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

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.

Laminin-332 coordinates mechanotransduction and growth cone bifurcation in sensory neurons

“…The proteins were run on SDS gels and Western blotting was carried out with antibodies against known matrix components to identify molecules exclusively present in keratinocyte-derived matrix. We, like others, 6 identified laminin-332 as one molecule specific to keratinocyte-derived matrix 2,[21][22] ( Fig. 2a, Supplementary Fig.…”

A Novel Subtractive Antibody Phage Display Method to Discover Disease Markers

“…So far, a few studies have successfully used scFv fragments to isolate the unknown target antigen and to determine its identity by mass spectrometry (5,6). We demonstrate that scFv fragments, isolated during the subtractive selection strategy presented here, can be applied successfully to immunoaffinity purify target proteins for identification by mass spectrometry.…”

“…In proteomics, phage display offers an elegant approach in the search for disease markers. Subtractive antibody selections have been successfully performed on whole cells (3)(4)(5)(6)(7)(8)(9)(10), tissue sections (11), biomaterials ex vivo (12), and cell extracts (13)(14)(15)(16)(17)(18)(19). A major advantage of subtractive phage display technology is the simultaneous generation of recombinant monoclonal antibodies recognizing potential disease markers.…”

An Efficient Method for Isolating Antibody Fragments Against Small Peptides by Antibody Phage Display

“…The KM13 helper phage has another trypsin cleavage site in the engineered pIII, which renders KM13 noninfective after trypsin treatment [23], whereas the scFv-pIII fusion loses scFv protein but remains infective. The nonspecific binding population in eluted phage is therefore dramatically reduced by trypsin treatment compared with the traditional eluting method (e.g., triethylamine eluting) in phage display panning [26,27].…”