of carnitine biosynthesis metabolites in urine by HPLC-electrospray tandem mass spectrometry. Clinical Chemistry, 48, 826–834.

    PubMed  CAS  Google Scholar 

  • Vaz, F. M., Ofman, R., Westinga, K., Back, J. W., & Wanders, R. J. A. (2001). Molecular and biochemical characterization of rat epsilon N-trimethyllysine hydroxylase, the first enzyme of carnitine biosynthesis. The Journal of Biological Chemistry, 276, 33512–33517. doi:10.1074/jbc.M105929200.

    Article  PubMed  CAS  Google Scholar 

  • Vaz, F. M., & Wanders, R. J. A. (2002). Carnitine biosynthesis in mammals. The Biochemical Journal, 361, 417–429. doi:10.1042/0264-6021:3610417.

    Article  PubMed  CAS  Google Scholar 

  • Vekey, K., Telekes, A., & Vertes, A. (2008). Medical Applications of Mass Spectrometry. AE Amsterdam, The Netherlands: Elsevier.

    Google Scholar 

  • Verhoeven, N. M., Roe, D. S., Kok, R. M., et al. (1998). Phytanic acid and pristanic acid are oxidized by sequential peroxisomal and mitochondrial reactions in cultured fibroblasts. Journal of Lipid Research, 39, 66–74.

    PubMed  CAS  Google Scholar 

  • Vertes, A., Nemes, P., Shrestha, B., et al. (2008). Molecular imaging by mid-IR laser ablation mass spectrometry. Applied Physics A Materials Science & Processing, 93, 885–891. doi:10.1007/s00339-008-4750-5.

    Article  CAS  Google Scholar 

  • Walsh, M. K., & Lichtman, J. W. (2003). In vivo time-lapse imaging of synaptic takeover associated with naturally occurring synapse elimination. Neuron, 37, 67–73. doi:10.1016/S0896-6273(02)01142-X.

    Article  PubMed  CAS  Google Scholar 

  • Wood, K. V., Bonham, C. C., Miles, D., et al. (2002). Characterization of betaines using electrospray MS/MS. Phytochemistry, 59, 759–765. doi:10.1016/S0031-9422(02)00049-3.

    Article  PubMed  CAS  Google Scholar 

  • Woodhull, A. M. (1973). Ionic blockage of sodium channels in nerve. The Journal of General Physiology, 61, 687–708. doi:10.1085/jgp.61.6.687.

    Article  PubMed  CAS  Google Scholar 

  • Yancey, P. H., Clark, M. E., Hand, S. C., Bowlus, R. D., & Somero, G. N. (1982). Living with water stress: Evolution of osmolyte systems. Science, 217, 1214–1222. doi:10.1126/science.7112124.

    Article  PubMed  CAS  Google Scholar 

  • Zito, K. (2003). The flip side of synapse elimination. Neuron, 37, 1–2. doi:10.1016/S0896-6273(02)01182-0.

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

The authors are grateful for the support of this work by the W. M. Keck Foundation (041904), the National Science Foundation under grant 0719232, and the Research Enhancement Fund of the George Washington University. The opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation. The authors thank Peter Nemes (George Washington University) for his help in setting up the LAESI experiments. One of the authors (P. S.) thanks the Director of the Indian Institute of Chemical Technology, Hyderabad, and the Council of Scientific and Industrial Research, India for granting leave.

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  1. Department of Chemistry, W. M. Keck Institute for Proteomics Technology and Applications, George Washington University, Washington, 20052, USA

    Prabhakar Sripadi & Akos Vertes

  2. Research Center for Genetic Medicine, Children’s National Medical Center, Washington, 20010, USA

    Javad Nazarian, Yetrib Hathout & Eric P. Hoffman

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  1. Prabhakar Sripadi
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Sripadi, P., Nazarian, J., Hathout, Y. et al. In vitro analysis of metabolites from the untreated tissue of Torpedo californica electric organ by mid-infrared laser ablation electrospray ionization mass spectrometry. Metabolomics 5, 263–276 (2009). https://doi.org/10.1007/s11306-008-0147-x

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