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.

What is claimed is: 
     
         1 . A method of identifying an altered post-translational modification (PTM) state of a protein in a patient, the method comprising:
 contacting a functional extract of a sample from the patient with an array comprising an ordered plurality of proteins that are representative of proteins in the patient;   establishing a specific PTM reaction on the array, whereby the reaction results in a PTM of one or more proteins in the array through the activity of one or more enzymes present in the extract;   determining a level of the PTM of said one or more proteins by detecting a signal from the array; and   comparing the level of PTM of said one or more proteins in the array with the level of PTM of the corresponding one or more proteins in a control sample to identify an altered PTM state of a protein in the patient.   
     
     
         2 . The method of  claim 1 , wherein the PTM is selected from the group consisting of ubiquitination, phosphorylation, sumoylation, and neddylation. 
     
     
         3 . The method of  claim 1 , wherein the patient sample is obtained from a source selected from the group consisting of plasma, cerebrospinal fluid, saliva, urine, and a biopsy specimen comprising cells or tissues of the patient. 
     
     
         4 . The method of  claim 3 , wherein the source is a biopsy specimen which is a tumor. 
     
     
         5 . The method of  claim 1 , wherein the patient is suspected of having cancer, a neurodegenerative disease, a metabolic disease, an immune disease, or an infectious disease. 
     
     
         6 . The method of  claim 5 , wherein the patient is suspected of having a neurodegenerative disease and the neurodegenerative disease is Alzheimer's disease or Huntington's disease. 
     
     
         7 . The method of  claim 1  wherein, prior to or during the step of establishing a specific PTM reaction, the extract is supplemented with a reagent, a substrate, an enzyme, an enzyme inhibitor, ATP, creatine phosphate, a drug, an antibody, or a combination thereof. 
     
     
         8 . The method of  claim 7 , wherein the extract is supplemented with a substrate of an enzyme that carries out said specific PTM reaction. 
     
     
         9 . The method of  claim 7 , wherein the extract is supplemented with an inhibitor of an enzyme that reverses said specific PTM reaction. 
     
     
         10 . The method of  claim 1 , wherein the functional extract is prepared by a method comprising hypotonic lysis or freeze-thawing of cells in the patient sample. 
     
     
         11 . The method of  claim 10 , wherein the extract is prepared by adding lysis buffer in an amount no more than 0.8 times the volume of cells. 
     
     
         12 . The method of  claim 1 , wherein the functional extract is essentially detergent-free. 
     
     
         13 . The method of  claim 1 , wherein the signal is a light signal. 
     
     
         14 . The method of  claim 13 , wherein, in the step of determining, the position and intensity of the light signal are measured. 
     
     
         15 . The method of  claim 1  further comprising, following the step of establishing a specific PTM reaction, washing the reaction components off the array and incubating the array with a first antibody that specifically binds proteins in the array bearing said specific PTM. 
     
     
         16 . The method of  claim 15 , further comprising washing unbound first antibody off the array and contacting the array with a second antibody that specifically binds the first antibody, wherein the second antibody is attached to a label moiety. 
     
     
         17 . The method of  claim 1  further comprising, prior to the step of comparing, performing the steps of establishing and determining using a control sample instead of the patient sample and a second array comprising said ordered plurality of proteins, wherein the results obtained from the step of determining using the control sample are compared with the results obtained from the step of determining using the patient sample in the step of comparing. 
     
     
         18 . The method of  claim 1  further comprising, prior to the step of contacting, preparing the functional extract. 
     
     
         19 . The method of  claim 1 , wherein two or more specific PTM reactions are established simultaneously. 
     
     
         20 . The method of  claim 1 , wherein the step of establishing a specific PTM reaction includes adding a biochemical energy source to the extract. 
     
     
         21 . The method of  claim 20 , wherein the biochemical energy source is a mixture of ATP and creatine phosphate. 
     
     
         22 . The method of  claim 1 , wherein the step of establishing a specific PTM reaction includes adding a protein modifying moiety to the extract. 
     
     
         23 . The method of  claim 22 , wherein the modifying moiety is a ubiquitin-like modifying moiety selected from the group consisting of ISG15, UCRP, FUB1, NEDD8, FAT10, SUMO-1, SUMO-2, SUMO-3, Apg8, Apg12, Urml, UBL5, and Ufm1. 
     
     
         24 . A method of identifying a protein PTM enzyme activity in a patient, the method comprising:
 contacting a functional extract of a sample from the patient with an array comprising an ordered plurality of proteins that are representative of proteins in the patient;   establishing a specific PTM reaction on the array, whereby the reaction results in a PTM of one or more proteins in the array through said enzyme activity present in the extract;   determining a level of the PTM of said protein by detecting a signal from the array; and   comparing the results of the step of determining with results obtained for a control reaction lacking said functional extract, wherein a PTM enzyme activity in the patient is identified when the level of the PTM of said protein is higher than the level of the PTM for said protein in the control.   
     
     
         25 . The method of  claim 24 , wherein the PTM is selected from the group consisting of ubiquitination, phosphorylation, sumoylation, and neddylation. 
     
     
         26 . The method of  claim 24 , wherein the patient sample is obtained from a source selected from the group consisting of plasma, cerebrospinal fluid, saliva, urine, and a biopsy specimen comprising cells or tissues of the patient. 
     
     
         27 . The method of  claim 26 , wherein the source is a tumor. 
     
     
         28 . The method of  claim 24 , wherein the patient is suspected of having cancer, a neurodegenerative disease, a metabolic disease, an immune disease, or an infectious disease. 
     
     
         29 . The method of  claim 28 , wherein the patient is suspected of having a neurodegenerative disease and the neurodegenerative disease is Alzheimer's disease or Huntington's disease. 
     
     
         30 . The method of  claim 24 , wherein prior to or during the step of contacting, the extract is mixed with a reagent, a substrate, an enzyme, an enzyme inhibitor, ATP, creatine phosphate, a drug, an antibody, or a combination thereof. 
     
     
         31 . The method of  claim 30 , wherein the extract is supplemented with a substrate for said PTM enzyme. 
     
     
         32 . The method of  claim 30 , wherein the extract is supplemented with an inhibitor of said PTM enzyme. 
     
     
         33 . The method of  claim 24 , wherein the functional extract is prepared by a method comprising hypotonic lysis or freeze-thawing of cells in the patient sample. 
     
     
         34 . The method of  claim 33 , wherein the extract is prepared by adding lysis buffer in an amount no more than 0.8 times the volume of cells. 
     
     
         35 . The method of  claim 24 , wherein the functional extract is essentially detergent-free. 
     
     
         36 . The method of  claim 24 , wherein the signal is a light signal. 
     
     
         37 . The method of  claim 36  wherein, in the step of determining, the position and intensity of the light signal are measured. 
     
     
         38 . The method of  claim 24  further comprising, following the step of establishing a specific PTM reaction, washing the reaction components off the array and incubating the array with a first antibody that specifically binds proteins in the array bearing said specific PTM. 
     
     
         39 . The method of  claim 38 , further comprising washing unbound first antibody off the micrroarray and contacting the array with a second antibody that specifically binds the first antibody, wherein the second antibody is attached to a label moiety. 
     
     
         40 . The method of  claim 24  further comprising, prior to the step of contacting, preparing the functional extract. 
     
     
         41 . The method of  claim 24 , wherein two or more specific PTM reactions are established simultaneously. 
     
     
         42 . The method of  claim 24 , wherein the step of establishing a specific PTM reaction includes adding a biochemical energy source to the extract. 
     
     
         43 . The method of  claim 42 , wherein the biochemical energy source is a mixture of ATP and creatine phosphate. 
     
     
         44 . The method of  claim 24 , wherein the step of establishing a specific PTM reaction includes adding a protein modifying moiety to the extract. 
     
     
         45 . The method of  claim 44 , wherein the modifying moiety is a ubiquitin-like modifying moiety selected from the group consisting of ISG15, UCRP, FUB1, NEDD8, FAT10, SUMO-1, SUMO-2, SUMO-3, Apg8, Apg12, Urml, UBL5, and Ufm1. 
     
     
         46 . A method of diagnosing a disease or medical condition in a patient, the method comprising:
 contacting a functional extract of a sample from the patient with an array comprising an ordered plurality of proteins that are representative of proteins in the patient;   establishing a specific PTM reaction on the array, whereby the reaction results in a PTM of one or more proteins in the array through the activity of one or more enzymes present in the extract;   identifying one or more post-translationally modified proteins in the array by detecting a signal from the array to yield a PTM state data set for the patient sample; and   comparing the PTM state data set with a standard data set comprising PTM state data that are diagnostic for the disease or medical condition, so as to diagnose the disease or medical condition in the patient.   
     
     
         47 . The method of  claim 46 , wherein the signal generated on the array indicates a PTM state selected from the group consisting of ubiquitination, phosphorylation, sumoylation, and neddylation. 
     
     
         48 . The method of  claim 46 , wherein the patient sample is obtained from a source selected from the group consisting of plasma, cerebrospinal fluid, saliva, urine, and a biopsy specimen comprising cells or tissues of the patient. 
     
     
         49 . The method of  claim 48 , wherein the source is a tumor. 
     
     
         50 . The method of  claim 46 , wherein the patient is suspected of having cancer, a neurodegenerative disease, a metabolic disease, an immune disease, or an infectious disease. 
     
     
         51 . The method of  claim 48 , wherein the patient is suspected of having a neurodegenerative disease and the neurodegenerative disease is Alzheimer's disease or Huntington's disease. 
     
     
         52 . The method of  claim 46  further comprising, following the step of contacting, washing the reaction components off the array and incubating the array with a first antibody that specifically binds proteins in the array bearing said specific PTM. 
     
     
         53 . The method of  claim 52 , further comprising washing unbound first antibody off the micrroarray and contacting the array with a second antibody that specifically binds the first antibody, wherein the second antibody is attached to a label moiety. 
     
     
         54 . The method of  claim 46  further comprising, prior to the step of contacting, preparing the functional extract. 
     
     
         55 . The method of  claim 46 , wherein two or more specific PTM reactions are established simultaneously. 
     
     
         56 . The method of  claim 46 , wherein the step of establishing a specific PTM reaction includes adding a biochemical energy source to the extract. 
     
     
         57 . The method of  claim 56 , wherein the biochemical energy source is a mixture of ATP and creatine phosphate. 
     
     
         58 . The method of  claim 46 , wherein the step of establishing a specific PTM reaction includes adding a protein modifying moiety to the extract. 
     
     
         59 . The method of  claim 58 , wherein the modifying moiety is a ubiquitin-like modifying moiety selected from the group consisting of ISG15, UCRP, FUB1, NEDD8, FAT10, SUMO-1, SUMO-2, SUMO-3, Apg8, Apg12, Urm1, UBL5, and Ufm1. 
     
     
         60 . A kit for the diagnosis of a disease or medical condition or the detection of a biological state by the analysis of a PTM state of a protein in a patient sample, the kit comprising:
 a standard comprising one or more functional extracts capable of producing a known pattern of protein PTM states on a protein microarray, wherein the pattern is diagnostic for the disease, medical condition, or biological state; and   instructions for carrying out the method of any one of  claims 1 ,  24 , and  46 .   
     
     
         61 . The kit of  claim 60 , further comprising a reagent selected from the group consisting of a substrate, an enzyme, an enzyme inhibitor, a drug, an antibody, ATP, creatine phosphate, and a protein modifying moiety. 
     
     
         63 . The kit of  claim 60 , further comprising a protein microarray. 
     
     
         64 . A method of identifying a set of biomarkers for a disease, medical condition, or biological state, comprising:
 comparing a protein PTM profile from one or more patients having the disease, medical condition, or biological state with a protein PTM profile from one or more control subjects who do not have the disease, medical condition, or biological state, wherein the profiles are obtained by analyzing functional extracts from the patients and control subjects with a microarray containing an ordered plurality of proteins according to the method of any one of  claims 1 ,  24 , and  46 ; and   determining one or more differences in a PTM state of one or more proteins between the patient and control profiles, wherein said one or more differences are identified as a set of biomarkers for the disease, medical condition, or biological state.