, 5:263 | Cite as

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

  • Prabhakar Sripadi
  • Javad Nazarian
  • Yetrib Hathout
  • Eric P. Hoffman
  • Akos VertesEmail author
Original Article


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.


Torpedo californica Electric organ Neuromuscular junction Metabolites Osmolytes Metabolomics Quantitation Laser ablation Electrospray ionization LAESI Collision activated dissociation 



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.

Supplementary material

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  1. 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.PubMedCrossRefGoogle Scholar
  2. 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.PubMedCrossRefGoogle Scholar
  3. 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.PubMedCrossRefGoogle Scholar
  4. 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.CrossRefGoogle Scholar
  5. 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.PubMedCrossRefGoogle Scholar
  6. Brass, E. P. (2002). Pivalate-generating prodrugs and carnitine homeostasis in man. Pharmacological Reviews, 54, 589–598. doi: 10.1124/pr.54.4.589.PubMedCrossRefGoogle Scholar
  7. 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.PubMedCrossRefGoogle Scholar
  8. Bremer, J. (1983). Carnitine—metabolism and functions. Physiological Reviews, 63, 1420–1480.PubMedGoogle Scholar
  9. 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.PubMedCrossRefGoogle Scholar
  10. Caldas, T., Demont-Caulet, N., Ghazi, A., & Richarme, G. (1999). Thermoprotection by glycine betaine and choline. Microbiology, 145, 2543–2548.PubMedGoogle Scholar
  11. 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.PubMedCrossRefGoogle Scholar
  12. 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.CrossRefGoogle Scholar
  13. 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.PubMedCrossRefGoogle Scholar
  14. Dettmer, K., Aronov, P. A., & Hammock, B. D. (2007). Mass spectrometry-based metabolomics. Mass Spectrometry Reviews, 26, 51–78. doi: 10.1002/mas.20108.PubMedCrossRefGoogle Scholar
  15. 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.PubMedGoogle Scholar
  16. 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.CrossRefGoogle Scholar
  17. 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.PubMedCrossRefGoogle Scholar
  18. 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.PubMedGoogle Scholar
  19. 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.Google Scholar
  20. 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.PubMedCrossRefGoogle Scholar
  21. 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.PubMedCrossRefGoogle Scholar
  22. 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.PubMedCrossRefGoogle Scholar
  23. 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.PubMedCrossRefGoogle Scholar
  24. 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.PubMedCrossRefGoogle Scholar
  25. 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.PubMedCrossRefGoogle Scholar
  26. 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.PubMedCrossRefGoogle Scholar
  27. Kent, G. C. (1992). Comparative anatomy of the vertebrates. St. Louis, MO: Mosby-Year Book.Google Scholar
  28. 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.CrossRefGoogle Scholar
  29. 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.
  30. 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.PubMedCrossRefGoogle Scholar
  31. 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.PubMedCrossRefGoogle Scholar
  32. 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.PubMedCrossRefGoogle Scholar
  33. 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.CrossRefGoogle Scholar
  34. 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.PubMedCrossRefGoogle Scholar
  35. 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.PubMedCrossRefGoogle Scholar
  36. 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.PubMedCrossRefGoogle Scholar
  37. Morris, D., Bull, G., & Hebb, C. O. (1965). Acetylcholine in the electric organ of Torpedo. Nature, 207, 1295. doi: 10.1038/2071295a0.PubMedCrossRefGoogle Scholar
  38. 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.PubMedCrossRefGoogle Scholar
  39. 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.PubMedCrossRefGoogle Scholar
  40. 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.PubMedCrossRefGoogle Scholar
  41. 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.PubMedCrossRefGoogle Scholar
  42. 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.PubMedCrossRefGoogle Scholar
  43. 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/ Scholar
  44. 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.CrossRefGoogle Scholar
  45. 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.CrossRefGoogle Scholar
  46. 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.PubMedCrossRefGoogle Scholar
  47. 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.PubMedCrossRefGoogle Scholar
  48. 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.PubMedGoogle Scholar
  49. 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.PubMedCrossRefGoogle Scholar
  50. 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.PubMedCrossRefGoogle Scholar
  51. 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.CrossRefGoogle Scholar
  52. 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.CrossRefGoogle Scholar
  53. Smutna, M., Vorlova, L., & Svobodova, Z. (2002). Pathobiochemistry of ammonia in the internal environment of fish. Acta Veterinaria, 71, 169–181. Review.Google Scholar
  54. Stadler, H., & Fuldner, H. H. (1980). Proton NMR detection of acetylcholine status in synaptic vesicles. Nature, 286, 293–294. doi: 10.1038/286293a0.PubMedCrossRefGoogle Scholar
  55. 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.PubMedCrossRefGoogle Scholar
  56. 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.PubMedCrossRefGoogle Scholar
  57. 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.PubMedCrossRefGoogle Scholar
  58. 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.PubMedGoogle Scholar
  59. 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.PubMedCrossRefGoogle Scholar
  60. Vaz, F. M., & Wanders, R. J. A. (2002). Carnitine biosynthesis in mammals. The Biochemical Journal, 361, 417–429. doi: 10.1042/0264-6021:3610417.PubMedCrossRefGoogle Scholar
  61. Vekey, K., Telekes, A., & Vertes, A. (2008). Medical Applications of Mass Spectrometry. AE Amsterdam, The Netherlands: Elsevier.Google Scholar
  62. 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.PubMedGoogle Scholar
  63. 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.CrossRefGoogle Scholar
  64. 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.PubMedCrossRefGoogle Scholar
  65. 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.PubMedCrossRefGoogle Scholar
  66. 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.PubMedCrossRefGoogle Scholar
  67. 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.PubMedCrossRefGoogle Scholar
  68. Zito, K. (2003). The flip side of synapse elimination. Neuron, 37, 1–2. doi: 10.1016/S0896-6273(02)01182-0.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Prabhakar Sripadi
    • 1
  • Javad Nazarian
    • 2
  • Yetrib Hathout
    • 2
  • Eric P. Hoffman
    • 2
  • Akos Vertes
    • 1
    Email author
  1. 1.Department of Chemistry, W. M. Keck Institute for Proteomics Technology and ApplicationsGeorge Washington UniversityWashingtonUSA
  2. 2.Research Center for Genetic MedicineChildren’s National Medical CenterWashingtonUSA

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