, 14:50 | Cite as

Organic washes of tissue sections for comprehensive analysis of small molecule metabolites by MALDI MS imaging of rat brain following status epilepticus

Original Article



In-situ detection and in particular comprehensive analysis of small molecule metabolites (SMMs, m/z < 500) using matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) remain a challenge, mainly due to ion suppression effects from more abundant molecules in tissue section like lipids.


A strategy based on organic washes to remove most ionization-suppressing lipids from tissue section was firstly explored for improved analysis of SMMs by MALDI MSI.


The tissue sections after rinse with different organic solvents were analyzed by MALDI MSI, and the results were compared for the optimized washing conditions.


The rinse with chloroform for 15 s at − 20 °C significantly removed most glycerophospholipids and glycerolipids from tissue section. Consequentially, ATP-related energy metabolites, amino acids and derivatives, glucose derivatives, glycolysis pathway metabolites and other SMMs were able to be well-visualized with enhanced ion intensity and good reproducibility. The organic washes-based MALDI MSI was applied to the metabolic pathway analysis in rat brain following status epilepticus (SE) model, which was, as far as we know, the first report about in-situ detection of a broad range of metabolites in the model of SE by MALDI MSI technique. The alterations of cyclic adenosine monophosphate (cyclic AMP), inosine, glutamine, glutathione, taurine and spermine during SE were observed.


A simple organic washing protocol enables comprehensive analysis of tissue SMMs in MALDI MSI by removing ionization-suppressing lipids. The application in the SE model indicates that MALDI MSI analysis potentially provides new insight for understanding the disease mechanism.


MALDI MSI Metabolites Organic wash Status epilepticus 



This work was supported by National Natural Science Foundation of China (Grant Nos. 21575146, 21635008 and 21621062).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest and no conflict of financial interest.

Ethical approval

The animal experiments were performed according to “the Guide for the Care and Use of Laboratory Animals” from the Association for Assessment and Accreditation of Laboratory Animal Care and ethical approval of the present study was obtained from the Animal Care and Use Committee at National Center for Nanoscience and Technology of China.

Supplementary material

11306_2018_1348_MOESM1_ESM.docx (2.9 mb)
Supplementary material 1 (DOCX 2995 KB)


  1. Alvestad, S., Hammer, J., Qu, H., Haberg, A., Ottersen, O. P., & Sonnewald, U. (2011). Reduced astrocytic contribution to the turnover of glutamate, glutamine, and GABA characterizes the latent phase in the kainate model of temporal lobe epilepsy. Journal of Cerebral Blood Flow and Metabolism, 31(8), 1675–1686. Scholar
  2. Angel, P. M., Spraggins, J. M., Baldwin, H. S., & Caprioli, R. (2012). Enhanced sensitivity for high spatial resolution lipid analysis by negative ion mode matrix assisted laser desorption ionization imaging mass spectrometry. Analytical Chemistry, 84(3), 1557–1564. Scholar
  3. Calligaris, D., Longuespee, R., Debois, D., Asakawa, D., Turtoi, A., Castronovo, V., et al. (2013). Selected protein monitoring in histological sections by targeted MALDI-FTICR in-source decay imaging. Analytical Chemistry, 85(4), 2117–2126. Scholar
  4. Dona, F., Conceicao, I. M., Ulrich, H., Ribeiro, E. B., Freitas, T. A., Abrahao Nencioni, A. L., et al. (2016). Variations of ATP and its metabolites in the hippocampus of rats subjected to pilocarpine-induced temporal lobe epilepsy. Purinergic Signalling, 12(2), 295–302. Scholar
  5. Eid, T., Thomas, M. J., Spencer, D. D., Runden-Pran, E., Lai, J. C. K., Malthankar, G. V., et al. (2004). Loss of glutamine synthetase in the human epileptogenic hippocampus: Possible mechanism for raised extracellular glutamate in mesial temporal lobe epilepsy. Lancet, 363(9402), 28–37. Scholar
  6. Esteve, C., Tolner, E. A., Shyti, R., van den Maagdenberg, A. M. J. M., & McDonnell, L. A. (2016). Mass spectrometry imaging of amino neurotransmitters: A comparison of derivatization methods and application in mouse brain tissue. Metabolomics
  7. Filibian, M., Frasca, A., Maggioni, D., Micotti, E., Vezzani, A., & Ravizza, T. (2012). In vivo imaging of glia activation using H-1-magnetic resonance spectroscopy to detect putative biomarkers of tissue epileptogenicity. Epilepsia, 53(11), 1907–1916. Scholar
  8. Folch, J., Lees, M., & SLOANE STANLEY, G. H. (1957). A simple method for the isolation and purification of total lipids from animal tissue. Journal of Biological Chemistry, 226(1), 497–509.PubMedGoogle Scholar
  9. Gatenby, R. A., & Gillies, R. J. (2004). Why do cancers have high aerobic glycolysis? Nature Reviews Cancer, 4(11), 891–899. Scholar
  10. Green, D. R., Galluzzi, L., & Kroemer, G. (2014). Metabolic control of cell death. Science, 345(6203), 1466–1490. Scholar
  11. Guo, S., Wang, Y., Zhou, D., & Li, Z. (2015). Electric field-assisted matrix coating method enhances the detection of small molecule metabolites for mass spectrometry imaging. Analytical Chemistry, 87(12), 5860–5865. Scholar
  12. Guo, Z., Zhang, Q. C., Zou, H. F., Guo, B. C., & Ni, J. Y. (2002). A method for the analysis of low-mass molecules by MALDI-TOF mass spectrometry. Analytical Chemistry, 74(7), 1637–1641. Scholar
  13. Harmsen, E., Detombe, P. P., Dejong, J. W., & Achterberg, P. W. (1984). Enhanced ATP and GTP synthesis from hypoxanthine or inosine after myocardial ischemia. American Journal of Physiology, 246(1), H37–H43.Google Scholar
  14. Hillert, M. H., Imran, I., Zimmermann, M., Lau, H., Weinfurter, S., & Klein, J. (2014). Dynamics of hippocampal acetylcholine release during lithium-pilocarpine-induced status epilepticus in rats. Journal of Neurochemistry, 131(1), 42–52. Scholar
  15. Hua, Y. M., Dagan, S., Wickramasekara, S., Boday, D. J., & Wysocki, V. H. (2010). Analysis of deprotonated acids with silicon nanoparticle-assisted laser desorption/ionization mass spectrometry. Journal of Mass Spectrometry, 45(12), 1394–1401. Scholar
  16. Karlsson, O., & Hanrieder, J. (2016). Imaging mass spectrometry in drug development and toxicology. Archives of Toxicology, 91(6), 2283–2294.CrossRefPubMedPubMedCentralGoogle Scholar
  17. Kovacs, Z., Kekesi, K. A., Juhasz, G., Barna, J., Heja, L., Lakatos, R., et al. (2015). Non-adenosine nucleoside inosine, guanosine and uridine as promising antiepileptic drugs: a summary of current literature. [Research Support, Non-U.S. Gov’t; Review]. Mini Reviews in Medicinal Chemistry, 14(13), 1033–1042.CrossRefPubMedGoogle Scholar
  18. Kovacs, Z., Kekesi, K. A., Juhasz, G., & Dobolyi, A. (2014). The antiepileptic potential of nucleosides. Current Medicinal Chemistry, 21(6), 788–821.CrossRefPubMedGoogle Scholar
  19. Lemaire, R., Wisztorski, M., Desmons, A., Tabet, J. C., Day, R., Salzet, M., et al. (2006). MALDI-MS direct tissue analysis of proteins: Improving signal sensitivity using organic treatments. Analytical Chemistry, 78(20), 7145–7153. Scholar
  20. Li, S., Zhang, Y., Liu, J. a., Han, J., Guan, M., Yang, H., et al. (2016). Electrospray deposition device used to precisely control the matrix crystal to improve the performance of MALDI MSI. Scientific Reports, 6, 37903. Scholar
  21. Lietsche, J., Imran, I., & Klein, J. (2016). Extracellular levels of ATP and acetylcholine during lithium-pilocarpine induced status epilepticus in rats. Neuroscience Letters, 611, 69–73. Scholar
  22. Liu, H., Chen, R., Wang, J., Chen, S., Xiong, C., Wang, J., et al. (2014). 1,5-Diaminonaphthalene hydrochloride assisted laser desorption/ionization mass spectrometry imaging of small molecules in tissues following focal cerebral ischemia. Analytical Chemistry, 86(20), 10114–10121. Scholar
  23. Meng, J., Shi, C., & Deng, C. (2011). Facile synthesis of water-soluble multi-wall carbon nanotubes and polyaniline composites and their application in detection of small metabolites by matrix assisted laser desorption/ionization mass spectrometry. Chemical Communications, 47(39), 11017–11019. Scholar
  24. Miura, D., Fujimura, Y., Tachibana, H., & Wariishi, H. (2010a). Highly sensitive matrix-assisted laser desorption ionization-mass spectrometry for high-throughput metabolic profiling. Analytical Chemistry, 82(2), 498–504. Scholar
  25. Miura, D., Fujimura, Y., Yamato, M., Hyodo, F., Utsumi, H., Tachibana, H., et al. (2010b). Ultrahighly sensitive in situ metabolomic imaging for visualizing spatiotemporal metabolic behaviors. Analytical Chemistry, 82(23), 9789–9796. Scholar
  26. Norris, J. L., & Caprioli, R. M. (2013). Analysis of tissue specimens by matrix-assisted laser desorption/ionization imaging mass spectrometry in biological and clinical research. Chemical Reviews, 113(4), 2309–2342. Scholar
  27. Nugent, A. C., Martinez, A., D’Alfonso, A., Zarate, C. A., & Theodore, W. H. (2015). The relationship between glucose metabolism, resting-state fMRI BOLD signal, and GABA(A)-binding potential: A preliminary study in healthy subjects and those with temporal lobe epilepsy. Journal of Cerebral Blood Flow and Metabolism, 35(4), 583–591. Scholar
  28. Padma, M. V., Simkins, R., White, P., Satter, M., Christian, B. T., Dunigan, K., et al. (2004). Clinical utility of 11C-flumazenil positron emission tomography in intractable temporal lobe epilepsy. Neurology India, 52(4), 457–462.PubMedGoogle Scholar
  29. Patti, G. J., Yanes, O., & Siuzdak, G. (2012). Metabolomics: The apogee of the omics trilogy. Nature Reviews Molecular Cell Biology, 13(4), 263–269. Scholar
  30. Paxinos, G., & Watson, C. (1986). The rat brain in stereotaxic coordinates. San Diego: Academic Press.Google Scholar
  31. Racine, R., Okujava, V., & Chipashvili, S. (1972). Modification of seizure activity by electrical stimulation: III. Mechanisms. Electroencephalography & Clinical Neurophysiology, 32(3), 295–299.CrossRefGoogle Scholar
  32. Rice, A. C., & DeLorenzo, R. J. (1998). NMDA receptor activation during status epilepticus is required for the development of epilepsy. Brain Research, 782(1–2), 240–247. Scholar
  33. Shi, C. Y., & Deng, C. H. (2016). Recent advances in inorganic materials for LDI-MS analysis of small molecules. Analyst, 141(10), 2816–2826. Scholar
  34. Smeland, O. B., Hadera, M. G., McDonald, T. S., Sonnewald, U., & Borges, K. (2013). Brain mitochondrial metabolic dysfunction and glutamate level reduction in the pilocarpine model of temporal lobe epilepsy in mice. Journal of Cerebral Blood Flow and Metabolism, 33(7), 1090–1097. Scholar
  35. Sumner, L. W., Amberg, A., Barrett, D., Beale, M. H., Beger, R., Daykin, C. A., et al. (2007). Proposed minimum reporting standards for chemical analysis. Metabolomics, 3(3), 211–221. Scholar
  36. Thomas, A., Lenglet, S., Chaurand, P., Deglon, J., Mangin, P., Mach, F., et al. (2011). Mass spectrometry for the evaluation of cardiovascular diseases based on proteomics and lipidomics. Thrombosis and Haemostasis, 106(1), 20–33. Scholar
  37. Thomas, A., Patterson, N. H., Charbonneau, J. L., & Chaurand, P. (2013). Orthogonal organic and aqueous-based washes of tissue sections to enhance protein sensitivity by MALDI imaging mass spectrometry. Journal of Mass Spectrometry, 48(1), 42–48. Scholar
  38. van der Hel, W. S., Notenboom, R. G. E., Bos, I. W. M., van Rijen, P. C., van Veelen, C. W. M., & de Graan, P. N. E (2005). Reduced glutamine synthetase in hippocampal areas with neuron loss in temporal lobe epilepsy. Neurology, 64(2), 326–333.CrossRefPubMedGoogle Scholar
  39. Vens-Cappell, S., Kouzel, I. U., Kettling, H., Soltwisch, J., Bauwens, A., Porubsky, S., et al. (2016). On-tissue phospholipase C digestion for enhanced MALDI-MS imaging of neutral glycosphingolipids. Analytical Chemistry, 88(11), 5595–5599. Scholar
  40. Wang, C., Xie, N., Wang, Y., Li, Y., Ge, X., & Wang, M. (2015). Role of the mitochondrial calcium uniporter in rat hippocampal neuronal death after pilocarpine-induced status epilepticus. Neurochemical Research, 40(8), 1739–1746. Scholar
  41. Wishart, D. S., Jewison, T., Guo, A. C., Wilson, M., Knox, C., Liu, Y., et al. (2013). HMDB 3.0-The human metabolome database in 2013. Nucleic Acids Research, 41(D1), D801–D807. Scholar
  42. Wishart, D. S., Knox, C., Guo, A. C., Eisner, R., Young, N., Gautam, B., et al. (2009). HMDB: A knowledgebase for the human metabolome. Nucleic Acids Research, 37, D603–D610. Scholar
  43. Wishart, D. S., Tzur, D., Knox, C., Eisner, R., Guo, A. C., Young, N., et al. (2007). HMDB: The human metabolome database. Nucleic Acids Research, 35, D521–D526. Scholar
  44. Wu, Q., Comi, T. J., Li, B., Rubakhin, S. S., & Sweedler, J. V. (2016). On-tissue derivatization via electrospray deposition for matrix assisted laser desorption/ionization mass spectrometry imaging of endogenous fatty acids in rat brain tissues. Analytical Chemistry, 88(11), 5988–5995. Scholar
  45. Wu, Y., Pearce, P. S., Rapuano, A., Hitchens, T. K., de Lanerolle, N. C., & Pan, J. W. (2015). Metabolic changes in early poststatus epilepticus measured by MR spectroscopy in rats. Journal of Cerebral Blood Flow and Metabolism, 35(11), 1862–1870. Scholar
  46. Xie, X. Y., Jiang, Y. C., Yuan, Y., Wang, P. Q., Li, X. Y., Chen, F. M., et al. (2016). MALDI imaging reveals NCOA7 as a potential biomarker in oral squamous cell carcinoma arising from oral submucous fibrosis. Oncotarget, 7(37), 59987–60004. Scholar
  47. Xu, G., Liu, S., Peng, J., Lv, W., & Wu, R. a. (2015). Facile synthesis of gold@graphitized mesoporous silica nanocomposite and its surface-assisted laser desorption/lonization for time-of-flight mass spectroscopy. ACS Applied Materials & Interfaces, 7(3), 2032–2038. Scholar
  48. Yaari, Y., Yue, C., Su, H.. Yaari, Y., Yue, C., & Su, H. (2007). Recruitment of apical dendritic T-type Ca2+ channels by backpropagating spikes underlies de novo intrinsic bursting in hippocampal epileptogenesis. Journal of Physiology, 580(Pt. 2), 435–450.CrossRefPubMedPubMedCentralGoogle Scholar
  49. Yagnik, G. B., Hansen, R. L., Korte, A. R., Reichert, M. D., Vela, J., & Lee, Y. J. (2016). Large scale nanoparticle screening for small molecule analysis in laser desorption ionization mass spectrometry. Analytical Chemistry, 88(18), 8926–8930. Scholar
  50. Yang, X., Lin, Z., Yan, X., & Cai, Z. (2016). Zeolitic imidazolate framework nanocrystals for enrichment and direct detection of environmental pollutants by negative ion surface-assisted laser desorption/ionization time-of-flight mass spectrometry. RSC Advances, 6(28), 23790–23793. Scholar
  51. Yuan, Y., Zhou, H., & Wei, Y. (2005). Effects of melatonin on contents of hippocampal cyclic AMP in rats witH L-glutamate-induced seizures. Chinese Journal of Histochemistry and Cytochemistry, 14(3), 331–334.Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Beijing National Laboratory for Molecular Sciences, CAS Research/Education Center for Excellence in Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Beijing Mass Spectrum CenterInstitute of Chemistry Chinese Academy of SciencesBeijingChina
  2. 2.Graduate SchoolUniversity of Chinese Academy of SciencesBeijingChina

Personalised recommendations