Diagnostic method based on large scale identification of post-translational modification of proteins

Abstract

Methods for the large scale identification of post-translational modification states of proteins and enzyme activities for carrying out post-translational modification reactions involve the analysis of functional extracts from fresh and frozen samples using protein arrays. The methods and kits of the present invention can be used to analyze and characterize compounds for their effects on post-translational modifications and their pathways. The methods and kits can also be used to diagnose and characterize a wide variety of diseases and medical conditions, including cancer, neurodegenerative diseases, immune diseases, infectious diseases, genetic diseases, metabolic conditions, and drug effects using cells or body fluids of a patient.

1 . A method of identifying at least one post-translational modification (PTM) or PTM alteration on at least one protein, the method comprising the steps of:
 (a) contacting a functional cell extract with a solid state array, the array comprising an ordered plurality of proteins under conditions that allow PTM to occur or that allow PTM to be modified;   (b) establishing at least one PTM reaction or PTM alteration reaction thereof on the array, whereby the reaction results in at least one PTM or PTM alteration of at least one protein on the array through the activity of one or more enzymes present in the cell extract; and   (c) detecting the at least one PTM or PTM alteration by detecting a signal from the array thereby identifying the PTM or PTM alteration on the at least one protein.   
     
     
         2 . The method of  claim 1 , further comprising identifying the effect of a test agent on the PTM or PTM alteration comprising the additional steps of:
 (a) contacting the functional cell extract with a test agent;   (b) establishing at least one PTM reaction or PTM alteration on the array in the presence of the test agent, whereby the PTM reaction results in at least one PTM or PTM alteration of at least one protein on the array through the activity of one or more enzymes present in the cell extract; and   (c) detecting the at least one PTM or PTM alteration and comparing the PTM reaction or PTM alteration reaction with a parallel reaction where a control agent has been added thereby allowing for detection of an effect of the test agent on at least one PTM or PTM alteration.   
     
     
         3 . The method of  claim 1 , wherein increase in the signal from the array compared to a background or the reaction with a control is indicative of increased PTM. 
     
     
         4 . The method of  claim 1 , wherein decrease in the signal from the array compared to a background or the control is indicative of PTM alteration. 
     
     
         5 . The method of  claim 1 , wherein the detecting is performed using an antibody or antigen-binding fragment thereof, a natural or recombinant ligand, a small molecule, a modifying moiety, or a biochemical analysis capable of detecting the PTM or PTM alteration. 
     
     
         6 . The method of  claim 5 , wherein the antibody or antigen-binding fragment thereof, the natural or recombinant ligand, the small molecule, or the modifying moiety is labeled with a tag. 
     
     
         7 . The method of  claim 6 , wherein the tag is a fluorescent molecule, a radioisotope, a nucleotide chromophore, an enzyme, a substrate, a chemiluminescent moiety, magnetic particle, bioluminescent moiety, or peptide. 
     
     
         8 . The method of  claim 5 , wherein the biochemical analysis is performed using mass spectroscopy, peptide mapping, or amino acid sequencing. 
     
     
         9 . The method of  claim 1 , wherein the functional cell extract is not diluted prior to said contacting with the solid state array. 
     
     
         10 . The method of  claim 1 , wherein the functional cell extract is obtained from a frozen or cryopreserved sample. 
     
     
         11 . The method of  claim 1 , wherein an additional cellular energy source in the form of ATP is provided to the functional cell extract. 
     
     
         12 . The method of  claim 1 , wherein the array comprising a plurality of proteins, comprises at least one protein, protein fragment or peptide attached to the array without an added tag. 
     
     
         13 . The method of  claim 1 , wherein the array comprising a plurality of proteins comprises at least one protein, protein fragment or peptide attached to the array with a C-terminal or N-terminal tag. 
     
     
         14 . The method of  claim 1 , wherein the functional cell extract is derived from a specified cellular compartment. 
     
     
         15 . The method of  claim 14 , wherein the cellular compartment is nucleus. 
     
     
         16 . The method of  claim 14 , wherein the cellular compartment is cytosol. 
     
     
         17 . The method of  claim 14 , wherein the cellular compartment is mitochondria. 
     
     
         18 . The method of  claim 1 , wherein the functional cell extract is derived from a biological sample. 
     
     
         19 . The method of  claim 18 , wherein the biological sample is selected from the group consisting of saliva, blood, serum, plasma, urine, cerebrospinal fluid, chorionic villus, placenta, solid tissue, amniotic fluid, a cell sample, and a tissue culture sample. 
     
     
         20 . The method of  claim 1 , wherein the PTM is selected from the group consisting of ubiquitination, phosphorylation, glycosylation, sumoylation, acetylation, S-nitrosylation or nitrosylation, citrullination or deimination, neddylation, OClcNAc, ADP-ribosylation, methylation, hydroxylation, fattenylation, ufmylation, prenylation, myristoylation, S-palmitoylation, tyrosine sulfation, formylation, carboxylation, and any combination thereof. 
     
     
         21 . The method of  claim 1 , wherein the PTM alteration is selected from the group consisting of deubiquitination (DUB), dephosphorylation, deglycosylation, desumoylation, deacetylation, de-S-nitrosylation or denitrosylation, decitrullination or dedeimination, deneddylation, removal of OClcNAc, de-ADP-ribosylation, demethylation, de-hydroxylation, defattenylation, deufmylation, deprenylation, demyristoylation, de-S-palmitoylation, tyrosine desulfation, deformylation, decarboxylation, deamidation, and any combination thereof. 
     
     
         22 . The method of  claim 1 , wherein the solid state array is selected from the group consisting of protein arrays on microchips, ELISA plates with immobilized proteins attached on the plates, protein-coated beads, and microfluidic chips coated with desired proteins. 
     
     
         23 . The method of  claim 1 , wherein 2-10 PTM or PTM alterations thereof are identified simultaneously.