Protein microarrays on mirrored surfaces for performing proteomic analyses

Abstract

Provided are protein microarrays, their manufacture, use, and application. Protein microarrays in accordance with the present invention are useful in a variety preoteomic analyses. Various protein arrays in accordance with the present invention may immobilize large arrays of proteins that may be useful for studying protein-protein interactions to improve understanding of disease processes, facilitating drug discovery, or for identifying potential antigens for vaccine development. The protein array elements of the invention are native or modified proteins (e.g., antibodies or fusion proteins). The protein array elements may be attached directly to a organic functionalized mirrored substrate by a binding reaction between functional groups on the substrate (e.g., amine) and protein (e.g., activated carboxylic acid). Techniques for chemical blocking of the arrays are also provided. The invention contemplates spotting of array elements onto solid planar substrates, labeling of complex protein mixtures, and the analysis of 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 protein-binding microarrays for proteomic analysis in accordance with the present invention are also provided.

1 . A method of detecting of antibodies in serum against potential protein antigens associated with a disease condition, comprising: 
 labeling proteins in a solution of a serum sample obtained from an individual;    contacting an aliquot of said labeled protein-containing serum sample solution with an array comprising, 
 a solid substrate having a substantially planar surface comprising an organic chemically-modified dielectric-coated reflective metal,  
 a plurality of array elements comprising proteins stably attached to the substrate surface, 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 antibody proteins in the sample to antigen protein array elements of the array.    
   
   
       2 . The method of  claim 1 , wherein the protein array elements are fusion proteins.  
   
   
       3 . The method of  claim 2 , wherein antigens identified by said assay are used in a vaccine development program.  
   
   
       4 . The method of  claim 1 , wherein the plurality of proteins are directly attached to the substrate surface.  
   
   
       5 . The method of  claim 1 , wherein the plurality of proteins are indirectly attached to the substrate surface via a chemical adapter.  
   
   
       6 . The method of  claim 1 , wherein the metal is selected from the group consisting of aluminum, gold, chromium, titanium and platinum.  
   
   
       7 . The method of  claim 1 , wherein the metal is aluminum.  
   
   
       8 . The method of  claim 1 , wherein the dielectric comprises a metal oxide selected from the group consisting of silicon oxide, silicon dioxide and aluminum oxide.  
   
   
       9 . The method of  claim 1 , wherein the dielectric is silicon dioxide about 800 angstroms thick.  
   
   
       10 . The method of  claim 1 , wherein the organic chemical modification comprises a functionalized silane molecule.  
   
   
       11 . The method of  claim 10 , wherein the functionalized silane molecule is an amino-modified silane molecule.  
   
   
       12 . The method of  claim 1 , wherein the reflective metal is disposed on a non-metallic solid substrate.  
   
   
       13 . The method of  claim 12 , wherein the non-metallic solid substrate is selected from the group consisting of glasses and plastics.  
   
   
       14 . The method of  claim 13 , wherein the non-metallic solid substrate is a glass microscope slide.  
   
   
       15 . The method of  claim 1 , wherein the protein blocking agent is selected from the group consisting of casein, non-fat milk and BSA.  
   
   
       16 . The method of  claim 15 , wherein the the protein blocking agent is casein.  
   
   
       17 . The method of  claim 15 , wherein the metal is aluminum disposed on a glass slide, the oxide coating is about 800 angstrom thick silicon dioxide, and the organic chemical modification comprises a functionalized silane molecule.  
   
   
       18 . The method of  claim 2 , wherein the proteins are directly bound to a functional group of the organic chemically-modified surface.  
   
   
       19 . The method of  claim 18 , wherein the organic chemically-modified surface displays an amine and the proteins have an exposed, activated carboxylic acid group.  
   
   
       20 . The method of  claim 3 , wherein the adapter comprises a homobifunctional organic linker designed or selected to stably attach to the substrate surface on one terminus and stably attach to a functional group on the plurality of proteins on the other terminus.  
   
   
       21 . The method of  claim 20 , wherein the adapter comprises a bis-NHS activated ester.  
   
   
       22 . The method of  claim 3 , wherein the adapter comprises a heterobifunctional organic linker designed or selected to stably attach to the substrate surface on one terminus and stably attach to a functional group on the plurality of proteins on the other terminus.  
   
   
       23 . The method of  claim 22 , wherein the adapter comprises a modified NHS activated ester.  
   
   
       24 . The method of  claim 23 , wherein the modification is a protein-binding functional group at a non-substrate bound terminus of the adapter.  
   
   
       25 . The method of  claim 24 , wherein the protein-binding functional group is selected from the group consisting of maleimide, biotin, avidin or avidin analog.  
   
   
       26 . The method of  claim 25 , wherein the protein-binding functional group is maleimide and the attached protein comprise an exposed, reduced thiol group.  
   
   
       27 . The method of  claim 25 , wherein the protein-binding functional group is biotin and the proteins are avidin conjugated.  
   
   
       28 . The method of  claim 25 , wherein the protein-binding functional group is avidin and the attached proteins are biotinylated.  
   
   
       29 . The method of  claim 25 , wherein the protein-binding functional group is a protein designed or selected to attach stably to a functional group on the protein array elements.  
   
   
       30 . The method of  claim 29 , wherein the protein-binding functional group is Protein A or Protein G and the attached proteins are antibodies.  
   
   
       31 . The method of  claim 30 , wherein the protein-binding functional group is glutathione and the attached proteins are GST-protein fusions.  
   
   
       32 . The method of  claim 1 , wherein the protein labels comprise fluorescent dyes.  
   
   
       33 . The method of  claim 32 , wherein the fluorescent dyes comprise amine-reactive cyanine dyes.  
   
   
       34 . The method of  claim 33 , wherein the amine-reactive cyanine dyes comprise Cyanine 3 and Cyanine 5 dyes.