Abstract
The neurotrauma field needs more accurate and sensitive tools to diagnose and evaluate damage to the brain or spinal cord. A controlled, in vitro mechanical trauma model to study protein changes during astrocyte activation after injury is used as a means to discover neurotrauma biomarkers. This model offers several advantages over analyzing cerebrospinal fluid (CSF) or microdialysates including increased sample amount, higher reproducibility between biological samples, enrichment of undegraded proteins due to fewer extracellular matrix proteases in the in vitro versus in vivo system and the ability to relate trauma changes to one cell type. This protocol describes an approach for preparing intact protein samples from conditioned medium (CM) and whole cell lysates of murine cortical astrocytes. The proteins from these samples are separated using 2-D gel electrophoresis, and the protein spot intensities are measured. Proteins showing statistically significant trauma induced changes are then identified using tandem mass spectrometry. This protocol contains optimization steps to enrich proteins of interest, to remove substances that interfere with isoelectric focusing (IEF) and to improve IEF conditions. With this procedure, the 2-D gels show high resolution spot separation and allow for high confidence protein identifications from 107 released and 22 whole cell lysates protein spots so far. Some proteins were identified from multiple spots, suggesting post-translational modification had occurred as a result of the mechanical trauma.
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References
Allen, N.J., and Barres, B.A. (2009). Neuroscience: Glia – more than just brain glue. Nature 457, 675–677.
Amruthesh, S.C., Boerschel, M.F., McKinney, J.S., Willoughby, K.A., and Ellis, E.F. (1993). Metabolism of arachidonic acid to epoxyeicosatrienoic acids, hydroxyeicosatetraenoic acids, and prostaglandins in cultured rat hippocampal astrocytes. J Neurochem 61, 150–159.
Berkelman, T., and Stenstedt, T. (1998). 2-D Electrophoresis Using Immobilized pH Gradients: Principles and Methods, Edition AB. In Handbooks from Amersham Biosciences (Piscataway, NJ).
Bowman, C.L., Ding, J.P., Sachs, F., and Sokabe, M. (1992). Mechanotransducing ion channels in astrocytes. Brain Res 584, 272–286.
Castle, L., Dale, E., Habers, A., Sadownick, B., Strong, W., Whitman, C., and Zhu, M. First Dimension Separation Methods. In 2-D Electrophoresis for Proteomics: A Methods and Product Manual, D. Garfin, and L. Heerdt, eds. Bulletin 2651 US/EG Rev B, (Hercules, CA, Bio-Rad Laboratories).
de Vellis, J., Ghiani, C., Wanner, I., and Cole, R. (2009). Preparation of Normal and Reactive Astrocyte Cultures. In Protocols for Neural Cell Culture, L. Doering, ed. (Totowa, NJ, Humana Press Inc. Springer), pp. 193–216.
Di, X., Goforth, P.B., Bullock, R., Ellis, E., and Satin, L. (2000). Mechanical injury alters volume activated ion channels in cortical astrocytes. Acta Neurochir Suppl 76, 379–383.
Eddleston, M., and Mucke, L. (1993). Molecular profile of reactive astrocytes – implications for their role in neurologic disease. Neuroscience 54, 15–36.
Ellis, E.F., McKinney, J.S., Willoughby, K.A., Liang, S., and Povlishock, J.T. (1995). A new model for rapid stretch-induced injury of cells in culture: Characterization of the model using astrocytes. J Neurotrauma 12, 325–339.
Friedman, D.B., Hoving, S., and Westermeier, R. (2009). Isoelectric focusing and two-dimensional gel electrophoresis. Methods Enzymol 463, 515–540.
Hanrieder, J., Wetterhall, M., Enblad, P., Hillered, L., and Bergquist, J. (2009). Temporally resolved differential proteomic analysis of human ventricular CSF for monitoring traumatic brain injury biomarker candidates. J Neurosci Methods 177, 469–478.
Hergenroeder, G.W., Redell, J.B., Moore, A.N., and Dash, P.K. (2008). Biomarkers in the clinical diagnosis and management of traumatic brain injury. Mol Diagn Ther 12, 345–358.
Keene, S.D., Greco, T.M., Parastatidis, I., Lee, S.H., Hughes, E.G., Balice-Gordon, R.J., Speicher, and D W, Ischiropoulos, H. (2009). Mass spectrometric and computational analysis of cytokine-induced alterations in the astrocyte secretome. Proteomics 9, 768–782.
Kochanek, P.M., Berger, R.P., Bayir, H., Wagner, A.K., Jenkins, L.W., and Clark, R.S. (2008). Biomarkers of primary and evolving damage in traumatic and ischemic brain injury: Diagnosis, prognosis, probing mechanisms, and therapeutic decision making. Curr Opin Crit Care 14, 135–141.
Lakshmanan, R., Loo, J.A., Drake, T., Leblanc, J., Ytterberg, A.J., McArthur, D.L., Etchepare, M., and Vespa, P.M. (2010). Metabolic crisis after traumatic brain injury is associated with a novel microdialysis proteome. Neurocrit Care 12, 324–836.
Lamb, R.G., Harper, C.C., McKinney, J.S., Rzigalinski, B.A., and Ellis, E.F. (1997). Alterations in phosphatidylcholine metabolism of stretch-injured cultured rat astrocytes. J Neurochem 68, 1904–1910.
Liu, T., Qian, W.J., Gritsenko, M.A., Xiao, W., Moldawer, L.L., Kaushal, A., Monroe, M.E., Varnum, S.M., Moore, R.J., Purvine, S.O., et al. (2006). High dynamic range characterization of the trauma patient plasma proteome. Mol Cell Proteomic 5, 1899–1913.
Montgomery, D.L. (1994). Astrocytes: Form, functions, and roles in disease. Vet Pathol 31, 145–167.
Neary, J.T., Kang, Y., Willoughby, K.A., and Ellis, E.F. (2003). Activation of extracellular signal-regulated kinase by stretch-induced injury in astrocytes involves extracellular ATP and P2 purinergic receptors. J Neurosci 23, 2348–2356.
O’Farrell, P.H. (1975). High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250, 4007–4021.
Ostrow, L.W., and Sachs, F. (2005). Mechanosensation and endothelin in astrocytes – hypothetical roles in CNS pathophysiology. Brain Res Brain Res Rev 48, 488–508.
Reier, P.J., and Houle, J.D. (1988). The glial scar: Its bearing on axonal elongation and transplantation approaches to CNS repair. Adv Neurol 47, 87–138.
Ridet, J.L., Malhotra, S.K., Privat, A., and Gage, F.H. (1997). Reactive astrocytes: Cellular and molecular cues to biological function. Trends Neurosci 20, 570–577.
Sanchez, J.C., Rouge, V., Pisteur, M., Ravier, F., Tonella, L., Moosmayer, M., Wilkins, M.R., and Hochstrasser, D.F. (1997). Improved and simplified in-gel sample application using reswelling of dry immobilized pH gradients. Electrophoresis 18, 324–327.
Sandvig, A., Berry, M., Barrett, L.B., Butt, A., and Logan, A. (2004). Myelin-, reactive glia-, and scar-derived CNS axon growth inhibitors: Expression, receptor signaling, and correlation with axon regeneration. Glia 46, 225–251.
Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. (1996). Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem 68, 850–858.
Sjödin, M.O., Bergquist, J., and Wetterhall, M. (2010). Mining ventricular cerebrospinal fluid from patients with traumatic brain injury using hexapeptide ligand libraries to search for trauma biomarkers. J Chromatogr B Anal Technol Biomed Life Sci 878, 2003–2012.
Smith, R. (2009). Two-Dimensional Electrophoresis: An Overview. In Methods in Molecular Biology, D. Sheehan, and R. Tyther, eds. (Totowa, NJ, Humana Press), pp. 3–17.
Sofroniew, M.V. (2005). Reactive astrocytes in neural repair and protection. Neuroscientist 11, 400–407.
Wanner, I.B., Deik, A., Torres, M., Rosendahl, A., Neary, J.T., Lemmon, V.P., and Bixby, J.L. (2008). A new in vitro model of the glial scar inhibits axon growth. Glia 56(15), 1691–1709. PubMed PMID: 18618667.
Westermeier, R., and Naven, T. (2002). Trouble Shooting. In Proteomics in Practice. (Weinheim, Germany, Wiley-VCH GmbH), pp. 283–293.
Westermeier, R. (2006). Critical Aspects for Sample Preparation in Proteome Analysis. GE Healthcare Seminar at Human Proteome Organisation Fifth Annual World Congress (Long Beach, CA).
Acknowledgements
We kindly acknowledge Dr. Puneet Souda for his help with the LC-MS/MS work and Reiner Westermeier for his expertise and in-depth discussions on running 2-D gels. The 2-D gel and LC-MS/MS work was performed in the W. M. Keck Proteomic Facility at the UCLA Molecular Instrumentation Center which was established with a grant from the W. M. Keck Foundation. This project is supported by the Craig Neilsen Foundation (for IBW, grant # 82776).
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Sondej, M.A., Doran, P., Loo, J.A., Wanner, IB. (2011). Sample Preparation of Primary Astrocyte Cellular and Released Proteins for 2-D Gel Electrophoresis and Protein Identification by Mass Spectrometry. In: Ivanov, A., Lazarev, A. (eds) Sample Preparation in Biological Mass Spectrometry. Springer, Dordrecht. https://doi.org/10.1007/978-94-007-0828-0_39
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