Identification of keratinocyte-specific markers using phage display and mass spectrometry.
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.
Jensen
Kim Bak
KB
Department of Molecular Biology, University of Aarhus, DK-8000 Aarhus, Denmark.
Jensen
Ole Nørregaard
ON
Ravn
Peter
P
Clark
Brian F C
BF
Kristensen
Peter
P
D000911:Antibodies, Monoclonal; D001940:Breast / Q000166:cytology; D002460:Cell Line; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D003090:Coliphages / Q000235:genetics; D000939:Epitopes / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics; D005819:Genetic Markers; D006367:HeLa Cells; D006801:Humans; D007381:Intermediate Filament Proteins / Q000235:genetics; D015603:Keratinocytes / Q000502:physiology*; D007797:Laminin / Q000235:genetics; D013058:Mass Spectrometry; D019151:Peptide Library; D051190:Plectin; D012680:Sensitivity and Specificity; D012879:Skin Physiological Phenomena; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000911:Antibodies, Monoclonal; D000939:Epitopes; D005819:Genetic Markers; D007381:Intermediate Filament Proteins; D007797:Laminin; C495332:PLEC protein, human; D019151:Peptide Library; D051190:Plectin