New methods to quantify protein kinase activities directly from complex cellular mixtures are critical for understanding biological regulatory pathways. Herein, a fluorescence-based chemosensor strategy for the direct measurement of kinase activities in crude mammalian cell lysates is described. We first designed a new fluorescent peptide reporter substrate for each target kinase. These kinase chemosensors were readily phosphorylated by recombinant target enzyme and underwent a several-fold fluorescence increase upon phosphorylation. Then, using unfractionated cell lysates, a homogeneous kinase assay was developed that was reproducible, linear and highly preferential for monitoring changes in cellular activity of the target kinase. The general protocol was developed for the kinase Akt and then easily extended to measure protein kinase A (PKA) and mitogen-activated protein kinase-associated protein kinase 2 (MK2) activities. This assay platform is immediately useful for studying protein kinase signaling in crude cellular extracts.

We introduce a versatile fluorescent peptide reporter of protein kinase activity. The probe can be modified to target a desired kinase by changing the kinase recognition motif in the peptide sequence. The reporter motif contains the Sox amino acid, which generates a fluorescence signal when bound to Mg2+ present in the reaction mixture. The phosphorylated peptide exhibits a much greater affinity for Mg2+ than its unphosphorylated analogue and, thus, a greater fluorescence intensity. Product formation during phosphorylation by the kinase is easily followed by the increase in fluorescence intensity over time. These probes exhibit a 3-5-fold increase in fluorescence intensity upon phosphorylation, the magnitude of which depends on the substrate. Peptides containing the reporter functionality are phosphorylated on serine by Protein Kinase C and cAMP-dependent protein kinase and are shown to be good substrates for these enzymes. The principle of this design extends to peptides phosphorylated on threonine and tyrosine.

A modular peptide scaffold has been developed for fluorescent sensing of divalent zinc. The signaling component of the chemosensor is the chelation-sensitive fluorophore 8-hydroxy-5-(N,N-dimethylsulfonamido)-2-methylquinoline, which is prepared as the protected amino acid derivative Fmoc-Sox-OH and integrated into peptide sequences. Nineteen synthetic peptides incorporating the signaling element exhibit a range of affinities for Zn(2+) through variation of the type and number of Zn(2+) ligands, ligand arrangement and the beta-turn sequence that acts as a preorganization element between the ligands. The stoichiometry of the peptide-Zn(2+) complexes is evaluated by several criteria. The fluorescence response of these peptides to pH and various important metal ions is reported. Eleven of these sequences form only 1:1 complexes with Zn(2+) and their affinities range from 10 nM to nearly 1 microM. When used in concert, these sensors can provide Zn(2+) concentration information in a valuable range.

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We report a novel protein kinase assay designed for high-throughput detection of one or many kinases in a complex mixture. A solution-phase phosphorylation reaction is performed on 900 different peptide substrates, each covalently linked to an oligonucleotide tag. After incubation, phosphoserine, phosphothreonine, and phosphotyrosine are chemically labeled, and the substrates are hybridized to a microarray with oligonucleotides complementary to the tags to read out the phosphorylation state of each peptide. Because protein kinases act on more than one peptide sequence, each kinase can be characterized by a unique signature of phosphorylation activity on multiple substrates. Using this method, we determined signatures for 26 purified kinases and demonstrated that enzyme mixtures can be screened for activity and selectivity of inhibition.

We have developed a miniaturized and multiplexed solution assay for the measurement of protease activity in complex samples. This technology can accelerate research in functional proteomics and enable biologists to carry out multiplexed protease inhibitor screens on a large scale. The assay readout is based on Illumina's universal Sentrix BeadArrays. The peptide sequences that serve as protease substrates are conjugated to oligonucleotide sequences complementary to the oligo tags on randomly assembled and decoded bead arrays. The peptide portion is C-terminally labeled with a biotin residue and contains a sequence of five histidine residues on the amino terminus. The unique oligonucleotide part of each oligonucleotide-peptide conjugate is attached to amino terminus of the peptide sequence. Upon protease cleavage, the biotin residue is cleaved from the oligonucleotide-peptide conjugate. Following the reaction, all biotin-containing species are captured and removed by incubation with streptavidin beads. The cleaved conjugates that remain in solution are captured by hybridization of their oligo sequence to Sentrix BeadArrays and detected using a labeled antibody against pentahistidine tag of the conjugate or by an antibody sandwich assay. We have generated multiple sets of oligonucleotide tagged peptide substrates of varying complexity (100 to 1000 substrates in a mixture) and show that the response of individual substrate is independent of the complexity of the mixture. Our initial results demonstrate the feasibility of assaying proteases in a multiplexed environment with high sensitivity.