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Research

Identification of Keratinocyte-specific Markers Using Phage Display and Mass Spectrometry^*

Kim Bak Jensen, Ole Nørregaard Jensen, Peter Ravn^,¶, Brian F. C. Clark and Peter Kristensen^,||

Department of Molecular Biology, University of Aarhus, DK-8000 Aarhus, Denmark
Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense, Denmark
^¶ Nemod Immunotherapie AG, B-13125 Berlin-Buch, Germany

Specific molecular markers for various normal and pathogenic cell states and cell types provide knowledge of basic biological systems and have a direct application in targeted therapy. We describe a proteomic method based on the combination of new and improved phage display antibody technologies and mass spectrometry that allows identification of cell type-specific protein markers. The most important features of the method are (i) reduction of experimental noise originating from background binding of phage particles and (ii) isolation of affinity binders after a single round of selection, which assures a high diversity of binders. The method demonstrates, for the first time, the ability to detect, identify, and analyze both secreted and membrane-associated extracellular proteins as well as a variety of different cellular structures including proteins and carbohydrates. The optimized phage display method was applied to analysis of human skin keratinocytes resulting in the isolation of a panel of antibodies. Fourteen of these antibodies were further characterized, half of which predominantly recognized keratinocytes in a screen of a range of different cell types. Three cognate keratinocyte antigens were subsequently identified by mass spectrometry as laminin-5, plectin, and fibronectin. The combination of phage display technology with mass spectrometry methods for protein identification is a general and promising approach for proteomic analysis of cell surface complexity.

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