Abstract
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.
Highlights
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
Primary cultures of keratinocytes have not undergone any transformational changes as is the case for the cell lines used in previous studies with phage display in direct selections (13, 14, 30 –32)
In recent years, phage display has established itself as an alternative method for the generation of monoclonal antibodies against individual antigens as well as components of complex mixtures
Summary
Cell Cultures—Primary human keratinocytes were isolated from mammary tissue and cultured according to Norsgaard et al [22]. After blocking the wells were briefly rinsed with PBS and incubated with phage repertoire for 1.5 h at room temperature in 3 ml of 50:50 volume % 4% MPBS and keratinocyte medium. The control selection with the M13K07 (Stratagene)-rescued Griffin repertoire was performed using the conditions described above and in Marks et al [23] except for elution of phage with 100 mM triethylamine and subsequent neutralization with 1 M Tris-HCl, pH 7.5 before infection into log-phase TG1. Phage-displayed antibodies D4 and 12 were incubated with the wells, and the ELISA was performed as described above for monoclonal phage ELISAs. Indirect immunofluorescence studies were performed on keratinocytes fixated and permeabilized using PBS supplemented with 1% formaldehyde and 0.1% Tween 20 for 5 min at room temperature. The gene segments were identified using the VBASE directory at the Medical Research Council, Cambridge, UK.
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