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Originally published In Press as doi:10.1074/mcp.M300033-MCP200 on June 23, 2003.
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Molecular & Cellular Proteomics 2:443-452, 2003.
© 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

Biomarker Discovery and Identification in Laser Microdissected Head and Neck Squamous Cell Carcinoma with ProteinChip® Technology, Two-dimensional Gel Electrophoresis, Tandem Mass Spectrometry, and Immunohistochemistry*

Christian Melle{ddagger}, Günther Ernst{ddagger}, Bettina Schimmel{ddagger}, Annett Bleul{ddagger}, Sven Koscielny§, Andreas Wiesner, Ralf Bogumil, Ursula Möller||, Dirk Osterloh**, Karl-Jürgen Halbhuber|| and Ferdinand von Eggeling{ddagger},{ddagger}{ddagger}

From the {ddagger} Core Unit Chip Application (CUCA), Institute of Human Genetics and Anthropology, Friedrich-Schiller-University, 07740 Jena, Germany, § Clinic of ENT Diseases, Friedrich-Schiller-University, 07740 Jena, Germany, Ciphergen Biosystems GmbH, 37085 Göttingen, Germany, || Institute of Anatomy II, Friedrich-Schiller-University, 07740 Jena, Germany, and ** SIRS-Lab GmbH, 07745 Jena, Germany

Head and neck cancer is a frequent malignancy with a complex, and up to now not clear etiology. Therefore, despite of improvements in diagnosis and therapy, the survival rate with head and neck squamous-cell carcinomas is poor. For a better understanding of the molecular mechanisms behind the process of tumorigenesis and tumor progression, we have analyzed changes of protein expression between microdissected normal pharyngeal epithelium and tumor tissue by ProteinChip® technology. For this, cryostat sections from head and neck tumors (n = 57) and adjacent mucosa (n = 44) were laser-microdissected and analyzed on ProteinChip arrays. The derived mass spectrometry profiles exhibited numerous statistical differences. One peak significantly higher expressed in the tumor (p = 0.000029) was isolated by two-dimensional gel electrophoresis and identified as annexin V by in-gel proteolytic digestion, peptide mapping, tandem mass spectrometry analysis, and immuno-deplete assay. The relevance of this single marker protein was further evaluated by immunohistochemistry. Annexin-positive tissue areas were re-analyzed on ProteinChip arrays to confirm the identity of this protein. In this study, we could show that biomarker in head and neck cancer can be found, identified, and assessed by combination of ProteinChip technology, two-dimensional gel electrophoresis, and immunohistochemistry. In our experience, however, such studies only make sense if a relatively pure microdissected tumor tissue is used. Only then minute changes in protein expression between normal pharyngeal epithelium and tumor tissue can be detected, and it will become possible to educe a tumor-associated protein pattern that might be used as a marker for tumorigenesis and progression.

{ddagger}{ddagger} To whom correspondence should be addressed: Institut für Humangenetik und Anthropologie, CUCA, 07740 Jena, Germany. Tel.: 0049(0)3641-935526; Fax: 0049(0)3641-935582; E-mail: fegg{at}mti.uni-jena.de


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