| Autor: |
Zamora-Olivares D; Department of Chemistry , The University of Texas at Austin , Austin , Texas 78712 , United States.; Texas Institute for Discovery Education in Science and Freshman Research Initiative , The University of Texas at Austin , Austin , Texas 78712 , United States., Kaoud TS; Division of Chemical Biology & Medicinal Chemistry, College of Pharmacy , The University of Texas at Austin , Austin , Texas 78712 , United States.; Medicinal Chemistry Department, Faculty of Pharmacy , Minia University , Minia 61519 , Egypt., Zeng L; College of Chemistry and Molecular Science , Wuhan University , Wuhan , Hubei 430072 , China., Pridgen JR; Division of Chemical Biology & Medicinal Chemistry, College of Pharmacy , The University of Texas at Austin , Austin , Texas 78712 , United States., Zhuang DL; Texas Institute for Discovery Education in Science and Freshman Research Initiative , The University of Texas at Austin , Austin , Texas 78712 , United States., Ekpo YE; Texas Institute for Discovery Education in Science and Freshman Research Initiative , The University of Texas at Austin , Austin , Texas 78712 , United States., Nye JR; Texas Institute for Discovery Education in Science and Freshman Research Initiative , The University of Texas at Austin , Austin , Texas 78712 , United States., Telles M; Texas Institute for Discovery Education in Science and Freshman Research Initiative , The University of Texas at Austin , Austin , Texas 78712 , United States., Anslyn EV; Department of Chemistry , The University of Texas at Austin , Austin , Texas 78712 , United States., Dalby KN; Division of Chemical Biology & Medicinal Chemistry, College of Pharmacy , The University of Texas at Austin , Austin , Texas 78712 , United States.; Department of Oncology, Dell Medical School , The University of Texas at Austin , Austin , Texas 78712 , United States. |
| Abstrakt: |
The understanding of complex biological systems requires an ability to evaluate interacting networks of genes, proteins, and cellular reactions. Enabling technologies that support the rapid quantification of these networks will facilitate the development of biological models and help to identify treatment targets and to assess treatment plans. The biochemical process of protein phosphorylation, which underlies almost all aspects of cell signaling, is typically evaluated by immunoblotting procedures (Western blot) or more recently proteomics procedures, which provide qualitative estimates of the concentration of proteins and their modifications in cells. However, protein modifications are difficult to correlate with activity, and while immunoblotting and proteomics approaches have the potential to be quantitative, they require a complex series of steps that diminish reproducibility. Here, a complementary approach is presented that allows for the rapid quantification of a protein kinase activity in cell lysates and tumor samples. Using the activity of cellular ERK (extracellular signal-regulated kinase) as a test case, an array sensing approach that utilizes a library of differential peptide-based biosensors and chemometric tools was used to rapidly quantify nanograms of active ERK in micrograms of unfractionated cell lysates and tumor extracts. This approach has the potential both for high-throughput and for quantifying the activities of multiple protein kinases in a single biological sample. The critical advantages of this differential sensing approach over others are that it removes the need for the addition of exogenous inhibitors to suppress the activities of major off-target kinases and allows us to quantitate the amount of active kinase in tested samples rather than measuring the changes in its activity upon induction or inhibition. |
