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Kinase-Substrate Profiling Services

Kinase Specificity

In Silico Kinase Specificity Prediction (IKSP) Services

Kinase Match

In Silico Kinase Specificity Prediction (IKMP) Services

Kinase Testing

In Vitro Kinase and Phosphopeptide Testing (IKPT) Services

Kinase-Substrate Discovery

In Vitro Antibody Microarray Analysis (CKSP) Services

Phosphoprotein Match

In Silico Phosphoprotein Specificity Prediction (IPMP) Services

Kinase-Substrate Specificity

Kinex™ Kinase Substrate Microarray
Analysis (KKSM) Services

Kinase-Substrate Discovery

In Vitro Mass Spectrometry Analysis (MSKS) Services
The vast majority of the proteins phosphorylated by specific protein kinases in humans and other species remain unknown despite more than four decades of intense effort. We have identified at least 10 thousand kinase-substrate phosphosite pairs from our literature searches, but we believe that actual number exceeds 10 million. With the emergence of protein kinases as one of the most promising families of drug targets in the pharmaceutical industry today, it is vital to define the proteins and their most critical phosphosites that are controlled by these important regulatory enzymes. We expect that many of the genetic mutations that facilitate disease arise from alterations in the amino acids that define the specificity of protein kinases and in the amino acids in phosphosites that provide for kinase recognition. With the sequencing of tens of thousands of human genomes that are anticipated in the coming decade, establishing linkages between kinases, phosphosites and gene mutations will provide a major advance in the application of personalized medicine. To help realize this potential, Kinexus offers five unique and cost effective services that permit the discovery and validation of kinase substrate connections.

The “in silico” prediction services offered by Kinexus have been created using proprietary software that has been developed by Kinexus and our academic partners at the University of British Columbia and Simon Fraser University. The algorithm for kinase substrate prediction uses our databases of the carefully aligned sequences of 492 human protein kinase catalytic domains and 10,000 known kinase-substrate phosphosite pairs to permit the elucidation of phosphosite amino acid frequency matrices for any classical protein kinase for which the catalytic domain sequence is
known.. With this algorithm, Kinexus has been able to predicted the sequences of optimal peptide substrates for most protein kinases. Conversely, Kinexus can take any known or putative phosphosite sequence and generate a score for each of 482 human protein kinases for their ability to target the sequence for phosphorylation.

Our In Silico Kinase Specificity Prediction (IKSP) Services was originally developed to predict the importance of each of the amino acids surrounding the phosphorylation sites of substrates of typical human protein kinases. However, our research has revealed that our aligorithm may have wider application to other species, including those as diverse as budding yeast. It is also useful for prediction of the possible effects of mutation of human kinases within their catalytic domains on their substrate specificities. With this service, clients provide the name and Uniprot ID or NCBI accession numbers for the desired protein kinase, and Kinexus provides back a table with the expected probability frequencies of each of the 20 amino acids 7 amino acids before and after the phospho-acceptor amino acid. This information can be used to design an optimal substrate peptide to assay this protein kinase. Such a substrate peptide can be synthesized for our clients with our Custom Peptide Synthesis Services, This information can also be used to predict the best physiological substrates of a kinase of interest.

Kinexus has produced a Kinex™ Kinase Substrate Microarray (KKSM) chip that features 445 peptides that correspond to the optimal predicted substrate sequences predicted for 492 human protein kinase catalytic domains using our proprietary algorithms. With the KKSM services, we can test 3 different kinases, sourced from our clients or from Kinexus, for their ability to phosphorylate these peptides. Alignment of the sequences of the best substrate peptides permits experimental deduction of the optimum substrate specificities for these kinases. We have observed as high degree of concordance with the results of our In Silico Kinase Specificity Prediction (IKSP) Services and the Kinex™ Kinase Substrate Microarray (KKSM) Services for kinase substrate specificity determination.

Our most complete phosphosite and kinase prediction for a protein of special interest is provided with our In Silico Phosphoprotein Specificity Prediction (IPMP) Services. Clients only have to provide a short name and the Uniprot ID number for the human protein that they wish to have analyzed. Kinexus will identify all of the best candidate phosphosites for this protein from our library of 650,000 putative and experimentally confirmed human phosphosites and reveal their evolutionary conservation in 20 other species.
In addition, the ranking scores for approximately 500 human protein kinase domains for each of these phosphosites will be provided. These analyses serves to define those phosphosites that probably play a critical role in the regulation of the target protein and the best candidate kinases that target these phosphosites. This provided valuable leads for follow that can be confirmed with the following in vitro proteomics service offered by Kinexus to identify physiological protein substrates and their phosphosites.

Our In Silico Kinase Match Prediction (IKMP) Services permits clients to take a known or putative phosphosite and find the most promising protein kinase candidates that target this phosphosite. Such information is freely available on-line for over 90,000 known and 560,000 predicted human phosphosites in our PhosphoNET KnowledgeBase. However, this custom service is necessary if clients wish to predict kinases for uncharacterized or mutated phosphosites in humans and other species. Our In Silico Kinase Match Prediction (IKMP) Services also allow our clients to start with a human protein kinase of interest and identify the top 1000 or 5000 known and predicted human phosphosites that are likely to be targeted by that kinase.

For over 360 different human protein kinases that score highly for targeting phosphosites of high interest, Kinexus offers an in vitro testing service to validate these predictions. With our In Vitro Kinase and Phosphopeptide Testing (IKPT) Services, Kinexus can test the ability of recombinant protein substrates for phosphorylation by a purified and active protein kinase. Phosphorylation is quantified in each assay by the incorporation of [gamma-33P]ATP into the substrate protein or production of ADP. To identify the sites that are phosphorylated in vitro on a target recombinant protein after this demonstration, Kinexus offers our Mass Spectrometry Protein Phosphosite (MSPS) Profiling Services for phosphosite profiling on individual proteins. Interested clients in this follow up service should contact our Technical Services Representatives for more information. Alternatively, Kinexus can synthesize peptides that feature the putative phosphorylation sites for desired target proteins and test these in vitro as substrates for prospective kinases that are predicted from our algorithms. The advantage of this later approach is reduced costs to establish the most likely kinases that target a specific region of interest in a substrate protein.

As alternative higher throughput strategies to identify physiological substrates of specific kinases of interest, Kinexus is pleased to offer two more unique approaches. Our Custom Kinase Substrate Profiling (CKSP) Services uniquely permits the rapid simultaneous identification of a panel of physiological substrates for most known protein kinases in any tissues or cells using our Kinex™ Antibody Microarray, which features over 270 phosphosite antibodies. This cost effective method also provides for the identification of the specific sites at which these substrates are phosphorylated, as well as commercial antibodies that can be used to specifically track and quantify these phosphosites in follow up studies.

The second strategy for kinase substrate identification exploits the power of mass spectrometry to identify phosphosites in cell lysate proteins that are phosphorylated in vitro by an exogenously added protein kinase. With our Mass Spectrometry Kinase-Substrate (MSKS) Profiling Services we use differentially labeled cell lysates with light and heavy isotopes to identify phosphopeptides that demonstrate enhanced phosphorylation following incubation of one of the lysate preparations with a kinase of interest in the presence of ATP. With this approach, a wider range of potential substrates can be identified than by our Kinex™ Antibody Microarray-based method, but it may be necessary to develop custom phosphosite specific antibodies for follow up. With our Custom Peptide Synthesis Services, Kinexus can also assist in such antibody development.

Please view the specific webpages and our Customer Information Packages for more descriptions about these services.