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New astrocyte cell surface markers Mass-spectrometric identification of the astrocyte cell surface proteome reveals new classification markers Melanie Jungblut1, Andrea Jacobs1,2, Thomas Bock 2, Andreas Bosio1, and Bernd Wollscheid2 1 Miltenyi Biotec GmbH, 51429 Bergisch Gladbach, Germany 2 Institute of Molecular Sytems Biology, NCCR Neuro Center for Proteomics, ETH Zurich, Switzerland 4. 1. 5. Introduction Astrocytes are the most abundant cell type among cells of the central nervous system. They are involved in the control of synaptogenesis, synaptic transmission, neurogenesis, and maintenance of neuronal metabolism. Despite the importance of astrocytes, little is known about their phenotype at the cell surface protein level and this limits our understanding of the cell’s interactions with its environment. The isolation of astrocytes and their different subpopulations for functional studies is currently hindered mainly due to a lack of specific cell surface protein markers. Magnetic cell sorting by MACS® Technology is a convenient approach for the fast and easy separation of a large amount of specific cells from a mixed cell suspension. However, a prerequisite for the selective isolation of a particular cell type via MACS Technology is the identification of specific cell surface proteins and availability of suitable antibodies. Therefore, we used the mass spectrometry–based cell surface capturing (CSC) technology to generate a snapshot of the astrocyte cell surface glycoproteome (fig. 1).1–3 The CSC technology enables phenotyping of cells without antibodies and allows the specific analysis of a wide variety of cell surface glycoproteins, leading to identification of previously unknown astrocyte cell surface markers. 2. 6. 3. Figure 1 Identification of cell surface glycoproteins using the CSC technology. The CSC technology was used to detect cell surface N-glycoproteins of astrocytes. The steps involve specific labeling of oxidized cell surface polysaccharides using the bi-functional linker molecule biocytin hydrazide (1), followed by cell homogenization and protein digestion (2), affinity enrichment of biocytin hydrazide–labeled peptides (3), enzymatic peptide release (4), peptide analysis by tandem mass spectrometry (LC-MS/MS) (5), and peptide or protein identification (6). Results Figure 2 Astrocyte cultures. Astrocytes were cultured for 21 days after plating and then immunostained using an anti-GFAP antibody (green). Nuclei were visualized with DAPI (blue). GLAST 25% 8,5% 8% Marker 1 Conclusion • The identified markers should represent ideal targets for the ongoing development of novel astrocyte-specific cell separation reagents. References 1. Wollscheid, B. et al. (2009) Nat. Biotechnol. 27: 378–386. 2. Doerr, A. (2009) Nat. Methods 6: 401. 3. Gundry, R.L. et al. (2008) Proteomics Clin. Appl. 2: 892–903. 4. Pennartz, S. et al. (2009) J. Vis. Exp. 29 pii:1267; doi: 10.3791/1267.d CD45 21% 4% Marker 2 Marker 2 • The CSC analysis identified 482 astrocyte cell surface glycoproteins and revealed a high complexity in the astrocyte cell surface proteome. 14% GLAST • New astrocyte cell surface markers were identified and antibody generation, as well as functional studies, are ongoing. 2% 22% 12% CD45 The antibody-independent experimental approach created a qualitative ‘snapshot’ of the astrocyte cell surface proteome used as the basis for MACS Cell Separation and quantitative studies. Marker 1 GLAST • 0.5% CD45 GLAST As astrocytes cannot be isolated from primary brain tissue of wild-type mice, astrocytes in vitro were used for mass spectrometric analysis of the surface proteome. Therefore, cortical tissue obtained from P1 mice was dissociated using the Neural Tissue Dissociation Kit (P) and single-cell suspensions were plated onto poly-D-lysine–coated culture flasks. After three weeks, the cultures consisted predominantly of astrocytes, which was confirmed by immunocytochemical staining of the intracellular astrocyte marker glial fibrillary acidic protein (GFAP) (fig. 2). The astrocyte cultures were then analyzed by the CSC technology to identify cell-specific surface markers. This experimental approach revealed 482 astrocyte cell surface– exposed glycoproteins. To test whether some of these antigens could serve as useful astrocyte markers, brain tissue derived from P7 mice was dissociated using a Neural Tissue Dissocation Kit (T or P) and single-cell suspensions were labeled with commercially available antibodies specific for the identified proteins and analyzed by flow cytometry. A monoclonal antibody directed against the astrocyte-specific glutamate transporter (GLAST, sometimes referred to as EAAT1) was used for co-staining, as well as an antibody specific for the leukocyte marker CD45 (fig. 3). We found that Markers 1 and 2, for example, are expressed by GLAST+ astrocytes but not by CD45+ leukocytes and may serve as astrocyte-specific surface markers. Marker 3, in contrast, is also expressed by CD45+ leukocytes. 23% 20% Marker 3 Marker 3 Figure 3 Validation of promising astrocyte cell surface markers. Brain tissue from P7 mice was dissociated using a Neural Tissue Dissociation Kit and single-cell suspensions were labeled with antibodies specific for astrocyte cell surface antigens identified by CSC technology, as well as with antibodies against either the astrocyte marker GLAST or the leukocyte marker CD45. MACS® Product Order no. Neural Tissue Dissociation Kit (P) 130-092-628 Neural Tissue Dissociation Kit (T) 130-093-231 Anti-Astrocyte (GLAST) MicroBeads Miltenyi Biotec provides products and services worldwide. Visit www.miltenyibiotec.com/local to find your nearest Miltenyi Biotec contact. MACS is a registered trademark of Miltenyi Biotec GmbH. Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for therapeutic or diagnostic use. Copyright © 2010 Miltenyi Biotec GmbH. All rights reserved.