Methods, Compositions and Kits for High Throughput Kinase Activity Screening Using Mass Spectrometry and Stable Isotopes

Abstract

A mass-spectrometry-based method and substrates are provided herein for large scale kinome activity profiling directly from crude lysates using 90 chemically synthesized peptide substrates with amino acid sequences derived from known phosphoproteins. Quantification of peptide phosphorylation rates was achieved via the use of stable isotope labeled synthetic peptides. Half of these peptides immediately or rapidly showed robust and site-specific phosphorylation after incubation with serum-starved HEK293 cell lysate. A method and substrates for obtaining 90 simultaneous activity measurements in a single-reaction format were developed and validated. Activating kinase pathways through insulin or EGF stimulation reproducibly altered the phosphorylation rates of peptides derived from known pathway protein substrates. While examining cell-cycle-specific activities with the panel, a peptide derived from phosphoinositide 3-kinase regulatory subunit demonstrated mitotic and tyrosine-specific phosphorylation, which was confirmed to be a Src kinase site in vivo. The kinome activity profiling strategy was successfully applied with lysates of each of: cells manipulated by various combination of mitogen stimulation, pharmacological perturbation and siRNA-directed kinase knockdown; seven different breast cancer cell lines treated with gefitinib; and each of normal and cancerous tissue samples from renal cell carcinoma patients. This method concurrently measures multiple peptide phosphorylation rates to provide a diagnostic fingerprint pattern for activated kinases, protein phosphatases, modulators of these enzymes, and pathways (kinome) from as little starting material as a few cells.

1 . A composition comprising an optimized oligopeptide substrate having an amino acid sequence, wherein a site in the amino acid sequence is recognized and phosphorylated by a protein kinase, or is recognized and dephosphoiylated by a protein phosphatase and further comprising at least one modification for purification and analysis by mass spectrometry (MS). 
     
     
         2 . The composition according to  claim 1 , wherein the modification for purification comprises at least one hydrophobic amino acid at a terminus of or at an interior position within the amino acid sequence, wherein the hydrophobic amino acid is selected from the group comprising phenylalanine, leucine, tryptophan, valine, and isoleucine. 
     
     
         3 - 4 . (canceled) 
     
     
         5 . The composition according to  claim 1 , wherein the modification for analysis by MS comprises a charged amino acid, wherein the charged amino acid is selected from the group of arginine, lysine and histidine. 
     
     
         6 - 7 . (canceled) 
     
     
         8 . The composition according to  claim 1 , wherein the modification comprises amino acids arginine-phenylalanine located at the carboxy terminus of the amino acid sequence of the oligopeptide. 
     
     
         9 . The oligopeptide composition according to  claim 1  further comprising an amino acid sequence of a protein kinase substrate chemically bound to a tri-peptide sequence proline-phenylalanine-arginine (PFR), wherein the kinase substrate comprises at least one amino acid for phosphorylation selected from the group serine, threonine, and tyrosine (S, T or Y). 
     
     
         10 . The oligopeptide composition according to  claim 9 , wherein the PFR tripeptide is located at the carboxy terminal end. 
     
     
         11 . (canceled) 
     
     
         12 . The oligopeptide according to  claim 9 , wherein the sequence is selected from the group as shown in Table 2. 
     
     
         13 - 19 . (canceled) 
     
     
         20 . A method for simultaneously measuring a plurality of kinase-related enzyme activities in at least one biological sample, the method comprising:
 contacting an aliquot of the at least one sample with a plurality of optimized peptide substrates under reaction conditions suitable for the plurality of kinase-related enzyme activities, each optimized substrate comprising an amino acid sequence including a phosphorylation site, and amino acid modifications for enrichment and for mass spectrometry (MS);   terminating the reaction and adding a plurality of internal standards, wherein the internal standards comprise amino acid sequences corresponding to amino acid sequences of the peptide substrates, wherein at least one end terminal amino acid of each internal standard further comprises label with a heavy stable isotope;   enriching phosphopeptide reaction products by immobilized metal ion affinity chromatography or titanium dioxide interaction chromatography, wherein prior to enriching the sample is passaged through a C18 solid phase extraction cartridge; and,   analyzing reaction products by ultra-high resolution MS, wherein a plurality of reaction products and internal standards are detected and measured.   
     
     
         21 . The method according to  claim 20 , wherein a single incubation measuring the plurality of kinase-related enzyme activities is performed in a single container, wherein the plurality is at least 10 enzyme activities, at least about 50 enzyme activities, or at least about 100 enzyme activities. 
     
     
         22 . The method according to  claim 21  further comprising reducing a cross-phosphorylation of the peptide substrates. 
     
     
         23 . The method according to  claim 22  wherein an optimized peptide substrate concentration is less than about 5 μM or less than about 1 μM. 
     
     
         24 - 27 . (canceled) 
     
     
         28 . The method according to  claim 20 , wherein the at least one biological sample is a mixture of at least five samples or a mixture of at least ten samples. 
     
     
         29 . (canceled) 
     
     
         30 . The method according to  claim 20 , wherein the at least one biological sample is from a mammalian or avian subject and is selected from the group of biological fluids comprising: a cell lysate, a tissue homogenate, urine, saliva, tears, sweat, blood, lymph, serum, spinal fluid, vaginal fluid, semen, and milk. 
     
     
         31 - 32 . (canceled) 
     
     
         33 . The method according to  claim 20 , wherein kinase-related enzyme activities comprises a profile of at least one selected from the group of protein kinases, protein phosphatases, and inhibitors and modulators of activities thereof. 
     
     
         34 . The method according to  claim 33 , wherein the enzyme activities are protein kinases, wherein the substrates are unphosphorylated and the internal standards are phosphorylated, and wherein the method further comprises after terminating the reaction, enriching by depleting the sample of unphosphorylated substrates by performing the immobilized metal affinity ion chromatography. 
     
     
         35 - 36 . (canceled) 
     
     
         37 . The method according to  claim 33 , wherein the enzyme activities are protein phosphatases, wherein the substrates are phosphorylated and the internal standards are unphosphorylated, and wherein the method further comprises after terminating the reaction, enriching by depleting the sample of phosphorylated substrates by immobilized metal ion affinity chromatography. 
     
     
         38 - 39 . (canceled) 
     
     
         40 . The method according to  claim 33  further comprising associating at least one protein kinase with at least one specific substrate in the kinase-related enzyme profile. 
     
     
         41 . A method for determining a kinase activation pattern for a disease condition, the method comprising:
 contacting an aliquot of a first biological sample with a plurality of optimized peptide substrates under conditions suitable for reaction of the plurality of kinase activities, wherein each optimized substrate comprises an amino acid sequence including a kinase phosphorylation site and an end terminal amino acid sequence modification for enhanced enrichment and mass spectrometry;   adding a plurality of internal standards to the reaction, each having at least one phosphorylated amino acid, and corresponding in sequence to the peptide substrates and further comprising an end terminal amino acid labeled with a heavy stable isotope;   enriching phosphopeptide reaction products and internal standards by immobilized metal ion affinity chromatography of the reaction, titanium dioxide affinity chromatography or the like; and,   analyzing reaction products by ultra-high resolution mass spectrometry, wherein a plurality of reaction products and internal standards are detected and measured, thereby generating a first kinase activation pattern for the sample, and comparing the first kinase activation pattern to second kinase activation pattern for a second biological sample, wherein the second biological sample is from a subject in need of diagnosis or prognosis of disease —  conditions selected from the group consisting of cardiac disease, inflammation, an early stage dystrophic tissue, a polyp, a potential tumor, and an advanced stage cancer tissue, and wherein the first biological sample is obtained from a normal tissue.   
     
     
         42 . The method according to  claim 41 , wherein the second biological sample is selected from the group of: a biopsy, an autopsy, an archival sample, a cell culture, and a tissue culture. 
     
     
         43 . The method according to  claim 41 , wherein the first sample and the second sample are selected respectively from: different members of a family; from cell cultures grown under different conditions wherein the different conditions are presence and absence, respectively, of at least one agent selected from the group of chemotherapeutic agent, mitogen, tumor promoter, kinase inhibitor, phosphatase inhibitor, protease inhibitor, modulator of kinase expression, and modulator of phosphatase expression; wherein the first and second samples are from cell cultures obtained at different time points; and wherein the first and second samples are from the same subject taken at different time points in the course of treatment and the method further comprises prognosis of success of the treatment or further comprises altering a course of chemotherapy. 
     
     
         44 - 47 . (canceled) 
     
     
         48 . The method according to  claim 43 , wherein analyzing reaction products is analyzing at least about five enzyme activities. 
     
     
         49 - 50 . (canceled) 
     
     
         51 . A kit for kinome activity assay for measuring a plurality of enzymes involved in kinase pathways (KAYAK), the kit comprising a plurality of optimized oligopeptide kinase substrates for the plurality of enzymes, each oligopeptide having an amino acid sequence comprising a protein kinase substrate and an end terminal modification for enrichment of a reaction product and enhanced mass spectrometry, the kit further comprising a plurality of internal standards, the internal standards having an amino acid sequences corresponding to the respective substrates, wherein the respective internal standard is phosphorylated and further comprises an end terminal amino acid labeled with a heavy isotope. 
     
     
         52 . The kit according to  claim 51 , wherein the kit is further characterized by at least one selected from:
 the end terminal modification comprises at least one hydrophobic amino acid located at the carboxy terminal end;   the amino acid sequence is selected from the group shown in Tables 1 and 2; and   the kit further comprising a container and instructions for use.   
     
     
         53 - 54 . (canceled) 
     
     
         55 . The kit according to  claim 51  wherein the plurality of optimized kinase substrates and corresponding internal standards is selected as prognostic and diagnostic of a course of a cancer, a cardiac condition, or an inflammatory condition, wherein the plurality of kinases are assayed simultaneously and provide a profile of the kinome of a sample.