Summary
Calcium dyshomeostasis is involved in neuropathological conditions such as traumatic brain injury (TBI), stroke, and neurodegenerative diseases. Under such conditions in the brain, calmodulin (CaM), a Ca2+ sensor, mediates critical signaling functions through binding and regulating a diverse population of downstream targets referred to as calmodulin-binding proteins (CaMBPs). We developed a CaM-affinity capture method followed by reversed-phase liquid chromatography tandem mass spectrometry (RPLC-MSMS) to study the calcium-dependent CaM-binding proteome in rat brain. A total of 69 potential CaMBPs were identified by this proteomic technique, of which 26 were known CaMBPs and 43 were putative novel CaMBPs. This study shows that the CaM-affinity capture when coupled with tandem mass spectrometry may serve as an effective tool toward constructing a brain CaM-binding proteomic network. The general methods described here can be applied to study possible alternations of calmodulin-binding proteome in neurological, neurodegenerative, and psychiatric disorders.
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References
Haeseleer, F., Palczewski, K. (2002) Calmodulin and Ca2+-binding proteins (CaBPs): variations on a theme. Adv Exp Med Biol. 514, 303–317.
Bouché, N., Yellin, A., Snedden, W.A., Fromm, H. (2004) Plant-specific calmodulin-binding proteins. Annu Rev Plant Biol. 56, 435–466.
Benaim, G., Villalobo, A. (2002) Phosphorylation of calmodulin. Functional implications. Eur J Biochem. 269, 3619–3631.
Xia, Z., Storm, D.R. (2005) The role of calmodulin as a signal integrator for synaptic plasticity. Nat Rev Neurosci. 6, 267–276.
Xia, Z., Choi, E.J., Wang, F., Blazynski, C., Storm, D.R. (1993) Type I calmodulin-sensitive adenylyl cyclase is neural specific. J Neurochem. 60, 305–311.
O’Day, D.H. (2003) CaMBOT: profiling and characterizing calmodulin-binding proteins. Cell Signal. 15, 347–354.
Reddy, V.S., Ali, G.S., Reddy, A.S. (2002) Genes encoding calmodulin-binding proteins in the Arabidopsis genome. J Biol Chem. 277, 9840–9852.
Zhu, H., Bilgin, M., Bangham, R., Hall, D., Casamayor, A., Bertone, P., Lan, N., et al. (2001) Global analysis of protein activities using proteome chips. Science 293, 2101–2105.
Shen, X., Valencia, C.A., Szostak, J.W., Dong, B., Liu, R. (2005) Scanning the human proteome for calmodulin-binding proteins. Proc Natl Acad Sci USA 102, 5969–5974.
Zhang, Z., Ottens, A.K., Golden, E.C., Hayes, R.L., Wang, K.K.W. (2006) Using calmodulin-affinity capture to study the rat brain calmodulin binding proteome and its vulnerability to calpain and caspase proteolysis. Calcium Binding Proteins 2, 125–134.
Ottens, A.K., Kobeissy, F.H., Wolper, R.A., Haskins, W.E., Hayes, R.L., Denslow, N.D., Wang, K.K.W. (2005) A multidimensional differential proteomic platform using dual-phase ion-exchange chromatography – polyacrylamide gel electrophoresis/reversed-phase liquid chromatography tandem mass spectrometry. Anal Chem. 77, 4836–4845.
Tabb, D.L., McDonald, W.H., Yates, J.R. (2002) DTASelect and contrast: tools for assembling and comparing protein identifications from shotgun proteomics. J Proteome Res. 1, 21–26.
Wang, K.K.W., Villalobo, A., Roufogalis, B.D. (1989) Calmodulin-binding proteins as calpain substrates. Biochem J. 262, 693–706.
O’Day, D.H., Myre, M.A. (2004) Calmodulin-binding domains in Alzheimer’s disease proteins: extending the calcium hypothesis. Biochem Biophys Res Commun. 320, 1051–1054.
Acknowledgments
We wish to thank Stephen F. Larner for insightful discussion and editing assistance. This work was supported by the Department of Defense grant DAMMED-03-1-0066, and NIH grants R01 NS049175-01-A1 and R01 NS051431.
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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Zhang, Z., Kobeissy, F.H., Ottens, A.K., MartÃnez, J.A., Wang, K.K.W. (2009). Calmodulin-Binding Proteome in the Brain. In: Ottens, A., Wang, K. (eds) Neuroproteomics. Methods in Molecular Biology, vol 566. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-562-6_12
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DOI: https://doi.org/10.1007/978-1-59745-562-6_12
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Publisher Name: Humana Press, Totowa, NJ
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