Proteomic Analysis Reveals Drug Accessible Cell Surface N‑Glycoproteins of Primary and Established Glioblastoma Cell Lines Thomas Bock, †,‡,△ Hansjoerg Moest, †,‡ Ulrich Omasits, †,‡ Silvia Dolski, § Emma Lundberg, ∥ Andreas Frei, †,‡ Andreas Hofmann, †,‡,□ Damaris Bausch-Fluck, †,‡ Andrea Jacobs, †,‡ Niklaus Krayenbuehl, § Mathias Uhlen, ∥,⊥ Ruedi Aebersold, †,# Karl Frei, § and Bernd Wollscheid* ,†,‡ † Department of Biology, Institute of Molecular Systems Biology and ‡ National Center of Competence in Research (NCCR)−Neuro Center for Proteomics, Swiss Federal Institute of Technology (ETH) Zurich, Zurich, Switzerland § Department of Neurosurgery, University Hospital Zurich, Zurich, Switzerland ∥ Science for Life Laboratory and ⊥ School of Biotechnology, AlbaNova University Center, Royal Institute of Technology, Stockholm, Sweden # Faculty of Science, UZH, Zurich, Switzerland * S Supporting Information ABSTRACT: Glioblastoma is the most common primary brain tumor in adults with low average survival time after diagnosis. In order to improve glioblastoma treatment, new drug-accessible targets need to be identified. Cell surface glycoproteins are prime drug targets due to their accessibility at the surface of cancer cells. To overcome the limited availability of suitable antibodies for cell surface protein detection, we performed a comprehensive mass spectrometric investigation of the glioblastoma surfaceome. Our combined cell surface capturing analysis of primary ex vivo glioblastoma cell lines in combination with established glioblastoma cell lines revealed 633 N-glycoproteins, which vastly extends the known data of surfaceome drug targets at subcellular resolution. We provide direct evidence of common glioblastoma cell surface glycoproteins and an approximate estimate of their abundances, information that could not be derived from genomic and/or transcriptomic glioblastoma studies. Apart from our pharmaceutically valuable repertoire of already and potentially drug- accessible cell surface glycoproteins, we built a mass-spectrometry-based toolbox enabling directed, sensitive, and repetitive glycoprotein measurements for clinical follow-up studies. The included Skyline Glioblastoma SRM assay library provides an elevated starting point for parallel testing of the abundance level of the detected glioblastoma surfaceome members in future drug perturbation experiments. KEYWORDS: glioblastoma, N-glycoproteins, cell surface accessible drug targets, cell surface capturing, N-glycoproteome SRM library ■ INTRODUCTION Glioblastoma (GBM) is a highly aggressive and infiltrating type of brain tumor, which shows an average post-treatment survival of approximately 15 months. 1 GBM therefore belongs to one of the most severe diseases of the human brain. Of all glia-derived tumors, the primary GBM presents the most frequent tumor of the human brain. 2 Histopathological examination of GBM is currently the main diagnostic procedure, 3 and only little supportive information for detailed classification 4 and targeted molecular therapy is available. 5 Despite advances in radio- therapy, chemotherapy, array-based screening tools, and neuro- imaging, the GBM tumor biology is still poorly understood. There remains a significant medical need for effective therapy. 6 Currently, the main limitation is the lack of accessible and targetable molecular features in GBM. 5 The cell surface proteome (surfaceome 7 ) is the most accessible subproteome of the cell for affinity-based probes. The surfaceome mediates cellular interactions with the local microenvironment and further contributes to key functions of the plasma membrane such as the transport of information and biochemical molecules. In GBM, the surfaceome constitutes a pool of accessible proteins that can be used as direct molecular targets in drug therapy, as cell surface markers for delivery of drugs to the tumor tissue and as marker molecules for the monitoring of the disease. Due to its accessibility, the surfaceome represents the largest group of clinically used drug targets. 8 Currently used GBM drugs against cell surface proteins mainly target proteins of the receptor tyrosine kinase (RTK) and integrin signaling pathways. 5 Commonly targeted RTK in GBM are EGFR and PDGFRA 9,10 and are related to the regulation of cell growth. Integrins have been shown to play Received: April 14, 2012 Published: August 21, 2012 Article pubs.acs.org/jpr © 2012 American Chemical Society 4885 dx.doi.org/10.1021/pr300360a | J. Proteome Res. 2012, 11, 4885−4893