Characterization of mature rat oligodendrocytes: a proteomic approach Debora Dumont, Jean-Paul Noben, Marjan Moreels, Joris Vanderlocht, Niels Hellings, Frank Vandenabeele, Ivo Lambrichts, Piet Stinissen and Johan Robben Hasselt University, Biomedical Research Institute (BIOMED) and transnationale Universiteit Limburg, School of Life Sciences, Diepenbeek, Belgium Abstract Oligodendrocytes are glial cells responsible for the synthesis and maintenance of myelin in the central nervous system (CNS). Oligodendrocytes are vulnerable to damage occurring in a variety of neurological diseases. Understanding oligo- dendrocyte biology is crucial for the dissemination of de- and remyelination mechanisms. The goal of the present study is the construction of a protein database of mature rat oligo- dendrocytes. Post-mitotic oligodendrocytes were isolated from mature Wistar rats and subjected to immunocytochem- istry. Proteins were extracted and analyzed by means of two- dimensional gel electrophoresis and two-dimensional liquid chromatography, both coupled to mass spectrometry. The combination of the gel-based and gel-free approach resulted in confident identification of a total of 200 proteins. A minority of proteins were identified in both proteomic strategies. The identified proteins represent a variety of functional groups, including novel oligodendrocyte proteins. The results of this study emphasize the power of the applied proteomic strategy to study known or to reveal new proteins and to investigate their regulation in oligodendrocytes in different disease models. Keywords: mass spectrometry, oligodendrocytes, proteo- mics, rat brain, two-dimensional gel electrophoresis, two- dimensional liquid chromatography. J. Neurochem. (2007) 102, 562–576. Oligodendrocytes synthesize and maintain myelin in the central nervous system. Oligodendrocytes are vulnerable to injury mediated by oxidative stress (Rosenberg et al. 1999), cytotoxicity (Griot-Wenk et al. 1991; Scolding and Comp- ston 1991; Jurewicz et al. 1998; Russell and Ley 2002) excitotoxicity (Matute et al. 2001; Werner et al. 2001), trophic factor deprivation and activation of apoptotic path- ways (Vartanian et al. 1995; Vanderlocht et al. 2006). Oligodendrocyte damage is observed in a variety of diseases including multiple sclerosis (Buntinx et al. 2002), stroke (Aboul-Enein et al. 2003), dementia (Kurz et al. 2003), spinal cord trauma (Gomes-Leal et al. 2004), encephalopa- thies (El Hachimi et al. 1998), leukodystrophies (Ip et al. 2006) and Alzheimer’s disease (Ness et al. 2005). Although the composition of the myelin sheath has been investigated by several proteomic strategies (Persson and Overholm 1990; Yamaguchi and Pfeiffer 1999; Taylor and Pfeiffer 2003;Taylor et al. 2004; Vanrobaeys et al. 2005), no comprehensive proteomic study of oligodendrocytes has been published. Characterization of the oligodendrocyte proteome is on one hand crucial for a better understanding of molecular mechanisms in oligodendrocytes under normal and pathological conditions. On the other hand, because of the limited availability of human brain biopsy material, animal models for disease (mainly rodents) are of utmost importance for CNS research. Therefore, a comprehensive proteomic map of rodent oligodendrocytes is expected to be a useful new tool aiding research of oligodendrocyte pathology. Received September 18, 2006; revised manuscript received December 17, 2006; accepted February 6, 2007. Address correspondence and reprint requests to Johan Robben, Ph.D., Hasselt University, Biomedical Research Institute, Agoralaan building, A 3590 Diepenbeek,Belgium. E-mail: johan.robben@uhasselt.be Abbreviations used: 2D-GE, two-dimensional gel electrophoresis; 2D-LC, two-dimensional liquid chromatography; ACN, acetonitrile; EAE, experimental autoimmune encephalomyelitis; FCS, fetal calf serum; GalC, galactosylceramide; HAc, acetic acid; HSP, heat-shock protein; IPG, immobilized pH-gradient; LC-ESI-MS/MS, liquid chro- matography-electrospray ionization-tandem mass spectrometry; MAG, myelin-associated glycoprotein; MBP, myelin basic protein; MIF, macrophage migration inhibitory factor; MS, mass spectrometry; Mw, molecular weight; PBS, phosphate buffered saline; pI, isoelectric point; SCX, strong cation exchange. Journal of Neurochemistry , 2007, 102, 562–576 doi:10.1111/j.1471-4159.2007.04575.x 562 Journal Compilation Ó 2007 International Society for Neurochemistry, J. Neurochem. (2007) 102, 562–576 Ó 2007 The Authors