Microarrays on mirrored substrates for performing proteomic analyses

Abstract

Provided are peptidomimetic protein-binding arrays, their manufacture, use, and application. The protein-binding array elements of the invention include a peptidomimetic segment linked to a solid support via a stable anchor. The invention contemplates peptidomimetic array element library synthesis, distribution, and spotting of array elements onto solid planar substrates, labeling of complex protein mixtures, and the analysis of differential protein binding to the array. The invention also enables the enrichment or purification, and subsequent sequencing or structural analysis of proteins that are identified as differential by the array screen. Kits including proteomic microarrays in accordance with the present invention are also provided.

1 . A method of performing a differential binding assay, comprising: 
 labeling proteins in a protein-containing biological sample solution;    contacting an aliquot of said labeled protein-containing biological sample solution with an array of protein-binding agents stably attached to the surface of a solid support, said array comprising, 
 a solid substrate having a substantially planar surface comprising an organic chemically-modified dielectric-coated reflective metal;  
 a plurality of protein-binding agents bound to said substrate, each of said protein-binding agents comprising, 
 an anchoring segment stably bound to said substrate surface,  
 a peptidomimetic protein-binding segment, and  
 a linker segment connecting and separating the anchoring and peptidomimetic segments; and  
 
 a di-thiol modified polyethylene glycol non-protein chemical blocking agent and a protein blocking agent bound to the substrate surface not occupied by protein-binding agent array elements bound to the substrate surface; and  
   analyzing the array to determine differential binding of proteins in the sample to protein-binding agents of the array.    
     
     
         2 . The method of  claim 1 , wherein in the array the metal is aluminum, the dielectric is SiO 2  and said modifying organic chemical is an aminosilane.  
     
     
         3 . The method of  claim 2 , wherein the aminosilane is functionalized with a maleimide.  
     
     
         4 . The method of  claim 3 , wherein in the array the peptidomimetic segment is a peptoid.  
     
     
         5 . The method of  claim 4 , wherein the protein blocking agent is selected from the group consisting of casein, non-fat milk and BSA.  
     
     
         6 . The method of  claim 5 , wherein the chemical blocking agent is casein.  
     
     
         7 . The method of  claim 6 , wherein the protein labels comprise fluorescent dyes.  
     
     
         8 . The method of  claim 7 , wherein the fluorescent dyes comprise amine-reactive cyanine dyes.  
     
     
         9 . The method of  claim 8 , wherein the amine-reactive cyanine dyes comprise Cyanine 3 and Cyanine 5 dyes.  
     
     
         10 . The method of  claim 9 , wherein in the array the SiO 2  coating is about 800 angstroms thick.  
     
     
         11 . The method of  claim 10 , wherein in the array the aluminum is disposed on a glass slide.  
     
     
         12 . The method of  claim 11 , wherein the assay is used to compare the binding profiles of a first biological sample untreated with a particular chemical agent and a second biological sample treated with the particular chemical agent.  
     
     
         13 . The method of  claim 12 , wherein the biological samples are cell lines.  
     
     
         14 . The method of  claim 13 , wherein proteins in the lyastes of the cell lines are labeled.  
     
     
         15 . The method of  claim 14 , wherein proteins in the lyastes are in their native states.  
     
     
         16 . The method of  claim 14 , wherein proteins in the lyastes are in their denatured states.  
     
     
         17 . The method of  claim 14 , wherein the assay is part of a screening process to identify drug candidates.  
     
     
         18 . The method of  claim 1 , wherein the protein blocking agent is selected from the group consisting of casein, non-fat milk and BSA.  
     
     
         19 . The method of  claim 18 , wherein the chemical blocking agent is casein.  
     
     
         20 . The method of  claim 1 , wherein in the array the SiO 2  coating is about 800 angstroms thick.  
     
     
         21 . The method of  claim 1 , wherein the protein labels comprise fluorescent dyes.  
     
     
         22 . The method of  claim 1 , wherein the assay is used to compare the binding profiles of a first biological sample untreated with a particular chemical agent and a second biological sample treated with the particular chemical agent.  
     
     
         23 . The method of  claim 22 , wherein the biological samples are cell lines.  
     
     
         24 . The method of  claim 23 , wherein proteins in the lyastes of the cell lines are labeled.  
     
     
         25 . The method of  claim 24 , wherein proteins in the lyastes are in their native states.  
     
     
         26 . The method of  claim 24 , wherein proteins in the lyastes are in their denatured states.  
     
     
         27 . The method of  claim 1 , wherein the assay is part of a screening process to identify drug candidates.