Find out how we provide both researchers timely and effective lab services!
Find out how we provide both researchers timely and effective lab services!
Kinases are specialized enzymes that transfer phosphate groups from ATP to a substrate. The most often studied kinases are protein kinases, which are generally subdivided into tyrosine, and serine/threonine-directed kinases.
As such, kinases are key signal transduction molecules relevant in almost every cellular process from cellular proliferation to DNA repair. Kinases alterations have been identified in many cancers (often as oncogenes) but also in several non-cancer diseases.
Kinomic profiling is achieved using the PamStation®12 Platform Technology (PamGene, International, Den Bosch, The Netherlands) that is a high-content phospho-peptide substrate microarray system.
In the PamStation®12 system, active kinases within a cellular/tissue lysate will phosphorylate specific peptide substrates within the microarray which survey the particular kinome (tyrosine or serine/threonine kinome) of interest. These phosphorylation events are detected using fluorescently-labeled anti-phospho-tryosine or anti-phospho-serine/threonine antibodies in real time with kinetic evaluation. Cell lysates are prepared using M-Per lysis buffer with protease and phosphatase inhibitor cocktails (Pierce). The PamChips® are blocked in 2% Bovine Serum Albumin (BSA). Following protein concentration determination (by BCA assay), 5-10 μg of protein for the PTK chip (~1 μg for the STK) will be loaded per well of the PamChip along with standard kinase buffer (Supplied by PamGene), 100 μM ATP, and FITC-labeled anti-phospho-tyrosine (for PTK) or phosphoserine/threonine (for STK) antibodies. For each PamChip®, 4 separate samples can be assayed since each of the wells contains the peptide substrate microarray surveying the tyrosine kinome (PTK) or serine/threonine (STK) kinome. The PamChips® can be assayed 3 chips at a time allowing for 4 samples in triplicate to be assayed at once.
The assay mix is pumped through the PamChips® with a kinetic image capture program (Evolve software, PamGene) in which exposures of phosphorylated peptide substrates are taken as frequently as every 6 seconds for the length of the program (45-90 minutes). The BioNavigator software platform (PamGene) provides a means to analyze the raw data and perform virtually any manipulation of the data. The BioNavigator Microarray software works seamlessly with the open source microarray statistical package, R, commercial products such as GeneSpring as well as knowledge bases such as MetaCore (GeneGo, Inc., a Thomson-Reuters Company), to develop pathway maps and biological networks.
Kinomics is the study of the kinome, a global description of kinases and kinase signaling. Since kinases drive numerous signaling pathways in biology (both normal and disease), determining the pertinent kinases in a biological system is of high importance. There are several different ways to study the kinome: RNA interference, mass spectrometry, and antibody arrays, just to name a few.
In our facility, we utilize the PamStation®12 (PamGene International) Kinomic Array platform to study the kinome. This system utilizes a high content peptide microarray containing phosphorylatable tyrosine (PTK chip) or serine/threonine (STK chip) residues within kinase substrates. Active kinases within a sample will phosphorylate specific peptides on the microarray to generate a kinomic profile, or fingerprint.
Kinomic profiles can help elucidate cellular signaling pathways driving particular biological processes and phenotypes and be used to identify and develop biomarkers.
Contact us prior to planning your experiments to we can help you best design your experiments to get the most from our profiling platform. Once we are in contact with you, cellular or tissue lysates can be prepared according to the "Sample Prep" protocol.
1. Shinde A, Hardy SD, Kim D, Akhand SS, Jolly MK, Wang WH, Anderson JC, Khodadadi RB, Brown WS, George JT, Liu S, Wan J, Levine H, Willey CD, Krusemark CJ, Geahlen RL, Wendt MK. Spleen tyrosine kinase-mediated autophagy is required for epithelial-mesenchymal plasticity and metastasis in breast cancer. Cancer Res. 2019. Epub 2019/02/09. doi: 10.1158/0008-5472.CAN-18-2636. PubMed PMID: 30733195.
2. Ibrahim AN, Yamashita D, Anderson JC, Abdelrashid M, Alwakeal A, Estevez-Ordonez D, Komarova S, Markert JM, Goidts V, Willey CD, Nakano I. Intratumoral spatial heterogeneity of BTK kinomic activity dictates distinct therapeutic response within a single glioblastoma tumor. J Neurosurg. 2019:1-12. Epub 2019/10/20. doi: 10.3171/2019.7.JNS191376. PubMed PMID: 31628288.
3. Aye JM, Andreas J, Lenzo FL, Burgher B, Hagen J, Giamo V, Nesline MK, Wang Y, Gardner M, Conroy JM, Papanicolau-Sengos A, Morrison C, Glenn ST. The effects of focal adhesion kinase and platelet-derived growth factor receptor beta inhibition in a patient-derived xenograft model of primary and metastatic Wilms tumor. Oncotarget. 2019;10(53). doi: 10.18632/oncotarget.27165.
4. Walker K, Boyd NH, Anderson JC, Willey CD, Hjelmeland AB. Kinomic profiling of glioblastoma cells reveals PLCG1 as a target in restricted glucose. Biomark Res. 2018;6:22. Epub 2018/06/28. doi: 10.1186/s40364-018-0136-9. PubMed PMID: 29946469; PMCID: PMC6001119.
5. Gilbert AN, Anderson JC, Duarte CW, Shevin RS, Langford CP, Singh R, Gillespie GY, Willey CD. Combinatorial Drug Testing in 3D Microtumors Derived from GBM Patient-Derived Xenografts Reveals Cytotoxic Synergy in Pharmacokinomics-informed Pathway Interactions. Scientific reports. 2018;8(1):8412. Epub 2018/06/01. doi: 10.1038/s41598-018-26840-4. PubMed PMID: 29849102; PMCID: PMC5976646.
6. Dussaq AM, Kennell T, Jr., Eustace NJ, Anderson JC, Almeida JS, Willey CD. Kinomics toolbox-A web platform for analysis and viewing of kinomic peptide array data. PLoS One. 2018;13(8):e0202139. Epub 2018/08/22. doi: 10.1371/journal.pone.0202139. PubMed PMID: 30130366; PMCID: PMC6103510.
7. Yang ES, Willey CD, Mehta A, Crowley MR, Crossman DK, Chen D, Anderson JC, Naik G, Della Manna DL, Cooper TS, Sonpavde G. Kinase analysis of penile squamous cell carcinoma on multiple platforms to identify potential therapeutic targets. Oncotarget. 2017;8(13):21710-8. Epub 2017/04/21. doi: 10.18632/oncotarget.15558. PubMed PMID: 28423512; PMCID: PMC5400617.
8. Ghosh AP, Willey CD, Anderson JC, Welaya K, Chen D, Mehta A, Ghatalia P, Madan A, Naik G, Sudarshan S, Sonpavde G. Kinomic profiling identifies focal adhesion kinase 1 as a therapeutic target in advanced clear cell renal cell carcinoma. Oncotarget. 2017;8(17):29220-32. Epub 2017/04/19. doi: 10.18632/oncotarget.16352. PubMed PMID: 28418903; PMCID: PMC5438725.
9. Gilbert AN, Shevin RS, Anderson JC, Langford CP, Eustace N, Gillespie GY, Singh R, Willey CD. Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing. J Vis Exp. 2016(112). doi: 10.3791/54026. PubMed PMID: 27341166.
10. Dussaq A, Anderson JC, Willey CD, Almeida JS. Mechanistic Parameterization of the Kinomic Signal in Peptide Arrays. J Proteomics Bioinform. 2016;9(5):151-7. doi: 10.4172/jpb.1000401. PubMed PMID: 27601856; PMCID: PMC5010871.
11. Stoltz K, Sinyuk M, Hale JS, Wu Q, Otvos B, Walker K, Vasanji A, Rich JN, Hjelmeland AB, Lathia JD. Development of a Sox2 reporter system modeling cellular heterogeneity in glioma. Neuro Oncol. 2015;17(3):361-71. doi: 10.1093/neuonc/nou320. PubMed PMID: 25416826; PMCID: PMC4483103.
12. Isayeva T, Xu J, Ragin C, Dai Q, Cooper T, Carroll W, Dayan D, Vered M, Wenig B, Rosenthal E, Grizzle W, Anderson J, Willey CD, Yang ES, Brandwein-Gensler M. The protective effect of p16(INK4a) in oral cavity carcinomas: p16(Ink4A) dampens tumor invasion-integrated analysis of expression and kinomics pathways. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc. 2015;28(5):631-53. doi: 10.1038/modpathol.2014.149. PubMed PMID: 25523612.
13. Anderson JC, Willey CD, Mehta A, Welaya K, Chen D, Duarte CW, Ghatalia P, Arafat W, Madan A, Sudarshan S, Naik G, Grizzle WE, Choueiri TK, Sonpavde G. High Throughput Kinomic Profiling of Human Clear Cell Renal Cell Carcinoma Identifies Kinase Activity Dependent Molecular Subtypes. PLoS One. 2015;10(9):e0139267. doi: 10.1371/journal.pone.0139267. PubMed PMID: 26406598.
14. Anderson JC, Taylor RB, Fiveash JB, de Wijn R, Gillespie GY, Willey CD. Kinomic Alterations in Atypical Meningioma. Med Res Arch. 2015;2015(3). doi: 10.18103/mra.v0i3.104. PubMed PMID: 27158663; PMCID: PMC4856299.
15. Duverger A, Wolschendorf F, Anderson JC, Wagner F, Bosque A, Shishido T, Jones J, Planelles V, Willey C, Cron RQ, Kutsch O. Kinase control of latent HIV-1 infection: PIM-1 kinase as a major contributor to HIV-1 reactivation. J Virol. 2014;88(1):364-76. doi: 10.1128/JVI.02682-13. PubMed PMID: 24155393; PMCID: PMC3911731.
16. Anderson JC, Minnich DJ, Dobelbower MC, Denton AJ, Dussaq AM, Gilbert AN, Rohrbach TD, Arafat W, Welaya K, Bonner JA, Willey CD. Kinomic profiling of electromagnetic navigational bronchoscopy specimens: a new approach for personalized medicine. PLoS One. 2014;9(12):e116388. doi: 10.1371/journal.pone.0116388. PubMed PMID: 25549342; PMCID: 4280210.
17. Anderson JC, Duarte CW, Welaya K, Rohrbach TD, Bredel M, Yang ES, Choradia NV, Thottassery JV, Yancey Gillespie G, Bonner JA, Willey CD. Kinomic exploration of temozolomide and radiation resistance in Glioblastoma multiforme xenolines. Radiother Oncol. 2014;111(3):468-74. Epub 2014/05/13. doi: 10.1016/j.radonc.2014.04.010. PubMed PMID: 24813092; PMCID: PMC4119546.
18. Jarboe JS, Jaboin JJ, Anderson JC, Nowsheen S, Stanley JA, Naji F, Ruijtenbeek R, Tu T, Hallahan DE, Yang ES, Bonner JA, Willey CD. Kinomic profiling approach identifies Trk as a novel radiation modulator. Radiother Oncol. 2012;103(3):380-7. Epub 2012/05/09. doi: 10.1016/j.radonc.2012.03.014. PubMed PMID: 22561027; PMCID: 3368103.
We provide fee-for-service work for academic and industry investigators through our core service. This can be found at this website.
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