TY - JOUR

T1 - Identification of keratinocyte-specific markers using phage display and mass spectrometry

AU - Jensen, Kim Bak

AU - Jensen, Ole Nørregaard

AU - Ravn, Peter

AU - Clark, Brian F C

AU - Kristensen, Peter

PY - 2003/2

Y1 - 2003/2

N2 - 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.

AB - 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.

KW - Antibodies, Monoclonal

KW - Breast/cytology

KW - Cell Line

KW - Cell Line, Tumor

KW - Cells, Cultured

KW - Coliphages/genetics

KW - Epitopes/analysis

KW - Escherichia coli/genetics

KW - Genetic Markers

KW - HeLa Cells

KW - Humans

KW - Intermediate Filament Proteins/genetics

KW - Keratinocytes/physiology

KW - Laminin/genetics

KW - Mass Spectrometry

KW - Peptide Library

KW - Plectin

KW - Sensitivity and Specificity

KW - Skin Physiological Phenomena

KW - Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

U2 - 10.1074/mcp.M200049-MCP200

DO - 10.1074/mcp.M200049-MCP200

M3 - Journal article

C2 - 12644568

VL - 2

SP - 61

EP - 69

JO - Molecular and Cellular Proteomics

JF - Molecular and Cellular Proteomics

SN - 1535-9476

IS - 2

ER -